WO2016210352A2

WO2016210352A2 - Synthesis of pheromones and other oxy-functionalized products via enzymatic hydroxylation of carboxylic acids

Info

Publication number: WO2016210352A2
Application number: PCT/US2016/039399
Authority: WO
Inventors: Vu Bui; Pedro COELHO; Mike M.Y. Chen; Peter Meinhold; Thomas HEEL
Original assignee: Provivi, Inc.
Priority date: 2015-06-24
Filing date: 2016-06-24
Publication date: 2016-12-29
Also published as: WO2016210352A3

Abstract

The present invention provides a method for synthesizing an oxyfunctionalized product. The method includes incubating an enzyme substrate with an enzyme capable of hydroxylating one terminal carbon of the enzyme substrate to form a hydroxylated product and converting at least a portion of the hydroxylated product to the oxyfunctionalized product. The enzyme substrate is an unsaturated or saturated carboxylic acid or an ester thereof, and the hydroxylated product can be a terminal hydroxy-substituted unsaturated carboxylic acid or an ester thereof. The oxy-functionalized products include insect pheromones and other useful compounds.

Description

SYNTHESIS OF PHEROMONES AND OTHER OXY- FUNCTIONALIZED PRODUCTS VIA ENZYMATIC HYDROXYLATION OF CARBOXYLIC ACIDS

CROSS-REFERENCES TO RELATED APPLICATIONS

[0001] The present application claims priority to U. S. Provisional Pat. Appl. No. 62/184,068, filed on June 24, 2015, which application is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

[0002] Insect pheromones can be used in a variety of insect control strategies that include mating disruption and attract-and-kill, as well as mass trapping. These strategies have proven to be effective, selective (e.g., they do not harm beneficial insects, such as bees and lady bugs), and safe (e.g., the compounds are generally biodegradable and do not accumulate in the food chain). Even the very stringent USDA Organic Program lists insect pheromones as one of the few synthetic organic compounds allowed in organic crop production, another important recognition of the high safety of these products. Accordingly, pheromones already form the basis of integrated pest management (IPM) practices in fruit production on the U. S. west coast, and their use in organic farming is growing worldwide.

[0003] Despite these advantages, pheromones are not widely used today because of the high cost of about $500 to $14,000 per kg of active ingredient (AI). Even though thousands of insect pheromones have been identified, less than about twenty insect pests worldwide are currently controlled using pheromone strategies, and only 0.05% of global agricultural land employs pheromones.

[0004] Lepidopteran pheromones, which are naturally occurring compounds, or identical or substantially similar synthetic compounds, are designated by an unbranched aliphatic chain (between 9 and 18 carbons) ending in an alcohol, aldehyde, or acetate functional group and containing up to 3 double bonds in the aliphatic backbone. [0005] The present invention provides methods by which lepidopteran insect pheromones as well as structurally related compounds are prepared using synthetic strategies that are enabled by a biocatalytic step.

BRIEF SUMMARY OF THE INVENTION

[0006] The present invention provides a method for synthesizing an oxy-functionalized product. The method includes incubating an enzyme substrate with an enzyme capable of hydroxylating one terminal carbon of the enzyme substrate to form a hydroxylated product and converting at least a portion of the hydroxylated product to the oxy-functionalized product. The enzyme substrate is a carboxylic acid or an ester thereof, and the hydroxylated product is a terminal hydroxy-substituted carboxylic acid or ester thereof.

[0007] In some embodiments, the enzyme used in the methods of the invention is a non- heme diiron monooxygenase. In some embodiments, the enzyme is a long-chain alkane hydroxylase. In some embodiments, the enzyme is a cytochrome P450. In some embodiments, the oxy-functionalized product is a pheromone. In some embodiments, the pheromone is a lepidopteran insect pheromone.

[0008] Other objects, features, and advantages of the present invention will be apparent to one of skill in the art from the following detailed description.

DETAILED DESCRIPTION OF THE INVENTION

I. Definitions [0009] The following definitions and abbreviations are to be used for the interpretation of the invention. The term "invention" or "present invention" as used herein is a non-limiting term and is not intended to refer to any single embodiment but encompasses all possible embodiments.

[0010] As used herein, the terms "comprises," "comprising," "includes," "including," "has," "having, "contains," "containing," or any other variation thereof, are intended to cover a non-exclusive inclusion. A composition, mixture, process, method, article, or apparatus that comprises a list of elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, mixture, process, method, article, or apparatus. Further, unless expressly stated to the contrary, "or" refers to an inclusive "or" and not to an exclusive "or." [0011] The terms "about" and "around," as used herein to modify a numerical value, indicate a close range surrounding that explicit value. If "X" were the value, "about X" or "around X" would indicate a value from 0.9X to 1.1X, and more preferably, a value from 0.95X to 1.05X. Any reference to "about X" or "around X" specifically indicates at least the values X, 0.95X, 0.96X, 0.97X, 0.98X, 0.99X, 1.01X, 1.02X, 1.03X, 1.04X, and 1.05X. Thus, "about X" and "around X" are intended to teach and provide written description support for a claim limitation of, e.g., "0.98X."

[0012] The terms "engineered enzyme" and "enzyme variant" include any enzyme comprising at least one amino acid mutation with respect to wild-type and also include any chimeric protein comprising recombined sequences or blocks of amino acids from two, three, or more different enzymes.

[0013] The terms "engineered heme enzyme" and "heme enzyme variant" include any heme-containing enzyme comprising at least one amino acid mutation with respect to wild- type and also include any chimeric protein comprising recombined sequences or blocks of amino acids from two, three, or more different heme-containing enzymes.

[0014] The terms "engineered cytochrome P450" and "cytochrome P450 variant" include any cytochrome P450 enzyme comprising at least one amino acid mutation with respect to wild-type and also include any chimeric protein comprising recombined sequences or blocks of amino acids from two, three, or more different cytochrome P450 enzymes. [0015] The term "whole cell catalyst" includes microbial cells expressing hydroxylase enzymes, wherein the whole cell catalyst displays hydroxylation activity.

[0016] As used herein, the term "metathesis reaction" refers to a catalytic reaction which involves the interchange of alkylidene units (i.e., R₂C= units) among compounds containing one or more carbon-carbon double bonds (e.g., olefinic compounds) via the formation and cleavage of the carbon-carbon double bonds. Metathesis can occur between two like molecules (often referred to as self-metathesis) and/or between two different molecules (often referred to as cross-metathesis).

[0017] As used herein, the term "metathesis catalyst" refers to any catalyst or catalyst system that catalyzes a metathesis reaction. One of skill in the art will appreciate that a metathesis catalyst can participate in a metathesis reaction so as to increase the rate of the reaction, but is itself not consumed in the reaction. [0018] As used herein, the term "metathesis product" refers to an olefin containing at least one double bond, the bond being formed via a metathesis reaction.

[0019] As used herein, the terms "microbial," "microbial organism," and "microorganism" include any organism that exists as a microscopic cell that is included within the domains of archaea, bacteria or eukarya. Therefore, the term is intended to encompass prokaryotic or eukaryotic cells or organisms having a microscopic size and includes bacteria, archaea, and eubacteria of all species as well as eukaryotic microorganisms such as yeast and fungi. Also included are cell cultures of any species that can be cultured for the production of a chemical.

[0020] As used herein, the term "non-naturally occurring", when used in reference to a microbial organism or enzyme activity of the invention, is intended to mean that the microbial organism or enzyme has at least one genetic alteration not normally found in a naturally occurring strain of the referenced species, including wild-type strains of the referenced species. Genetic alterations include, for example, modifications introducing expressible nucleic acids encoding metabolic polypeptides, other nucleic acid additions, nucleic acid deletions and/or other functional disruption of the microbial organism's genetic material. Such modifications include, for example, coding regions and functional fragments thereof, for heterologous, homologous, or both heterologous and homologous polypeptides for the referenced species. Additional modifications include, for example, non-coding regulatory regions in which the modifications alter expression of a gene or operon. Exemplary non-naturally occurring microbial organism or enzyme activity includes the hydroxylation activity described above.

[0021] As used herein, the term "exogenous" is intended to mean that the referenced molecule or the referenced activity is introduced into the host microbial organism. The term as it is used in reference to expression of an encoding nucleic acid refers to the introduction of the encoding nucleic acid in an expressible form into the microbial organism. When used in reference to a biosynthetic activity, the term refers to an activity that is introduced into the host reference organism.

[0022] The term "heterologous" as used herein with reference to molecules, and in particular enzymes and polynucleotides, indicates molecules that are expressed in an organism other than the organism from which they originated or are found in nature, independently of the level of expression that can be lower, equal or higher than the level of expression of the molecule in the native microorganism. [0023] On the other hand, the terms "native" and/or "endogenous" as used herein with reference to molecules, and in particular enzymes and polynucleotides, indicate molecules that are expressed in the organism in which they originated or are found in nature, independently of the level of expression that can be lower equal or higher than the level of expression of the molecule in the native microorganism. It is to be understood that expression of native enzymes or polynucleotides may be modified in recombinant microorganisms.

[0024] The term "homolog," as used herein with respect to an original enzyme or gene of a first family or species, refers to distinct enzymes or genes of a second family or species which are determined by functional, structural, or genomic analyses to be an enzyme or gene of the second family or species which corresponds to the original enzyme or gene of the first family or species. Homologs most often have functional, structural, or genomic similarities. Techniques are known by which homologs of an enzyme or gene can readily be cloned using genetic probes and PCR. Identity of cloned sequences as homologs can be confirmed using functional assays and/or by genomic mapping of the genes. [0025] A protein has "homology" or is "homologous" to a second protein if the amino acid sequence encoded by a gene has a similar amino acid sequence to that of the second gene. Alternatively, a protein has homology to a second protein if the two proteins have "similar" amino acid sequences. Thus, the term "homologous proteins" is intended to mean that the two proteins have similar amino acid sequences. In certain instances, the homology between two proteins is indicative of its shared ancestry, related by evolution.

[0026] The terms "analog" and "analogous" include nucleic acid or protein sequences or protein structures that are related to one another in function only and are not from common descent or do not share a common ancestral sequence. Analogs may differ in sequence but may share a similar structure, due to convergent evolution. For example, two enzymes are analogs or analogous if the enzymes catalyze the same reaction of conversion of a substrate to a product, are unrelated in sequence, and irrespective of whether the two enzymes are related in structure.

[0027] The term "oxy-functionalized," as used herein to refer to synthetic intermediates and synthetic products, refers to a compound having at least one oxygen-containing functional group. Examples of oxygen-containing functional groups include hydroxyl groups (i.e., alcohol groups), alcohol ester groups (e.g., acetate groups), aldehyde groups, carboxylic acid groups, and carboxylic acid ester groups. Oxy-functionalized products provided by the methods of the invention include, but are not limited to, terminally hydroxylated alkenes; terminally hydroxylated alkanes; unsaturated carboxylic acids (including unsaturated fatty acids) and esters thereof; saturated carboxylic acids (including saturated fatty acids) and esters thereof; terminally hydroxylated unsaturated carboxylic acids (including unsaturated fatty acids) and esters thereof; terminally hydroxylated saturated carboxylic acids (including saturated fatty acids) and esters thereof; unsaturated aldehydes; and saturated aldehydes.

[0028] As used herein, the term "alkane" refers to a straight or branched, saturated, aliphatic hydrocarbon having the number of carbon atoms indicated. The term "alkyl" refers to a straight or branched, saturated, aliphatic radical having the number of carbon atoms indicated. Alkyl can include any number of carbons, such as C_1-2, C_1-3, C_1-4, Ci_-5, Ci_-6, C₁.7, Ci_-8, C2-3, C₂-4, C_2-5, C_2-6, C_3-4, C_3-5, C_3-6, C_4-5, C4-6 and C_5-6. For example, C_1-6 alkyl includes, but is not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, hexyl, etc. Alkyl can refer to alkyl groups having up to 20 carbons atoms, such as, but not limited to heptyl, octyl, nonyl, decyl, etc. Alkanes and alkyl groups can be optionally substituted with one or more moieties selected from halo, alkenyl, and alkynyl.

[0029] As used herein, the term "alkene" refers to a straight chain or branched hydrocarbon having at least 2 carbon atoms and at least one double bond. A "terminal" alkene refers to an alkene wherein the double bond is between two carbon atoms at the end of the hydrocarbon chain (e.g., hex-l-ene). An "internal" alkene refers to an alkene wherein the double bond is between two carbon atoms that are not at the end of the hydrocarbon chain (e.g., (£)-hex-3- ene and (Z)-hex-3-ene). An "α,ω-alkenol" refers to a hydroxy-substituted terminal alkene having the formula (CH₂=CH)(CH₂)mOH, wherein m is an integer ranging from 1-30, such as 2-18. The term "alkenyl" refers to a straight chain or branched hydrocarbon radical having at least 2 carbon atoms and at least one double bond. Alkenyl can include any number of carbons, such as C₂, C_2-3, C₂. , C₂.₅, C₂.₆, C₂.₇, C_2-8, C₂.₉, C_2-io, C₃, C_3-4, C_3-5, C_3-6, C₄, C4-5, C_4-6, C₅, C5.6, and C₆. Alkenyl groups can have any suitable number of double bonds, including, but not limited to, 1, 2, 3, 4, 5 or more. Examples of alkenyl groups include, but are not limited to, vinyl (ethenyl), propenyl, isopropenyl, 1-butenyl, 2-butenyl, isobutenyl, butadienyl, 1-pentenyl, 2-pentenyl, isopentenyl, 1,3-pentadienyl, 1,4-pentadienyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 1,3-hexadienyl, 1,4-hexadienyl, 1,5-hexadienyl, 2,4-hexadienyl, or 1,3,5-hexatrienyl. Alkenes and alkenyl groups can be optionally substituted with one or more moieties selected from halo, alkyl, and alkynyl. [0030] As used herein, the term "selective" refers to preferential reaction of one site on a chemical compound over another site on the compound. As a non-limiting example, selectively hydroxylating oleic acid can refer to preferentially hydroxylating the terminal carbon of the acid to form more (Z)-18-hydroxyoctadec-9-enoic acid than other hydroxylated products (or forming exclusively (Z)-18-hydroxyoctadec-9-enoic acid without forming other hydroxylated products).

[0031] As used herein, the term "alkyne" refers to either a straight chain or branched hydrocarbon having at least 2 carbon atoms and at least one triple bond. A "terminal" alkyne refers to an alkyne wherein the triple bond is between two carbon atoms at the end of the hydrocarbon chain (e.g., hex-l-yne). An "internal" alkyne refers to an alkyne wherein the triple bond is between two carbon atoms that are not at the end of the hydrocarbon chain (e.g., hex-3-yne). The term "alkynyl" refers to either a straight chain or branched hydrocarbon radical having at least 2 carbon atoms and at least one triple bond. Alkynyl can include any number of carbons, such as C₂, C₂.₃, C₂.₄, C₂.₅, C₂.₆, C₂.₇, C₂.₈, C₂.₉, C₂.₁₀, C₃, C_3-4, C_3-5, C_3-6, C₄, C_4-5, C_4-6, C₅, C_5-6, and C₆. Examples of alkynyl groups include, but are not limited to, acetylenyl, propynyl, 1-butynyl, 2-butynyl, isobutynyl, sec-butynyl, butadiynyl, 1-pentynyl, 2-pentynyl, isopentynyl, 1,3-pentadiynyl, 1,4-pentadiynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 1,3-hexadiynyl, 1,4-hexadiynyl, 1,5-hexadiynyl, 2,4-hexadiynyl, or 1,3,5-hexatriynyl. Alkynes and alkynyl groups can be optionally substituted with one or more moieties selected from halo, alkyl, and alkenyl.

[0032] As used herein, the term "aryl" refers to an aromatic carbon ring system having any suitable number of ring atoms and any suitable number of rings. Aryl groups can include any suitable number of carbon ring atoms, such as, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15 or 16 ring atoms, as well as from 6 to 10, 6 to 12, or 6 to 14 ring members. Aryl groups can be monocyclic, fused to form bicyclic or tricyclic groups, or linked by a bond to form a biaryl group. Representative aryl groups include phenyl, naphthyl and biphenyl. Other aryl groups include benzyl, having a methylene linking group. Some aryl groups have from 6 to 12 ring members, such as phenyl, naphthyl or biphenyl. Other aryl groups have from 6 to 10 ring members, such as phenyl or naphthyl. Some other aryl groups have 6 ring members, such as phenyl. Aryl groups can be optionally substituted with one or more moieties selected from halo, hydroxy, amino, alkylamino, alkoxy, haloalkyl, carboxy, amido, nitro, oxo, and cyano. [0033] As used herein, the terms "halo" and "halogen" refer to fluorine, chlorine, bromine and iodine.

[0034] As used herein, the term "carboxy" refers to a moiety -C(0)OH. The carboxy moiety can be ionized to form the carboxylate anion. "Carboxylic acid" refers to a compound having a carboxy moiety. Unsaturated carboxylic acids are carboxylic acids having at least one carbon-carbon double bond or at least one carbon-carbon triple bond. Examples of unsaturated carboxylic acids include, but are not limited to, oleic acid, palmitoleic acid, linoleic acid, and the like. Saturated carboxylic acids are carboxylic acids having no carbon- carbon multiple bonds (e.g., carbon-carbon double bonds or carbon-carbon triple bonds). Examples of unsaturated carboxylic acids include, but are not limited to, palmitic acid, stearic acid, myristic acid, and the like.

[0035] As used herein, the term "hydroxy" refers to a moiety -OH.

[0036] As used herein, the term "amino" refers to a moiety - R₃, wherein each R group is H or alkyl. An amino moiety can be ionized to form the corresponding ammonium cation. [0037] As used herein, the term "amido" refers to a moiety - RC(0)R or -C(0) R₂, wherein each R group is H or alkyl.

[0038] As used herein, the term "nitro" refers to the moiety -N0₂.

[0039] As used herein, the term "oxo" refers to an oxygen atom that is double-bonded to a compound (i.e., 0=). [0040] As used herein, the term "cyano" refers to the moiety -CN.

II. Description of the Embodiments

[0041] Traditionally, straight chain monoene alcohols, acetates, and aldehydes are synthesized via multi-step syntheses. Scheme 1 represents an example of such synthesis. Scheme 1. General synthesis strategy for straight chain monoene alcohols, acetates, and aldehydes.

)

Scheme 1— cont'd.

Na Pd (Lindlar) Na Pd (Lindlar) NH₃ (I) H₂ NH₃ (I) H₂

o.

' _m ^wn ^' ' m \ / n [0042] The present disclosure describes several methods for the synthesis of terminally oxy-functionalized alkenes, as well as terminally oxy-functionalized unsaturated carboxylic acids, including fatty acids, and derivatives thereof such as terminally oxy-functionalized unsaturated carboxylic acid esters and terminally oxy-functionalized unsaturated aldehydes. Said methods are described in detail below and are generally applicable to the synthesis of various compounds, including but not limited to those shown in Table 1.

Table 1. Exemplary compounds that can be synthesized using methods described

present disclosure.

Example of Biological

Name Structure

importance

(Z)-3-hexanol See, Sugimoto et al. (2014)

West Indian Fruity Fly male

(Z)-3-nonen-l-ol HO sex pheromone

(Z)-5-decen-l-ol

(Z)-5-decenyl acetate AcCX Agrotis segetum sex

pheromone component

Anarsia lineatella sex

(E)-5-decen-l-ol

pheromone component

Anarsia lineatella sex

(E)-5-decenyl acetate

pheromone component

(Z)-7-dodecen- 1 -ol HO.

Pseudoplusia includens sex

(Z)-7-dodecenyl AcO. pheromone

acetate Agrotis segetum sex

pheromone component

Citrus Fruit Moth sex

(E)-8-dodecen- 1 -ol

pheromone

Grapholitha molesta,

(E)-8-dodecenyl

Ecdytolopha aurantiana sex acetate

AcO pheromone component

Grapholitha molesta,

(Z)-8-dodecen- 1 -ol Ecdytolopha aurantiana sex

pheromone component

(Z)-8-dodecenyl Grapholitha molesta sex

AcO

acetate pheromone component

(Z)-9-dodecen- 1 -ol

(Z)-9-dodecenyl Eupoecilia ambiguella sex

AcO

acetate pheromone

(Z)-9-tetradecen- 1 -ol

Pandemis pyrusana, Naranga

(Z)-9-tetradecenyl

AcO aenescens, Agrotis segetum acetate

sex pheromone component

(Z)- 11 -tetraceden- 1 -ol

(Z)-l 1-tetracedenyl Pandemis pyrusana,

AcO

acetate Choristoneura roseceana sex Example of Biological

Name Structure

importance

pheromone component

(E)- 11 -tetradecen- 1 -ol

Choristoneura roseceana,

(E)-l 1-tetradecenyl

Crocidolomia pavonana sex acetate

pheromone component

(Z)-7-hexadecen- 1 -ol

Diatraea considerata sex

(Z)-7-hexadecenal

pheromone component

(Z)-9-hexadecen- 1 -ol

Helicoverpa zea, Helicoverpa

(Z)-9-hexadecenal o ⁼ armigera, Heliothis virescens sex pheromone component

(Z)-9-hexadecenyl Naranga aenescens sex acetate pheromone component

(Z)- 11 -hexadecen- 1 -ol

Platyptila carduidactyla, Heliothis virescens sex pheromone

Helicoverpa zea, Helicoverpa armigera, Plutella xylostella,

(Z)-l 1-hexadecenal

Diatraea considerate,

Diatraea grandiosella, Diatraea saccharalis,

Acrolepiopsis assectella sex pheromone component

Discestra trifolii sex pheromone

Heliothis virescens, Plutella

(Z)-l 1-hexadecenyl

acetate

xylostella, Acrolepiopsis

assectella, Crocidolomia pavonana, Naranga aenescens sex pheromone component

(Z)- 13 -octadecen- 1 -ol

Diatraea considerata,

(Z)- 13 -octadecenal

Diatraea grandiosella sex pheromone component

Ac = -(CO)CH;

[0043] The synthetic strategies disclosed herein chiefly rely on the ability of hydroxylases to terminally hydroxylate unsaturated or saturated carboxylic acids, including fatty acids, and derivatives thereof, including unsaturated or saturated fatty acid esters. The unsaturated or saturated acids and esters can be obtained by any suitable means. For example, unsaturated fatty acids can be obtained via synthetic routes such as olefin metathesis, Wittig olefination, or alkyne substitution followed by partial hydrogenation. Saturated and unsaturated fatty acids and esters can also be obtained from commodity seed oils or other sources. The hydroxylation products can further be modified via any method, including but not limited to, oxidation, esterification, and olefin metathesis, to produce the desired end products. Deviations from this general scheme are also contemplated, as described herein.

Synthesis of Oxy-Functionalized Products and Enzymatic Hydroxylation Of Carboxylic Acids

[0044] In one aspect, the invention provides a method for synthesizing an oxy- functionalized product. The method includes incubating an enzyme substrate with an enzyme capable of selectively hydroxylating one terminal carbon of the enzyme substrate to form a hydroxylated product, and converting at least a portion of the hydroxylated product to the oxy-functionalized product. The enzyme substrate is an unsaturated carboxylic acid or an ester thereof, and the hydroxylated product is a terminal hydroxy-substituted unsaturated carboxylic acid or an ester thereof.

[0045] In general, the synthetic method includes an enzyme-catalyzed hydroxylation step. For example, the method can include the hydroxylation of an unsaturated carboxylic acid or ester as summarized in Scheme 2.

Scheme 2

R = H, Me, Ethyl, alkyl

[0046] Accordingly, some embodiments of the invention provide a method that includes:

incubating an enzyme substrate according to formula I

[0047] In some embodiments, R is Ci_-6 alkyl. In some embodiments, R is methyl. [0048] Unsaturated carboxylic acids and esters of any suitable length can be used in the methods of the invention. For example, an unsaturated carboxylic acid or ester can contain from about 4 carbons to about 22 carbon atoms, such as 4-20 carbon atoms, or 8-20 carbon atoms. In some embodiments, the unsaturated carboxylic acid or ester has a structure according to formula A:

wherein a is 0 and b is 4; or a is 1 and b is 3; or a is 2 and b is 2; or a is 3 and b is 1; or a is 4 and b is 0; or a is 0 and b is 5; or a is 1 and b is 4; or a is 2 and b is 3; or a is 3 and b is 2; or a is 4 and b is 1; or a is 5 and b is 0; or a is 0 and b is 6; or a is 1 and b is 5; or a is 2 and b is 4; or a is 3 and b is 3; or a is 4 and b is 2; or a is 5 and b is 1; or a is 6 and b is 0; or a is 0 and b is 7; or a is 1 and b is 6; or a is 2 and b is 5; or a is 3 and b is 4; or a is 4 and b is 3; or a is 5 and b is 2; or a is 6 and b is 1 ; or a is 7 and b is 0; or a is 0 and b is 8; or a is 1 and b is 7; or a is 2 and b is 6; or a is 3 and b is 5; or a is 4 and b is 4; or a is 5 and b is 3; or a is 6 and b is 2; or a is 7 and b is 1; or a is 8 and b is 0; or a is 0 and b is 9; or a is 1 and b is 8; or a is 2 and b is 7; or a is 3 and b is 6; or a is 4 and b is 5; or a is 5 and b is 4; or a is 6 and b is 3; or a is 7 and b is 2; or a is 8 and b is 1; or a is 9 and b is 0; or a is 0 and b is 10; or a is 1 and b is 9; or a is 2 and b is 8; or a is 3 and b is 7; or a is 4 and b is 6; or a is 5 and b is 5; or a is 6 and b is 4; or a is 7 and b is 3; or a is 8 and b is 2; or a is 9 and b is 1; or a is 10 and b is 0; or a is 0 and b is 11; or a is 1 and b is 10; or a is 2 and b is 9; or a is 3 and b is 8; or a is 4 and b is 7; or a is 5 and b is 6; or a is 6 and b is 5; or a is 7 and b is 4; or a is 8 and b is 3; or a is 9 and b is 2; or a is 10 and b is 1; or a is 11 and b is 0; or a is 0 and b is 12; or a is 1 and b is 11; or a is 2 and b is 10; or a is 3 and b is 9; or a is 4 and b is 8; or a is 5 and b is 7; or a is 6 and b is 6; or a is 7 and b is 5; or a is 8 and b is 4; or a is 9 and b is 3; or a is 10 and b is 2; or a is 11 and b is 1; or a is 12 and b is 0; or a is 0 and b is 13; or a is 1 and b is 12; or a is 2 and b is 11; or a is 3 and b is 10; or a is 4 and b is 9; or a is 5 and b is 8; or a is 6 and b is 7; or a is 7 and b is 6; or a is 8 and b is 5; or a is 9 and b is 4; or a is 10 and b is 3; or a is 11 and b is 2; or a is 12 and b is 1; or a is 13 and b is 0; or a is 0 and b is 14; or a is 1 and b is 13; or a is 2 and b is 12; or a is 3 and b is 11; or a is 4 and b is 10; or a is 5 and b is 9; or a is 6 and b is 8; or a is 7 and b is 7; or a is 8 and b is 6; or a is 9 and b is 5; or a is 10 and b is 4; or a is 11 and b is 3; or a is 12 and b is 2; or a is 13 and b is 1; or a is 14 and b is 0; or a is 0 and b is 15; or a is 1 and b is 14; or a is 2 and b is 13; or a is 3 and b is 12; or a is 4 and b is 11; or a is 5 and b is 10; or a is 6 and b is 9; or a is 7 and b is 8; or a is 8 and b is 7; or a is 9 and b is 6; or a is 10 and b is 5; or a is 11 and b is 4; or a is 12 and b is 3; or a is 13 and b is 2; or a is 14 and b is 1; or a is 15 and b is 0; or a is 0 and b is 16; or a is 1 and b is 15; or a is 2 and b is 14; or a is 3 and b is 13; or a is 4 and b is 12; or a is 5 and b is 11; or a is 6 and b is 10; or a is 7 and b is 9; or a is 8 and b is 8; or a is 9 and b is 7; or a is 10 and b is 6; or a is 11 and b is 5; or a is 12 and b is 4; or a is 13 and b is 3; or a is 14 and b is 2; or a is 15 and b is 1; or a is 16 and b is 0; or a is 0 and b is 17; or a is 1 and b is 16; or a is 2 and b is 15; or a is 3 and b is 14; or a is 4 and b is 13; or a is 5 and b is 12; or a is 6 and b is 11; or a is 7 and b is 10; or a is 8 and b is 9; or a is 9 and b is 8; or a is 10 and b is 7; or a is 11 and b is 6; or a is 12 and b is 5; or a is 13 and b is 4; or a is 14 and b is 3; or a is 15 and b is 2; or a is 16 and b is 1; or a is 17 and b is 0; or a is 0 and b is 17; or a is 1 and b is 17; or a is 2 and b is 16; or a is 3 and b is 15; or a is 4 and b is 14; or a is 5 and b is 13; or a is 6 and b is 12; or a is 7 and b is 11; or a is 8 and b is 10; or a is 10 and b is 8; or a is 1 1 and b is 7; or a is 12 and b is 6; or a is 13 and b is 5; or a is 14 and b is 4; or a is 15 and b is 3; or a is 16 and b is 2; or a is 17 and b is 1; or a is 18 and b is 0.

[0049] In some embodiments, the enzyme substrate has a structure according to formula I

wherein a and b are independently selected integers ranging from 1 to 9. In some embodiments, a and b are different integers ranging from 1 to 9.

[0050] The method of the invention can include a number of other synthetic steps, including those summarized in Scheme 3. In general, at least a portion of the hydroxylation product is converted to the final oxy-functionalized product in one or more steps. In cases where a portion of the hydroxylated product is converted to the final oxy-functionalized product, another portion of the hydroxylated product is left unreacted or is converted to one or more others compounds. As described in more detail below, for example, a portion of the hydroxylated product can be converted back to the enzyme substrate starting material. In other words, the enzyme substrate can be reconstituted from the hydroxylated product. Typically, a large portion of the hydroxylation product is converted to the final oxy- functionalized product. For example, at least 50% of the hydroxylation product will be converted to the final oxy-functionalized product in various embodiments. Greater than about 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95% of the hydroxylated product can be converted to the final oxy-functionalized product. In some embodiments, at least about 99% of the hydroxylated product is converted to the final oxy-functionalized product. In some embodiments, about 100% of the hydroxylated product is converted to the final oxy- functionalized product. As described below, other products can be formed during conversion of the hydroxylated product to the oxy-functionalized product.

[0051] In the process shown in Scheme 3, co-hydroxy fatty acids can be made via bio- oxidation of corresponding alkenes or by terminal hydroxylation of corresponding unsaturated fatty acids. The resulting co-hydroxy fatty acids can then be esterified and the terminal hydroxyl group protected to provide corresponding esters that can be coupled with various internal and terminal alkenes through olefin metathesis process. Coupling of terminal alkenes with the co-hydroxy-protected esters provides terminal hydroxy-alkenes and unsaturated esters as products. The terminal hydroxy-alkenes can be further manipulated through oxidation and acylation processes to generate final oxy-functionalized products of interest, such as insect pheromones and other compounds.

Scheme 3. S nthetic routes according to embodiments of the invention.

1 . Esterification R = H, Me, Ethyl, alkyl

2. Protection

R'O k ^OR R = Me, Ethyl, alkyl

R' = Protective group

Z-selective H

metathesis

e = 0-17 recycle

1. Deprotection 1 . Deprote

2. Oxidation 2. Acylatio

Acylation

[0052] Any suitable alcohol protecting group can be used in the methods of the invention. Such protecting groups are well known to one of ordinary skill in the art, including those that are disclosed in Protective Groups in Organic Synthesis, 4th edition, T. W. Greene and P. G. M. Wuts, John Wiley & Sons, New York, 2006, which is incorporated herein by reference in its entirety. For example, the hydroxylation product can be protected via esterification and the protected hydroxylation product can be deprotected via hydrolysis. In some embodiments, the hydroxylation product is protected via esterification with an acid selected from the group consisting of formate and acetate. The omega-hydroxy fatty acid or ester produced in the biohydroxylation step can also be cyclized to the corresponding lactone, which can then be metathesized with various terminal alkenes to generate appropriate intermediates as shown in Scheme 3A.

[0053] As noted above and shown in Schemes 3 and 3A, metathesis of alkenes with the co- hydroxy-protected esters provides terminal hydroxy-alkenes and unsaturated esters as products. In the case of e = a + 1 as illustrated in Scheme 3, the starting material is regenerated and thus can be recycled. Accordingly, some embodiments of the invention provide a method that further includes converting at least a portion of the hydroxylated product to reconstituted enzyme substrate. In some embodiments, the methods further include recycling the reconstituted enzyme substrate. Recycling can include processes such as extraction, distillation, and other techniques. In some embodiments, recycling the reconstituted enzyme substrate includes distilling the reconstituted enzyme substrate from a mixture comprising the olefinic alcohol, the metathesis catalyst, and the reconstituted enzyme substrate.

[0054] In some embodiments, the invention provides a method as described wherein converting the hydroxylation product to the oxy-functionalized product includes:

metathesizing the hydroxylation product according to formula II and a terminal olefin according to formula III e (HI),

wherein e is an integer ranging from 0 to 17,

in the presence of a metathesis catalyst to form an olefinic alcohol according to formula IV:

(IV). [0055] Synthetic steps such as metathesis reactions can be conducted with unprotected hydroxylation products or protected hydroxylation products. In some embodiments, the invention provides a method that includes:

protecting the hydroxylation product according to formula II to form a protected hydroxylation product according to formula Ila

wherein R² is an alcohol protecting group;

metathesizing the protected hydroxylation product and a terminal olefin according to formula III

(HI),

wherein e is an integer ranging from 0 to 17,

in the presence of a metathesis catalyst to form a protected olefinic alcohol according to formula IVa:

R²CX

e^{' H} (IVa); and

deprotecting the protected olefinic alcohol to form an olefinic alcohol according to formula IV:

Scheme 3 A. Synthetic routes according to embodiments of the invention.

Z-selective

metathesis

e = 0-17

Acylation

Scheme 3B. Synthetic routes according to embodiments of the invention.

Z-selective

metathesis

e = 0-17

Oxidation

Acylation

In some embodiments, the method includes:

cyclizing the hydroxylation product to form a lactone according to formula lib

metathesizing the lactone and a terminal olefin according to formula III

(HI), wherein e is an integer ranging from 0 to 17,

in the presence of a metathesis catalyst to form an ester according to formula

IVb:

[0057] The hydroxylation product, the terminal olefin, the lactone, the ester, and the olefinic alcohol can have any suitable combination of subscripts a, b, and e, as described above for subscripts a and b. In some embodiments, a, b, and e are independently selected integers ranging from 1 to 9. In some embodiments, a, b, and e are different integers ranging from 1 to 9.

[0058] In some embodiments, the method of the invention further includes oxidizing the olefinic alcohol to form the oxy-functionalized product, wherein the oxy-functionalized product has a structure according to formula V:

[0059] In some embodiments, the method of the invention further includes acylating the olefinic alcohol to form the oxy-functionalized product, wherein the oxy-functionalized product has a structure according to formula VI:

(VI),

wherein R is selected from H, Ci_-6 alkyl, and C₆-io aryl. In some embodiments, R is selected from H and Ci_-6 alkyl. In some embodiments, R³ is selected from H and methyl. In some embodiments, R³ is H. In some embodiments, R³ is methyl.

[0060] The hydroxylation products prepared via the method of the invention can be elaborated via olefin metathesis using a variety of internal alkenes as shown, e.g., in Schemes 4, 4A, and 4B, or polyenes as shown, e.g., in Scheme 5. In the process shown in Scheme 4A, the omega-hydroxy fatty acid or ester produced in the biohydroxylation step is cyclized to the corresponding lactone, which can then be metathesized with various internal alkenes to generate desired intermediates.

Scheme 4. Synthetic routes according to embodiments of the invention.

Scheme 4A. S nthetic routes according to embodiments of the invention.

Acylation

Scheme 4B. S nthetic routes according to embodiments of the invention.

Acylation

[0061] In some embodiments, the invention provides a method that includes:

(Ha),

wherein R² is an alcohol protecting group;

metathesizing the protected hydroxylation product and an internal olefin according to formula VII

(VII)

wherein each c is independently selected from integers ranging from 0 to 15, in the presence of a metathesis catalyst to form a protected olefinic alcohol according to formula LIVa:

deprotecting the protected olefinic alcohol to form an olefinic alcohol according to formula LIV:

HO.

^{C ' H} (LIV).

[0062] In some embodiments, the method includes:

cyclizing the hydroxylation product according to formula II to form a lactone according to formula lib

metathesizing the lactone and an internal olefin according to formula VII c^c H (VII)

wherein each c is independently selected from integers ranging from 0 to 15, in the presence of a metathesis catalyst to form an ester according to formula

hydrolyzing the ester to form an olefinic alcohol according to formula IV: HO^ ^'

(LIV).

[0063] In some embodiments, the method includes:

LIVb:

reducing the ester to form an olefinic alcohol according to formula IV:

HO.

(LIV).

[0064] Reduction of fatty acids and esters (e.g., compounds according to formulae IVb, IVc, IVd, IVe, XXIII, XXVII, LIVb, LV, as set forth herein) to the corresponding aldehydes or alcohols can be conducted with a suitable stoichiometric reducing agent. Examples of stoichiometric reducing agents include, but are not limited to, sodium bis(2- methoxyethoxy)aluminumhydride (trade names Red-Al, Vitride, SMEAH) and diisobutylaluminumhydride (DIBAL). Selectivity for the aldehyde can be increased by the inclusion of a bulky cyclic nitrogen Lewis base to modify the reducing agent (see, e.g., Shin, et al. Bull. Korean Chem. Soc. 35, 2169 (2014)).

[0065] In some embodiments, the method further includes oxidizing the olefinic alcohol according to formula LIV to form an aldehyde according to formula LV:

[0066] In some embodiments, the method further includes oxidizing the olefinic alcohol according to formula LIV to form an ester according to formula LVI:

(LVI), wherein R is selected from H, Ci_-6 alkyl, and C₆-io aryl. In some embodiments, R is selected from H and Ci_-6 alkyl. In some embodiments, R³ is selected from H and methyl. In some embodiments, R³ is H. In some embodiments, R³ is methyl.

[0067] The hydroxylation product, the internal olefin, the lactone, the ester, and the olefinic alcohol can have any suitable combination of subscripts a, b, and c, as described above for subscripts a and b. In some embodiments, a, b, and c are independently selected integers ranging from 1 to 9. In some embodiments, a, b, and c are different integers ranging from 1 to 9.

Scheme 5. A general approach to the synthesis of multi -unsaturated products.

2. Oxidation

[0068] In some embodiments, the method of the invention includes metathesizing the hydroxylation product according to formula II and an olefin according to formula 3

^{9 h} ' ' (3),

wherein i is an integer ranging from 1 to 10 and g, h, and j are independently- selected integers ranging from 0 to 15,

in the presence of a metathesis catalyst to form an olefinic alcohol according to formula 4:

HO

^{8 h 1} (4).

[0069] In some embodiments, the method includes protecting the hydroxylation product to form a protected hydroxylation product according to formula Ila

(Ha), wherein R² is an alcohol protecting group;

metathesizing the protected hydroxylation product and an olefin according to formula 3

^{9 h} ' ' (3),

in the presence of a metathesis catalyst to form a protected olefinic alcohol according to formula 4a:

R²0

^{3 h 1} (4a); and

deprotecting the protected olefinic alcohol to form an olefinic alcohol according to formula 4:

HO

^{8 h 1} (4).

[0070] In some embodiments, the method comprises:

cyclizing the hydroxylation product to form a lactone to formula lib

metathesizing the lactone and an olefin according to formula 3

^{9 h} ' ' (3),

in the presence of a metathesis catalyst to form an ester according to formula

4b:

hydrolyzing the protected olefinic alcohol to form an olefinic alcohol according to formula 4:

HO

^{8 h 1} (4).

[0071] The hydroxylation product, the olefin, the lactone, the ester, and the olefinic alcohol can have any suitable combination of subscripts a, b, g, h, i, and j, as described above for subscripts a and b. In some embodiments, a, b, g, h, i, and j are independently selected integers ranging from 1 to 9. In some embodiments, a, b, g, h, i, and j are different integers ranging from 1 to 9.

[0072] In some such embodiments, the method further includes oxidizing the olefinic alcohol to form the oxy-functionalized product, wherein the oxy-functionalized product has a structure according to formula 5 :

[0073] In some embodiments, the method further includes acylating the olefinic alcohol to form the oxy-functionalized product, wherein the oxy-functionalized product has a structure according to formula 6:

[0074] In certain embodiments, unsaturated carboxylic acids can be reacted with internal or terminal alkenes in the presence of a metathesis catalyst to generate various hydrocarbon chain lengths as part of the general synthesis route, as shown in Scheme 6. Scheme 6. A general approach for the synthesis of fatty acids of varying length from unsaturated carbox lates.

oxy-functionalized oxy-functionalized

products products

[0075] Accordingly, some embodiments of the invention provide a method for synthesizing an oxy-functionalized product as described above that includes:

metathesizing an enzyme substrate precursor according to formula I

(I),

wherein a and b are independently-selected from integers ranging from 0 to 15, and R is H, Ci_-6 alkyl, or C₆-io aryl,

and an olefin according to formula VII: c^c H (VII)

wherein each c is independently selected from integers ranging from 0 to 15, in the presence of a metathesis catalyst to form an enzyme substrate according formula VIII:

_H o_Rl

O (VIII); and incubating the enzyme substrate with an enzyme capable of hydroxylating the terminal carbon of the enzyme substrate to form a hydroxylation product according to formula IX

.OR¹

O (IX). [0076] In some such embodiments, the method includes metathesizing the hydroxylation product according to formula IX and a terminal olefin according to formula III

(HI),

wherein e is an integer ranging from 0 to 17,

HO.

e^H (XIII).

[0077] In some embodiments, the method includes protecting the hydroxylation product according to formula IX to form a protected hydroxylation product according to formula IXa

(IXa),

wherein R² is an alcohol protecting group;

(HI),

wherein e is an integer ranging from 0 to 17,

in the presence of a metathesis catalyst to form a protected olefinic alcohol according to formula XHIa:

R²O

e^{" H} (XIIIa); and

deprotecting the protected olefinic alcohol to form an olefinic alcohol according to formula XIII:

HO.

(XIII)

[0078] In some embodiments, the method comprises: cyclizing the hydroxylation product according to formula IX to form a lactone according to formula IXb

metathesizing the lactone and a terminal olefin according to formula III e (HI),

wherein e is an integer ranging from 0 to 17,

in the presence of a metathesis catalyst to form an ester according to formula

Xlllb:

(Xlllb); and

hydrolyzing the ester to form an olefinic alcohol according to formula XIII:

HO c _e H _(xm)

[0079] The hydroxylation product, the olefin, the lactone, the ester, and the olefinic alcohol can have any suitable combination of subscripts a, b, c, and e, as described above for subscripts a and b. In some embodiments, a, b, c, and e are independently selected integers ranging from 1 to 9. In some embodiments, a, b, c, and e are different integers ranging from 1 to 9.

[0080] In some embodiments, the method further includes oxidizing the olefinic alcohol according to formula XIII to form the oxy-functionalized product, wherein the oxy- functionalized product has a structure according to formula XV: e H (XV).

[0081] In some embodiments, the method further includes acylating the olefinic alcohol according to formula XIII to form the oxy-functionalized product, wherein the oxy- functionalized product has a structure according to formula XVI:

O

^{0 c e} H (XVI), wherein R³ is selected from H, Ci_-6 alkyl, and C₆-io aryl. In some embodiments, R³ is selected from H and Ci_-6 alkyl. In some embodiments, R³ is selected from H and methyl. In some embodiments, R³ is H. In some embodiments, R³ is methyl.

[0082] In some embodiments, the method includes:

metathesizing an enzyme substrate precursor according to formula I

(I),

and an olefin according to formula X: d (X)

wherein d is an integer ranging from 0 to 17,

in the presence of a metathesis catalyst to form an enzyme substrate according formula XI:

incubating the enzyme substrate with an enzyme capable of hydroxylating the terminal carbon of the enzyme substrate to form a hydroxylation product according to formula XII

[0083] In some such embodiments, the method includes metathesizing the hydroxylation product according to formula XII and a terminal olefin according to formula III

(HI),

wherein e is an integer ranging from 0 to 17,

in the presence of a metathesis catalyst to form an olefinic alcohol according to formula XI

[0084] In some embodiments, the method includes protecting the hydroxylation product according to formula XII to form a protected hydroxylation product according to formula Xlla

wherein R² is an alcohol protecting group;

(HI),

wherein e is an integer ranging from 0 to 17,

in the presence of a metathesis catalyst to form a protected olefinic alcohol according to formula XlVa:

_(XIVa). _and deprotecting the protected olefinic alcohol to form an olefinic alcohol according to formula IV:

[0085] In some embodiments, the method comprises:

cyclizing the hydroxylation product according to formula XII to form a lactone according to formula Xllb

(Xllb),

wherein e is an integer ranging from 0 to 17,

in the presence of a metathesis catalyst to form an ester according to formula

XI Vb:

O

cr

' e^H (XlVb); and hydrolyzing the ester to form an olefinic alcohol according to formula IV:

[0086] The hydroxylation product, the olefin, the lactone, the ester, and the olefinic alcohol can have any suitable combination of subscripts a, b, d, and e, as described above for subscripts a and b. In some embodiments, a, b, d, and e are independently selected integers ranging from 1 to 9. In some embodiments, a, b, d, and e are different integers ranging from 1 to 9.

[0087] In some embodiments, the method further includes oxidizing the olefinic alcohol according to formula XIV to form the oxy-functionalized product, wherein the oxy- functionalized product has a structure according to formula XVII:

^ ^{e H} (XVII).

[0088] In some embodiments, the method further includes acylating the olefinic alcohol according to formula IV to form the oxy-functionalized product, wherein the oxy- functionalized product has a structure according to formula XVIII:

O

° ^{d e} H (XVIII),

wherein R³ is selected from H, Ci_-6 alkyl, and C₆-io aryl. In some embodiments, R³ is selected from H and Ci_-6 alkyl. In some embodiments, R³ is selected from H and methyl. In some embodiments, R³ is H. In some embodiments, R³ is methyl.

[0089] The synthetic method can also include an enzyme-catalyzed hydroxylation of a saturated carboxylic acid or ester as summarized in Scheme 7.

Scheme 7

R⁴CX HO

x n ^■ ΌΗ

Hydroxylation II ^A

° NAD(P)H + 0₂ °

R⁴ = H, alkyl, aryl

X = 0, 1 -22

[0090] Accordingly, some embodiments of the invention provide a method that includes: incubating an enzyme substrate according to formula XIX R⁴C

O (XIX),

wherein x is an integer ranging from 0 to 22, and R⁴ is H, Ci_-6 alkyl, or

C₆-io aryl,

with an enzyme capable of hydroxylating the terminal carbon of the enzyme substrate to form a hydroxylation product according to formula XX

(XX).

[0091] In some embodiments, R⁴ is Ci_-6 alkyl. In some embodiments, R⁴ is methyl.

[0092] Saturated carboxylic acids and esters of any suitable length can be used in the methods of the invention. For example, a saturated carboxylic acid or ester can contain from about 4 carbons to about 22 carbon atoms, such as 4-20 carbon atoms, or 8-20 carbon atoms.

[0093] The method of the invention can include a number of other synthetic steps, including those summarized in Scheme 8. In the process shown in Scheme 8, saturated co- hydroxy fatty acids can be made via biohydroxylation of corresponding saturated carboxylic acids or ester derivatives. The resulting co-hydroxy fatty acids can then be esterified to provide corresponding esters. The resulting co-hydroxy fatty esters can then be dehydrated to form terminal alkene esters that can be coupled with various internal or terminal alkenes through an olefin metathesis process. Coupling of internal alkenes with the ω-hydroxy fatty esters provides internal hydroxyl-alkenes and saturated esters as products. The internal hydroxyl alkenes can be further manipulated through reduction, oxidation, and acylation processes to generate insect pheromones and other compounds of interest.

Synthetic routes according to embodiments of the invention.

R⁴0^ J~L / R⁴ = H, alkyl

X = 1 -16

O

Hydroxylation

NAD(P)H + 0₂

HO

OH

MeO

y R

O

Reduction

x y ^K R

Esterification

[0094] In some embodiments, converting the hydroxylation product of formula XX to the oxy-functionalized product comprises:

dehydrating the hydroxylation product to form a terminal olefin according to formula XXI:

HO.

(XXI);

metathesizing the terminal olefin and an alkene according to formula XXII

wherein y and z are each independently integers ranging from 0 to 18, and R⁵ is H, Ci-16 alkyl, Ci-i6 alkenyl, or Ci-i6 alkynyl,

in the presence of a metathesis catalyst to form an olefinic acid according to formula XXIII

(XXIII); and

reducing the olefinic acid to form an olefinic alcohol according to formula

XXIV x^{y R} (xxiv).

[0095] In some embodiments, converting the hydroxylation product to the oxy- functionalized product comprises:

esterifying the hydroxylation product to form an ester according to formula

XXV

R⁶0

x OH

⁰ (XXV),

wherein R⁶ is H, Ci_-6 alkyl, or C₆-₁₀ aryl;

dehydrating the esterification product to form a terminal olefin according to formula XXVI:

metathesizing the terminal olefin and an alkene according to formula XXII

^R y ^ (xxii),

in the presence of a metathesis catalyst to form an olefinic acid or ester according to formula XXVII

Y x ^{y K}

⁰ (XXVII); and

reducing the compound of formula XXVII to form an olefinic alcohol according to formula XXIV (XXIV).

[0096] In some such embodiments, R is selected from the group consisting of H and methyl.

[0097] In some embodiments, the olefinic alcohol of formula XXIV is the oxy- functionalized product. In some embodiments, the method further comprises oxidizing the olefinic alcohol of formula XXIV to form the oxy-functionalized product, wherein the oxy- functionalized product has a structure according to formula XXVIII:

(XXVIII).

[0098] In some embodiments, the method comprises acylating the olefinic alcohol of formula XXIV to form the oxy-functionalized product, wherein the oxy-functionalized product has a s ula XXIX:

(XXIX),

wherein R⁷ is selected from the group consisting of H, Ci_-6 alkyl, and

C₆-io aryl. Fatty Acid Starting Materials and Sources Thereof

[0099] The acids and esters used in the methods of the invention can be obtained by any suitable means. The fatty acid can be produced, for example, by plants or by microalgae. The fatty acid can be obtained from a commercially available oil or fat, such as soybean oil, cottonseed oil, peanut oil, sunflower seed oil, rapeseed oil, sesame seed oil, corn oil, olive oil, palm oil, palm kernel oil, coconut oil, butter, lard, fish oil, linseed oil, castor seed oil, or tallow. In some embodiments, the fatty acid is obtained from a commodity seed oil. Certain fatty acids that can be obtained from commodity seed oils are set forth in Table 2.

Table 2. Fatty Acid Content of Exemplary Commodity Seed Oils

Palm 39.2%

Olive 76.9%

Sesame 40.6%

Linoleic (18:2A9c,A12c) Soybean 53.2%

Cotton 53.3%

Sunflower 68.2%

Peanut 31.4%

Sesame 42.6%

Linolenic (18:3A9c,A12c,A15c) Linseed 47.4%.

[0100] Other crops— such as Calendula, Meadowfoam, Honesty, Caraway, Carrot, Coriander, Macadamia, Nutmeg, Oiticica, Tung, and Castor— produce unique unsaturated fatty acids including monoenes (e.g., 20: lA5c; 22: lA9c; 18: lA6c; and 16: lA9c) trienes (e.g, 18:3A8t,A10t,A12c; 18:3A9c,Al lt,A13t) and oxygenated trienes (e.g., 18:3A9c,Al 1,Δ13, 4- oxo). Fatty acid contents of various sources are summarized in Table 3, Table 4, and Table 5.

Table 3. Exemplary Fatty Acids and Seed Oil and Microalgal Sources Thereof

Table 4. Fatty acids compositions of major commodity seed oils

CottonSunPalm-

Fatty acid Δ notation Soybean Canola Peanut Palm Coconut Olive Sesame Linsee seed flower kernel oil

Hexanoic 6:0 - - - - - - 0.4 0.3 - - -

Octanoic

8:0 7.3 4.4

(Caprylic)

Decanoid

10:0 6.6 3.7

acid (Capric)

Laurie 12 0 - - - 0.5 - 0.2 47.8 48.4 - - -

My ri stic 14 0 0.1 - 0.9 0.2 0.1 1.1 18.1 15.6 0.02 0.1 -

Palmitic 16 0 11 3.9 24.7 6.8 11.6 44 8.9 7.7 10.5 9.2 6

Palmitoleic 16 lA9c 0.1 0.2 0.7 0.1 0.2 0.1 - - 0.6 0.1 1

Stearic 18 0 4 1.9 2.3 4.7 3.1 45 2.7 1.9 2.6 5.8 2.5

Oleic 18 lA9c 23.4 64.1 17.6 18.6 46.5 39.2 6.4 15 76.9 40.6 19

Linoleic 18 2A9c,A12c 53.2 18.7 53.3 68.2 31.4 10.1 1.6 2.7 7.5 42.6 24.1

18 3A9c,A12c,

Linolenic 7.8 9.2 0.3 0.5 - 0.4 - - 0.6 0.3 47.4

Δ15c

Arachidic 20:0 0.3 0.6 0.1 0.4 1.5 0.4 0.1 - 0.4 0.7 0.5

Gadoleic 20:A9c - 1 - - 1.4 - - - 0.3 0.2 -

20:2Al lc,A14

Eicosadienoic - - - - 0.1 - - - - - - c

Behenic 22:0 0.1 0.2 - - 3 - - - 0.2 0.2 -

Lignoceric 24:0 - 0.2 - - 1 - - - 0.1 - -

See, Gunstone, F.D. Structured and Modified Lipids. (CRC, 2001)

Table 5. Fatty acids compositions of modified Soybean and Canola seed oils

high- high- high- high- high- high-

Fatty acid Δ notation Soybean Canola

unsat palmitic stearic oleic oleic 1 auric

Hexanoic 6:0 - - - - - - - -

Octanoic (Caprylic) 8:0 - - - - - - - -

Decanoid acid

10:0

(Capric)

Laurie 12 0 - - - - - - - 38.8

Myristic 14 0 0.1 - - - - - 0.1 4.1

Palmitic 16 0 1 1 4 25 9 6 3.9 3.4 2.7

Palmitoleic 16 lA9c 0.1 - - - - 0.2 0.2 0.2

Stearic 18 0 4 3 4 26 3 1.9 2.5 1.6

Oleic 18 lA9c 23.4 28 16 18 86 64.1 77.8 32.8

Linoleic 18 2A9c,A12c 53.2 61 44 39 2 18.7 9.8 1 1.3

18 3A9c,A12c, Δ15

Linolenic 7.8 3 10 8 2 9.2 2.6 6.3

Arachidic 20:0 0.3 - - - - 0.6 0.9 0.4

Gadoleic 20:A9c - - - - - 1 1.6 0.8

Eicosadienoic 20:2Al lc, A14c - - - - - - - -

Behenic 22:0 0.1 - - - - 0.2 0.5 0.2

Lignoceric 24:0 - - - - - 0.2 - 0.2

See, Gunstone, F.D. Structured and Modified Lipids. (CRC, 2001)

[0101] In some embodiments, the unsaturated fatty acid is selected from Z-11-octadecenoic acid, Z-13-octadecenoic acid, and Z-l l-hexadecenoic acid. Plants that produce high quantities of these fatty acids include: Asclepias Syriaca (Z-11-octadecenoic acid, 44.9%), Cardwellia sublimis (Z-13 octadecenoic acid, 22%) and Kermadecia sinuate (Z-l l- hexadecenoic acid, 40.3%). See, Kuemmel and Chapman Lipids 3, 313 (1968); Vickery. Phytochemistry 10, 123-130 (1971).

[0102] In some embodiments, the unsaturated fatty acid is selected from Z-11-octadecenoic and Z-l l-hexadecenoic acid. Microalgae producing high quantities of these fatty acids include Pediastrum simplex (Z-11-octadecenoic acid, 68%) and Synechococcus elongates (Z- 11 -hexadecenoic acid, 35%).

[0103] In some embodiments, the unsaturated fatty acid is 2£^,,4Z,7Z-decatrienoic acid, which can be obtained from the juice of Noni fruit and isolated from Streptomyces viridochromogenes Tu 6105. See, Basar and Westendorf, Food Anal. Meth. 4, 57-65 (2011); Maier, et al. Pestic. Sci. 55, 733-739 (1999). Hydroxylation Catalysts

[0104] Various enzymes and/or whole cells comprising enzymes can be used to catalyze hydroxylation reactions described above.

[0105] Known enzyme families with terminal hydroxylation activity for medium and long chain alkanes and fatty acids include AlkB, CYP52, CYP153, and LadA (Bordeaux et al, 2012, Angew. Chem.-Int. Edit. 51 : 10712-10723; Ji et al, 2013, Front. Microbiol. 4). For example, Malca et al. describe terminal hydroxylation of mono-unsaturated fatty acid by cytochromes P450 of the CYP153 family (Malca et al, 2012, Chemical Communications 48: 5115-5117). Weissbart et al. describe the terminal hydroxylation of various cis and trans unsaturated lauric acid analogs (Weissbart et al, 1992, Biochimica et Biophysica Acta, Lipids and Lipid Metabolism 1124: 135-142). However, to date, none of these enzymes has been demonstrated to perform terminal hydroxylation of alkenes with internal olefins such as (E)- dec-5-ene. The presence of C=C bonds present competing sites of oxygen insertion and alters the 3 -dimensional orientation of the molecule. The regioselectivity of these enzymes for the terminal C-H bond of alkanes and fatty acid substrate may not extend to alkenes with internal olefins for these reasons. For asymmetric substrates, obtaining hydroxylation at the desired terminal C-H bond presents additional challenges compared to symmetric substrates. Finally, controlling the reaction selectivity to produce a single terminal alcohol instead of α-ω diols, acids, or diacids is also a major concern.

[0106] In particular embodiments, the search for a terminal hydroxylase with activity for unsaturated carboxylic acids having internal olefins starts with known terminal alkane and fatty acid hydroxylases. There are four families of enzymes with reported terminal alkane and fatty acid hydroxylation activity: (1) methane monooxygenases; (2) integral membrane diiron non-heme alkane hydroxylases (AlkB); (3) Cytochrome P450s (P450s); and (4) long chain alkane monoxygenases (LadA) (Bordeaux et al., 2012, Angew. Chem.-Int. Edit. 51 : 10712-10723; Ji et al, 2013, Front. Microbiol. 4). Methane monooxygenases are difficult to express in heterologous non-methanotrophic hosts and generally prefer small substrate (<C4). Of the remaining three families, the substrate specificity based on substrate chain length of representative members is summarized below in Table 6.

Table 6. Relative activities of terminal hydroxylases for alkanes and fatty acids with various chain lengths.

CYP15 LadA

AlkB CYP52 CYP52

CYP15 CYP52

CYP153 3A P. alkB2 (Feng et

3A16 P. putida al, A3 A4

A21

A6 sp. Gordonia (Scheller (Scheller

(Scheps et GPol 2007, (Kim et (Funhoff (Scheps et sp TF6* et al. , et al. ,

Alkane FA al, 2011, (Vanbeilen Proc. al. , 2007, et al. , al, 2011,

Org. (Fujii et al , 1996, 1996,

et al , 1994, Natl.

chain length Arch.

2006, J. Org. 2004, Biosci. Arch. Arch.

Biomol. Enzyme Acad. Biochem. Bacteriol. Biomol. Biotechnol. Biochem Biochem

Chem. 9: Microb. Sci. U. S. Biophys. 188: 5220- Chem. 9: Biochem. 68: Biophys. Biophys.

6727- Technol. 16: A. 104: 464: 5227) 6727- 2171-2177) 328: 328:

6733) 904-911) 5602- 213-220)

6733) 245-254) 245-254)

5607)

C8 100 100 100 95 72

C9 82 29 69 100 63

CIO 23 13 60 60 66

Cl l 1 <8 <6 6 48

C12 34 41 37

C12 FA

20 100 100 (lauric)

C14

C14 FA

86

(Myristic)

C15 83

C16 100 100 33

C16 FA

35 18 29 (Palmitic)

C18 78 48 20

C18 FA

30 1

(Stearic)

C22 74

C24 65

100% relative activity obtained with hexane [0107] In certain embodiments, depending on the chain length of the desired substrate, some members of these four enzyme families are better suited than others as candidates for evaluation. For C-10 substrates, the substrate specificity of characterized CYP153 and AlkB enzymes makes them candidate enzymes. Likewise, for longer substrates, members of the LadA and CYP52 families appear to have the closest substrate profile.

[0108] The most widely characterized member of the AlkB family is obtained from the Alk system of Pseudomonas putida GPol (van Beilen and Funhoff, 2005, Curr. Opin. Biotechnol. 16: 308-314). In addition to the integral membrane diiron non-heme hydroxylase AlkB, a rubredoxin (AlkG) and a rubredoxin reductase (AlkT) are required for hydroxylation function. The entire Alk system of P. putida GPol, alkBFGHJKL and alkST genes, which allows the strain to grown on alkanes as its sole carbon source, has been cloned into the broad host range vector pLAFRl (pGEc47) and is available from DSMZ in the host E. Coli K12 Gecl37 (Smits et al, 2001, Plasmid 46: 16-24). The other alk genes alkF, alkJ, alkH, alkK, alkL, and alkS encode an inactive rubredoxin, an alcohol dehydrogenase, an aldehyde dehydrogenase, an acyl-CoA synthase, an alkane transporter and a global pathway regulator, respectively (Smits et al, 2003, Antonie Van Leeuwenhoek 84: 193-200). These genes facilitate the use of the alcohol product from the AlkB reaction to generate the fatty acyl-CoA that is substrate for β-oxidation. To accumulate the alcohol product, a knockout strain of alkJ, E. coli GEC137 pGEc47AJ has been used in a whole-cell biotransformation to produce 1-dodecanol (Grant et al, 2011, Enzyme Microb. Technol. 48: 480-486). The presence of alkL appears to enhance substrate uptake and consequently improve the whole-cell activity for both Pseudomonas and E. coli (Cornelissen et al, 2013, Biotechnology and Bioengineering 110: 1282-1292; Julsing et al, 2012, Appl Environ. Microbiol. 78: 5724- 5733; Scheps et al, 2013, Microb. Biotechnol. 6: 694-707). A simplified version of pGEc47 containing only alkBFGST m the broad-host range vector pCOMlO, pBTIO, has also been used for the conversion of fatty-acid methyl esters to co-hydroxy fatty acid methyl esters in E. coli W3110 (Schrewe et al, 2011 , Advanced Synthesis & Catalysis 353 : 3485-3495).

[0109] CYP52 family members are membrane bound cytochrome P450s that require electron delivery from a reductase for function. CYP52 members have mainly been identified from alkane-degrading Candida species (Scheller et al, 1996, Arch. Biochem. Biophys. 328: 245-254; Craft et al, 2003, Appl. Environ. Microbiol. 69: 5983-5991; Scheller et al, 1998, J. Biol. Chem. 273 : 32528-32534; Seghezzi et al, 1992, DNA Cell Biol. 11 : 767- 780; Zimmer et al, 1996, Biochem. Biophys. Res. Commun. 224: 784-789). Thus far, expression and characterization of CYP52 enzymes have been performed in the native Candida host and other yeast hosts. Gene knockouts of (1) the β-oxidation pathways, (2) alcohol dehydrogenases and (3) select native CYP52s has resulted in strains that can accumulate co-hydroxy fatty acids when fatty acids are fed to the culture (Lu et al, 2010, J. Am. Chem. Soc. 132: 15451-15455). Of particular interest, DP428, DP522 and DP526 are C. tropicalis strains expressing a single CYP52 with the appropriate knockouts for catalyzing terminal hydroxylation of fatty acids (Lu et al, 2010, J. Am. Chem. Soc. 132: 15451-15455).

[0110] CYP153 family members are soluble and membrane associated cytochrome P450s that also depend on electron transfer from ferredoxin and ferredoxin reductase for function (Funhoff et al., 2007, Enzyme and Microbial Technology 40: 806-812). CYP153 members have been isolated from a range of alkane-degrading microorganisms. There are currently 56 annotated CYP153 sequences available from the Nelson P450 database, a BLAST search of CYP153A6 resulted in 221 identified homologs with >70% sequence identity. The use of CYP153 enzymes for terminal hydroxylation of octane and dodecanoic acid has been demonstrated with heterologous expression in E. coli. For the conversion of octane to octanol, the CYP153 operon from Mycobacterium sp. HXN-1500 was cloned into pET28b(+) and the biotransformation was performed in E. coli BL21(DE3) (Gudiminchi et al, 2012, Appl. Microbiol. Biotechnol. 96: 1507-1516). For the conversion of dodecanoic acid, an E. coli LEVIS 174 strain containing a fusion of a CYP153A_M.aq_. mutant with the CYP102A1 reductase domain in pColaDuet-1 along with alkL was used for the transformation (Scheps et al, 2013, Microb. Biotechnol. 6: 694-707).

[0111] Long chain alkane monooxygenase, LadA, isolated from G. thermodenitrificants NG80-2 catalyzes the terminal hydroxylation of CI 5 to C36 alkanes with a metal -free flavoprotein mechanism that differs from AlkB and CYP enzymes (Dong et al, 2012, Appl. Microbiol. Biotechnol. 94: 1019-1029). The LadA reaction requires FMNH₂ or NADPH and the native reductase partner has yet to be identified. Expression of the LadA gene in E. coli BL21 (DE3) using the pET-28a(+) plasmid yielded cell extracts with terminal hydroxylation activity for hexadecane (Dong et al, 2012, Appl. Microbiol. Biotechnol. 94: 1019-1029). Literature reports of LadA hydroxylation reactions have been performed using purified enzymes and examples of whole-cell biotransformation is lacking.

[0112] Coding sequences for enzymes that may be used herein may be derived from bacterial, fungal, or plant sources. Tables 3, 4, and 5 list enzymes for coding regions of representative non-heme diiron alkane monooxygenases, long-chain alkane hydroxylases, and cytochromes P450, respectively. Additional enzymes and their coding sequences may be identified by BLAST searching of public databases. Typically, BLAST searching of publicly available databases with known non-heme diiron alkane monooxygenases, cytochromes P450, and long-chain alkane hydroxylase sequences, such as those provided herein, is used to identify enzymes and their encoding sequences that may be used in the present invention. For example, enzymes having amino acid sequence identities of at least about 80-85%, 85%- 90%, 90%-95%, or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of the enzymes listed in Tables 3, 4, and 5 may be used. Hydroxylase enzymes can be codon-optimized for expression in certain desirable host organisms, such as yeast and E. coli.

[0113] In other embodiments, the sequences of the enzymes provided herein may be used to identify other homologs in nature. For example, each of the encoding nucleic acid fragments described herein may be used to isolate genes encoding homologous proteins. Isolation of homologous genes using sequence-dependent protocols is well known in the art. Examples of sequence-dependent protocols include, but are not limited to, (1) methods of nucleic acid hybridization, (2) methods of DNA and RNA amplification, as exemplified by various uses of nucleic acid amplification technologies {e.g., polymerase chain reaction (PCR), Mullis et al, U.S. Pat. No. 4,683,202; ligase chain reaction (LCR), Tabor, S. et al, Proc. Acad. Sci. USA 82: 1074 (1985); or strand displacement amplification (SDA), Walker et al, Proc. Natl. Acad. Sci. USA, 89:392 (1992)), and (3) methods of library construction and screening by complementation.

[0114] Hydroxylase enzymes or whole cells expressing hydroxylase enzymes can be further engineered for use in the methods of the invention. Enzymes can be engineered for improved hydroxylation activity, improved Z:E selectivity, improved regioselectivity, improved selectivity for hydroxylation over epoxidation and/or improved selectivity for hydroxylation over dehalogenation. The term "improved hydroxylation activity" as used herein with respect to a particular enzymatic activity refers to a higher level of enzymatic activity than that measured in a comparable non-engineered hydroxylase enzyme of whole cells comprising a hydroxylase enzyme. For example, overexpression of a specific enzyme can lead to an increased level of activity in the cells for that enzyme. Mutations can be introduced into a hydroxylase enzyme resulting in engineered enzymes with improved hydroxylation activity. Methods to increase enzymatic activity are known to those skilled in the art. Such techniques can include increasing the expression of the enzyme by increasing plasmid copy number and/or use of a stronger promoter and/or use of activating riboswitches, introduction of mutations to relieve negative regulation of the enzyme, introduction of specific mutations to increase specific activity and/or decrease the K_M for the substrate, or by directed evolution. See, e.g., Methods in Molecular Biology (vol. 231), ed. Arnold and Georgiou, Humana Press (2003).

[0115] Accordingly, some embodiments of the invention provide synthetic methods as described above, wherein the enzyme is a non-heme diiron monooxygenase. In some embodiments, the non-heme diiron monooxygenase is selected from Table 7 or a variant thereof having at least 90% identity thereto.

Table 7. Exemplary non-heme diiron monooxygenase enzymes.

Species Origin Gene Name Accession No

Ochrobactrum sp. ITRH1 alkB B5TVA3

Alcaligenaceae bacterium BTRH5 alkB B5TVA6

Pseudomonas sp. ITRH76 alkB B5TVA5

Pseudomonas sp. 7/156 alkB Q93LR8 uncultured Rhizobiales bacterium alkB D6NSH3 uncultured soil bacterium S5DSW0 uncultured bacterium alkB U3PYH2 uncultured prokaryote alkB C7EAT4 uncultured Rhizobiales bacterium alkB D6NSL1 uncultured Rhizobiales bacterium alkB D6NSJ4 uncultured prokaryote alkB C7EAZ5 uncultured Rhizobiales bacterium alkB D6NSK5 uncultured Rhizobiales bacterium alkB D6NSK3 uncultured Rhizobiales bacterium alkB D6NSJ7 uncultured Rhizobiales bacterium alkB D6NSK1 uncultured Rhizobiales bacterium alkB D6NSH4 uncultured Rhizobiales bacterium alkB D6NSJ2 uncultured Rhizobiales bacterium alkB D6NSI2 uncultured Rhizobiales bacterium alkB D6NSJ3 uncultured Rhizobiales bacterium alkB D6NSJ6

Pseudomonas sp. ITRI22 alkB B5TVB9 uncultured Rhizobiales bacterium alkB D6NSK7 uncultured soil bacterium S5DTG4

Pseudomonas putida (Arthrobacter siderocapsulatus) alkB Q9WWW6 uncultured Rhizobiales bacterium alkB D6NSI6 uncultured bacterium alkB B6Z2E6 uncultured bacterium alkB B1P6K4

Pseudomonas sp. G5(2012) PG5 40690 S2EW96

Alcanivorax dieselolei alkB B6Z2B7

Alcanivorax borkumensis alkB B6Z284 uncultured bacterium alkB B6Z2G9

Marinobacter sp. S17-4 alkB C7DLJ8 uncultured bacterium alkB B6Z2H0

Alcanivorax sp. S17-16 alkB B6Z2D8 uncultured organism alkB G3EBX7 uncultured bacterium alkB H9NJ23 uncultured bacterium alkB C8AYB7 uncultured bacterium alkB W0UB63 uncultured bacterium alkB U3Q1V0

Alcanivorax borkumensis alkB T1WPB9 Species Origin Gene Name Accession No uncultured organism alkB G3EBX5 uncultured Rhizobiales bacterium alkB D6NSK6 uncultured bacterium alkB U3Q5C8 uncultured bacterium alkB Q3HXE5

Xanthobacter flavus alkane- 1 -monooxygenase Q934J9 uncultured bacterium alkB Q3HXD6

Acidisphaera sp. C197 alkB Q5RLH8 uncultured bacterium alkB M9T624 uncultured bacterium alkB M9T8D1 uncultured bacterium alkB H9B8U8

Kordiimonas gwangyangensis alkB B6Z2E4 uncultured soil bacterium S5DPL2 uncultured bacterium alkB F0X332 uncultured bacterium alkB F0X324 uncultured bacterium alkB F0X334 uncultured organism alkB G3EBX2 uncultured bacterium alkB F0X328 uncultured soil bacterium S5DTI7 uncultured bacterium alkB Q3HXF7 uncultured bacterium alkB F0X327 uncultured bacterium alkB F0X335 uncultured bacterium alkB F0X329 uncultured bacterium alkB F0X342 uncultured bacterium alkB F0X300 uncultured bacterium alkB Q3HXE8 uncultured bacterium alkB U3Q1X0 uncultured bacterium alkB Q3HXD7

Ralstonia sp. PT11 alkB Q3HXC9 uncultured bacterium alkB Q3HXE6 uncultured bacterium alkB F0X305 uncultured bacterium alkB U3Q5A0 uncultured bacterium alkB F0X306

Marinobacter sp. P1-14D alkBl C6KEH4 uncultured Rhizobiales bacterium alkB D6NSI7 uncultured bacterium alkB F0X346 uncultured bacterium alkB F0X346 uncultured bacterium alkB F0X343 uncultured bacterium alkB F0X339 uncultured bacterium alkB F0X309 uncultured bacterium alkB F0X333 Species Origin Gene Name Accession No uncultured bacterium alkB F0X321 uncultured bacterium alkB Q3HXF0 uncultured bacterium alkB F0X312 uncultured bacterium alkB F0X303 uncultured bacterium alkB F0X331 uncultured bacterium alkB F0X302 uncultured bacterium alkB Q3HXE9 uncultured bacterium alkB F0X313 uncultured bacterium alkB F0X316 uncultured bacterium alkB M9TDK6 uncultured bacterium alkB H9B8V5 uncultured Rhizobiales bacterium alkB D6NSF4 uncultured Rhizobiales bacterium alkB D6NSF2 uncultured bacterium alkB B6Z2G8 uncultured Rhizobiales bacterium alkB D6NSF1 uncultured Rhizobiales bacterium alkB D6NSG4 uncultured Rhizobiales bacterium alkB D6NSG3 uncultured Rhizobiales bacterium alkB D6NSF3 uncultured Rhizobiales bacterium alkB D6NSI4 uncultured Rhizobiales bacterium alkB D6NSH9 uncultured Rhizobiales bacterium alkB D6NSG1 uncultured Rhizobiales bacterium alkB D6NSJ9 uncultured Rhizobiales bacterium alkB D6NSG6 uncultured soil bacterium S5DP42 uncultured bacterium alkB F0X323 uncultured bacterium alkB F0X318 uncultured bacterium alkB F0X317 uncultured bacterium alkB F0X325 uncultured bacterium alkB F0X308 uncultured bacterium alkB F0X336 uncultured soil bacterium S5E0W0 uncultured bacterium alkB F0X304

Bradyrhizobium sp. DFCI-1 C207 00091 U1HQ84 uncultured Rhizobiales bacterium alkB D6NSF9 uncultured Rhizobiales bacterium alkB D6NSH2 uncultured Rhizobiales bacterium alkB D6NSF6 uncultured Rhizobiales bacterium alkB D6NSG2 uncultured Rhizobiales bacterium alkB D6NSH7 uncultured bacterium alkB F0X322 uncultured soil bacterium S5DPY4 Species Origin Gene Name Accession No uncultured bacterium alkB F0X349 uncultured bacterium alkB F0X310 uncultured bacterium alkB F0X315 uncultured bacterium alkB F0X344 uncultured bacterium alkB F0X326 uncultured bacterium alkB W0UB94 uncultured bacterium alkB W0UAL7 uncultured soil bacterium S5DP84 uncultured soil bacterium S5E064 uncultured soil bacterium S5E0M5 uncultured bacterium alkB M9T7Y4 uncultured prokaryote alkB C7EAZ7

Thalassolituus oleivorans alkB Q8RSS6 uncultured prokaryote alkB C7EAZ8

Marinobacter sp. EVN1 Q672 13115 U7NVU4 uncultured Rhizobiales bacterium alkB D6NSF8

Marinobacter aquaeolei (strain ATCC 700491 / DSM

11845 / VT8) (Marinobacter hydrocarbonoclasticus Maqu_0610 A1TY92 (strain DSM 11845))

Marinobacter hydrocarbonoclasticus ATCC 49840 alkB MARHY2847 H8WCU7 uncultured Rhizobiales bacterium alkB D6NSG8

Alcanivorax borkumensis alkBl Q93UQ1

Alcanivorax borkumensis (strain SK2 / ATCC 700651 /

alkBl ABO_2707 Q0VKZ3 DSM 11573)

Marinobacter aquaeolei (strain ATCC 700491 / DSM

11845 / VT8) (Marinobacter hydrocarbonoclasticus Maqu_0440 A1TXS2 (strain DSM 11845))

Alcanivorax sp. 97CO-5 Y017_07510 W7AC06

Marinobacter sp. C1S70 Q667 13505 U7P171

Marinobacter sp. EVN1 Q672 13130 U7NYF9

Pseudoxanthomonas spadix (strain BD-a59) DSC_08960 G7UVX3

Marinobacter sp. EN3 Q673 04890 U7H9M7

Marinobacter sp. ES-1 Q666 09550 U7G9A6

Oceanicaulis sp. HTCC2633 OA2633 08724 A3UHL2

Citreicella sp. 357 C357 19621 I1AS33

Caulobacter sp. (strain K31) Caul_5439 B0TA04

Thalassolituus oleivorans MIL-1 TOL 1423 M5DQR5 uncultured bacterium alkB W0UAQ4 uncultured bacterium alkB W0UAL9 uncultured bacterium alkB W0UAQ9 Species Origin Gene Name Accession No gamma proteobacterium NOR5-3 NOR53 3428 B8KLY6 uncultured marine microorganism 21G8-5 A5CFX9 uncultured marine microorganism 9E7-8 A5CFU5

Alcanivorax pacificus Wl 1-5 S7S_02132 K2GLA3

Alcanivorax dieselolei C3W4W7

Alcanivorax sp. PN-3 Q668 06955 U7I1M1

Alcanivorax dieselolei (strain DSM 16502 / CGMCC

B5T 00721 K0C8Z6 1.3690 / B-5)

Alcanivorax dieselolei alkB2 D2JNY2 bacterium enrichment culture clone US3-MTBE mdpA L7T214 bacterium enrichment culture clone US2-MTBE mdpA L7SZY0

Marinobacter sp. ELB17 MELB17 10558 A3JHB9

Marinobacter sp. BSs20148 alkBl MRBBS_1602 M1FBW8

Pseudomonas alcaligenes NBRC 14159 alkB PA6 005 01830 U3AUD1

Simiduia agarivorans (strain DSM 21679 / JCM 13881 /

M5M 18065 K4KP06 BCRC 17597 / SA1)

gamma proteobacterium HTCC2207 GB2207 03060 Q1YPC4

Limnobacter sp. MED 105 LMED105 14555 A6GTF8

Alcanivorax sp. R8-12 alkB2 R9R6I2

Gammaproteobacteria bacterium MOLA455 alkBl U062 00014 W2UFM4

Alcanivorax hongdengensis A-l 1-3 A11A3 01150 L0WGR7

Acidovorax sp. KKS102 C380 12125 K0IAD8

Moritella sp. PE36 PE36 11657 A6FHH9

Ahrensia sp. R2A130 alkB R2A130 3229 E0MP68

Hoeflea phototrophica DFL-43 HPDFL43 04645 A9D3P4

Curvibacter putative symbiont of Hydra magnipapillata alkB Csp_A02180 C9Y7W7

Pseudovibrio sp. JE062 PJE062 1512 B6QXF8

Oxalobacteraceae bacterium IMCC9480 IMCC9480 2292 F1W4Y4

Methylibium petroleiphilum (strain PM1) alkB Mpe_B0606 A2SP81

Ralstonia sp. AU12-08 C404 01360 S9TME3

Burkholderia phytofirmans (strain DSM 17436 / PsJN) Bphyt_5401 B2TBV7 gamma proteobacterium BDW918 DOK 05250 I2JMD3

Pseudovibrio sp. (strain FO-BEG1) alkB PSE 3490 G8PKM2

Bradyrhizobium sp. DFCI-1 C207 06028 U1H8I8

Alcanivorax dieselolei (strain DSM 16502 / CGMCC

B5T 04393 K0CLJ4 1.3690 / B-5)

Alcanivorax sp. PN-3 Q668 04650 U7HLN0 Species Origin Gene Name Accession No

Alcanivorax dieselolei alkBl Q6B431

Burkholdena thailandensis E444 BTJ 212 W6C501

Burkholdena thailandensis 2002721723 BTQ 2100 W6BLA1

Burkholdena thailandensis H0587 BTL 1506 W6BA85

Burkholdena thailandensis (strain E264 / ATCC 700388 /

BTH I1814 Q2SXK3 DSM 13276 / CIP 106301)

Burkholdena pseudomallei 1026b BP1026B 10975 I1WH83

Burkholdena pseudomallei 1026a BP1026A 4019 I2KNJ5

Burkholdena pseudomallei MSHR305 BDL 3139 S5P5X7

Burkholdena pseudomallei 305 alkB BURPS305 7408 A4LDP5

Burkholdena pseudomallei Pasteur 52237 alkB BURPSPAST R0133 A8KVJ2

Burkholdena pseudomallei (strain K96243) BPSL2350 Q63SH1

Burkholdena pseudomallei (strain 1710b) BURPS 1710b_2801 Q3JQG8

Burkholdena pseudomallei BPC006 BPC006 I2776 K7Q7Y2

Burkholdena pseudomallei 1710a alkB BURPS 1710A 3234 C6TUD4

Burkholdena pseudomallei 1106b alkB_2BURPSl 106B A1957 C5ZKC8

Burkholdena pseudomallei (strain 1106a) alkB BURPS1106A 2735 A3NXB5

Burkholdena pseudomallei (strain 668) BURPS668 2678 A3NBI1

Burkholdena pseudomallei NCTC 13178 BBJ 481 V9Y591

Burkholdena pseudomallei MSHR1043 D512 14116 M7EHA3

Burkholdena pseudomallei 354a BP354A 0895 I2MQ94

Burkholdena pseudomallei 354e BP354E 0708 I2MD23

Burkholdena pseudomallei 1258b BP1258B 0905 I2LQQ4

Burkholdena pseudomallei 1258a BP1258A 0812 I2LKD3

Burkholdena pseudomallei 576 alkB BUC_2998 B7CM79

Burkholdena pseudomallei 1655 alkB BURPS1655 H0133 B2HAC8

Burkholdena pseudomallei S13 alkB BURPSS13_V0139 B1HDJ2

Burkholdena pseudomallei 406e alkB BURPS406E H0229 A8EBS1

Burkholdena pseudomallei MSHR146 BBN 1088 W0PXC8

Burkholdena pseudomallei MSHR511 BBQ 961 W0MCN0

Burkholdena pseudomallei NAU20B-16 BBS_2570 V9YGA1

Burkholdena pseudomallei MSHR346 GBP346 A2857 C4KQU6

Burkholdena pseudomallei MSHR338 M218 13015 W1M8G5

Burkholdena xenovorans (strain LB400) Bxe_B1208 Q13ME1

Burkholdena thailandensis MSMB43 A33K 14899 I6AHY8

Burkholdena sp. Chl-1 BChl lDRAFT_02054 I2IU52

Alcanivorax sp. R8-12 alkB3 R9R6Q8 gamma proteobacterium HTCC5015 GP5015 636 B5JV27

Alcanivorax pacificus Wl 1-5 S7S_03034 K2GFU4 Species Origin Gene Name Accession No

Actinoplanes sp. (strain ATCC 31044 / CBS 674.73 /

alkB ACPL 4910 G8SLX8 SE50/110)

Alcanivorax sp. DG881 ADG881 1174 B4X426

Methylibium sp. T29-B alkBl Y694 03823 W7WAG2

Methylibium sp. T29 mdpA X551 03232 W7VT91

Burkholderia thailandensis MSMB121 BTI 1284 N0AI18

Burkholderia sp. TJI49 B1M 44170 F0GKQ0

Burkholderia mallei (strain ATCC 23344) alkB BMA0635 Q62LK2

Burkholderia mallei (strain NCTC 10247) alkB BMA10247 1692 A3MLU7

Burkholderia mallei (strain NCTC 10229) alkB BMA10229_A2910 A2SA87

Burkholderia mallei (strain SAVP1) alkB BMASAVP1_A2377 A1V630

Burkholderia mallei PRL-20 alkB BMAPRL20 A0647 C5NLY3

Burkholderia mallei GB8 horse 4 BMAGB8 0674 C4AYJ3

Burkholderia mallei ATCC 10399 alkB BMA10399 E0136 A9KA35

Burkholderia mallei JHU alkB BMAJHU_C0140 A5XN41

Burkholderia mallei FMH alkB BMAFMH C0136 A5XJ42

Burkholderia mallei 2002721280 alkB BMA721280 A1345 A5TJ65

Burkholderia pseudomallei Pakistan 9 alkB BUH 2787 C0YFB6

Burkholderia sp. (strain 383) (Burkholderia cepacia

Bcepl8194_A4085 Q39IN4 (strain ATCC 17760 / NCIB 9086 / R18194))

Ralstonia sp. 5 2 56FAA HMPREF0989 00681 U3G9A8

Ralstonia sp. 5 7 47FAA HMPREF1004 00261 E2ST40

Burkholderia cenocepacia (strain AU 1054) Bcen_0501 Q1BY92

Burkholderia cenocepacia (strain HI2424) Bcen2424_0980 A0K5F6

Burkholderia sp. KJ006 MYA_0870 I2DKR1

Burkholderia vietnamiensis (strain G4 / LMG 22486)

Bcepl808_0897 A4JCA5 (Burkholderia cepacia (strain R1808))

Burkholderia cenocepacia KC-01 P355_2107 V5A0K9

Ralstonia pickettii (strain 12D) Rpicl2D_4221 C6BN09

Ralstonia pickettii (strain 12J) Rpic_4109 B2UI09

Ralstonia pickettii OR214 OR214 00862 R0CSD0

Mycobacterium thermoresistibile ATCC 19527 KEK 22639 G7CND0

Burkholderia cenocepacia PC184 BCPG_00786 A2VS55

Parvularcula bermudensis (strain ATCC BAA-594 /

PB2503 09204 E0TD71 HTCC2503 / KCTC 12087)

Rhodococcus triatomae BKS 15-14 G419 20650 M2WXQ1

Alcanivorax hongdengensis A-l 1-3 A11A3 01155 L0WH65

Alcanivorax hongdengensis G1C7G7

Micromonospora sp. ATCC 39149 MCAG_04553 C4REI2

Micromonospora lupini str. Lupac 08 alkB MILUP08 41795 I0KZ81 Species Origin Gene Name Accession No

Patulibacter medicamentivorans PAI11 23570 H0E6A7

Burkholderia cenocepacia (strain ATCC BAA-245 / DSM

16553 / LMG 16656 / NCTC 13227 / J2315 / CF5610) BCAL3029 B4EBR3 (Burkholderia cepacia (strain J2315))

Burkholderia cenocepacia BC7 BURCENBC7 AP5666 U1ZCU6

Burkholderia cenocepacia K56-2Valvano BURCENK562V_C5856 T0E860

Burkholderia cenocepacia HI 11 135 3695 G7HIJ0

Burkholderia cepacia GG4 GEM 2548 J7J4L5

Burkholderia ambifaria IOP40-10 BamIOP4010DRAFT 1629 B1FC70

Burkholderia vietnamiensis AU4i L810 3738 U2H0D0

Burkholderia ambifaria MEX-5 BamMEX5DRAFT_0109 B1SX43

Burkholderia cenocepacia (strain MCO-3) Bcenmc03_0941 B1JX99

Burkholderia cepacia (Pseudomonas cepacia) alkB Q9AEN3

Burkholderia multivorans CGD1 BURMUCGD 1 2488 B9BAK1

Burkholderia multivorans (strain ATCC 17616 / 249) alkB BMULJ_00816 B3CYB3

Burkholderia multivorans CGD2M BURMUCGD2M 2894 B9CFY2

Burkholderia multivorans CGD2 BURMUCGD2 2807 B9BSN6

Burkholderia glumae (strain BGR1) bglu_lg25240 C5AA12

Burkholderia multivorans CF2 BURMUCF2 0698 J5AST2

Burkholderia multivorans ATCC BAA-247 BURMUCF1 0763 J4JJJ2

Mycobacterium xenopi RIVM700367 MXEN 06581 I0RWI2

Alcanivorax sp. P2S70 Q670 07625 U7G3V1

Rhodococcus sp. p52 alkB U5S015

Rhodococcus pyridinivorans AK37 AK37_15478 H0JTS8

Micromonospora sp. M42 MCBG_00051 W7V9N0

Nocardia nova SH22a NONO_c63170 W5TPA6

Actinoplanes missouriensis (strain ATCC 14538 / DSM

43046 / CBS 188.64 / JCM 3121 / NCIMB 12654 / AMIS_28610 I0H4Z4 NBRC 102363 / 431)

Mycobacterium thermoresistibile ATCC 19527 KEK_04707 G7CD93

Streptomyces collinus Tu 365 B446 00650 B446 34640 S5VEV9

Mycobacterium smegmatis MKD8 alkB D806 1894 L8FH78

Mycobacterium smegmatis (strain ATCC 700084 / alkB MSMEG 1839

A0QTH1 mc(2)155) MSMEI_1797

Burkholderia gladioli (strain BSR3) bgla_lg28520 F2LCU4

Nocardia cyriacigeorgica (strain GUH-2) alkB NOCYR_2725 H6R6Y1

Mycobacterium sp. (strain Spyrl) Mspyrl_40540 E6TPD9

Mycobacterium gilvum (strain PYR-GCK)

(Mycobacterium flavescens (strain ATCC 700033 / PYR- Mflv_4721 A4TF88 GCK)) Species Origin Gene Name Accession No

Mycobacterium hassiacum DSM 44199 C731 1322 K5BKD8

Mycobacterium phlei RIVM601174 MPHLEI 02293 I0S2Q3

Burkholderia ambifaria (strain MC40-6) BamMC406_0853 B1YUL7

Conexibacter woesei (strain DSM 14684 / JCM 11494 /

Cwoe_5739 D3F1V9 NBRC 100937 / ID131577)

Burkholderia ambifaria (strain ATCC BAA-244 /

Bamb_0841 Q0BHH3 AMMD) (Burkholderia cepacia (strain AMMD))

Mycobacterium vaccae ATCC 25954 MVAC_06502 K0V939

Streptomyces sp. AA4 SSMG_06597 D9UYP9

Nocardia asteroides NBRC 15531 alkB NCAST 33 00580 U5EK43

Hydrocarboniphaga effusa AP103 WQQ_35830 I8T3V4

Mycobacterium sp. (strain Spyrl) Mspyrl_27000 E6TM45

Rhodococcus sp. EsD8 EBESD8 14280 N1M251

Rhodococcus pyridinivorans SB3094 Y013 10875 Y013 14995 V9XCI1 uncultured bacterium alk A7XY59

Dietzia sp. D5 W0C8S6

Gordonia amarae NBRC 15530 alkB GOAMR 34 00200 G7GP29 gamma proteobacterium BDW918 DOK 15269 I2JH75

Marinobacter sp. EVN1 Q672 03155 U7NQ32

Marinobacter santoriniensis NKSG1 MSNKSG1_09613 M7CV98

Marinobacter sp. ES-1 Q666 05770 U7GFG6 gamma proteobacterium HdNl alkM HDNlF_04190 E1VGR0

Nocardia farcinica (strain IFM 10152) NFA_33210 Q5YUH3

Mycobacterium chubuense (strain NBB4) Mycch_2783 I4BJT7

Acinetobacter towneri DSM 14962 = CIP 107472 F947 01315 N9CH84

Rhodococcus erythropolis CCM2595 O5Y 10330 T1VNI2

Rhodococcus erythropolis (strain PR4 / NBRC 100887) alkB RER 21620 C0ZWY5

Rhodococcus sp. P27 N806 20680 U0E9X4

Rhodococcus erythropolis DN1 N601 09550 T5IBP7

Rhodococcus erythropolis (Arthrobacter picolinophilus) alkB A4ZZL2

Mycobacterium fortuitum subsp. fortuitum DSM 46621 MFORT 07571 K0VIS2

Rhodococcus qingshengii BKS 20-40 G418 14624 M2XAS5

Rhodococcus erythropolis (Arthrobacter picolinophilus) alkB2 Q9AE68

Rhodococcus sp. (strain RHA1) alkB RHAl_ro02534 Q0SDP7

Rhodococcus sp. JVHl JVH1 3134 J1RMD5

Rhodococcus wratislaviensis IFP 2016 Rwratislav_18854 L2TK91 Species Origin Gene Name Accession No

Rhodococcus wratislaviensis alkBl K7WI49

Rhodococcus sp. (strain Q 15) alkB2 Q93DM7

Rhodococcus opacus M213 WSS_A20069 K8XV97

Rhodococcus erythropolis (Arthrobacter picolinophilus) alkB V5LET8

Streptomyces sp. AA4 SSMG_06805 D9V1L5

Geobacillus sp. MH-1 alkB-geo6 C5J0F7

Mycobacterium neoaurum VKM Ac-1815D D174 08465 V5X9E7

Rhodococcus imtechensis RKJ300 = JCM 13270 W59 13161 I0WSJ7

Prauserella rugosa alkB Q9XBM1

Rhodococcus erythropolis SK121 RHOER0001 4201 C3JG64

Amycolatopsis azurea DSM 43854 C791 5134 M2PZK0

Mycobacterium rhodesiae (strain NBB3) MycrhN_0412 G8RK27

Rhodococcus ruber alkB7 D3U111

Rhodococcus ruber BKS 20-38 G352_25762 M2XQQ3

Mycobacterium chubuense (strain NBB4) D2JYT1

Mycobacterium chubuense (strain NBB4) Mycch_1351 I4BFU6

Mycobacterium smegmatis JS623 Mycsm_01384 L0IUF4

Nocardia nova SH22a alkB NONO_c46180 W5TJL9

Rhodococcus sp. BCP1 alkB E5G6V9

Saccharomonospora marina XMU15 SacmaDRAFT_4417 H5X9W5

Mycobacterium sp. (strain JLS) Mjls_1369 A3PW94

Rhodococcus ruber alkB7 D3U119

Mycobacterium tuberculosis BT1 alkB HKBT1 3428 W6HJ76

Mycobacterium tuberculosis BT2 alkB HKBT2 3435 W6H3Z6

Mycobacterium tuberculosis HKBS1 alkB HKBSl_3438 W6GVB7

Mycobacterium tuberculosis EAI5 M943 16800 S5F023

Mycobacterium tuberculosis EAI5/NITR206 J114 17435 R4MLW1

Mycobacterium tuberculosis CAS/NITR204 J113 22685 R4MIF7

Mycobacterium bovis (strain ATCC BAA-935 /

alkB Mb3280c Q7TWW3 AF2122/97)

Mycobacterium tuberculosis (strain ATCC 25618 /

alkB Rv3252c RVBD_3252c 005895 H37Rv)

Mycobacterium tuberculosis str. Beijing/NITR203 J112 17475 M9UX97

Mycobacterium bovis BCG str. Korea 1168P K60 033810 M1IQ04

Mycobacterium liflandii (strain 128FXT) alkB MULP 01451 L7V4G7

Mycobacterium tuberculosis (strain CDC 1551 /

alkB MT3350 L7N540 Oshkosh)

Mycobacterium canettii CIPT 140070017 alkB BN45 60281 L0QZH1

Mycobacterium canettii CIPT 140070008 alkB BN43 60261 L0QC77 Species Origin Gene Name Accession No

Mycobacterium canettii CIPT 140060008 alkB BN44 70036 L0Q026

Mycobacterium tuberculosis 7199-99 MT7199 3294 L0NZI4

Mycobacterium tuberculosis KZN 605 TBXG_003280 I6RJV1

Mycobacterium tuberculosis KZN 4207 TBSG_03323 I1SDS8

Mycobacterium tuberculosis RGTB327 MRGA327 20020 H8HLB9

Mycobacterium tuberculosis (strain ATCC 35801 / TMC

alkB ERDMAN 3566 H8EY95 107 / Erdman)

Mycobacterium tuberculosis UT205 alkB UDA_3252c H6S7Q5

Mycobacterium bovis BCG str. Mexico alkB BCGMEX_3279c G7QY42

Mycobacterium tuberculosis CTRI-2 alkB MTCTRI2_3319 G2N7Q9

Mycobacterium canettii (strain CIPT 140010059) alkB MCAN_32711 G0THM9

Mycobacterium africanum (strain GM041182) alkB MAF 32630 F8M6G6 alkB CCDC5180 2963

Mycobacterium tuberculosis (strain CCDC5180) F7WQM1

CFBR 3446

alkB CCDC5079 3000

Mycobacterium tuberculosis (strain CCDC5079) F7WLN9

CFBS 3441

Mycobacterium tuberculosis (strain KZN 1435 / MDR) TBMG_03300 C6DXJ8

Mycobacterium bovis (strain BCG / Tokyo 172 / ATCC

alkB JTY 3277 C1AH26 35737 / TMC 1019)

Mycobacterium marinum (strain ATCC BAA-535 / M) alkB MMAR 1291 B2HEP2

Mycobacterium tuberculosis (strain Fl 1) TBFG_13281 A5WSG7

Mycobacterium tuberculosis (strain ATCC 25177 /

alkB MRA 3293 A5U7S6 H37Ra)

Mycobacterium tuberculosis str. Haarlem TBHG_03188 A4KLE9

Mycobacterium bovis (strain BCG / Pasteur 1173P2) alkB BCG_3281c A1KNQ4

Mycobacterium bovis 04-303 O216 17560 V2W1E0

Mycobacterium bovis AN5 O217_17270 V2VQT4

Mycobacterium tuberculosis GuangZ0019 alkB GuangZ0019_1145 T5HDB1

Mycobacterium tuberculosis FJ05194 alkB FJ05194_2026 T5H4I2

Mycobacterium tuberculosis '98-R604 INH-RIF-EM' TBKG_02259 T0EL87

Mycobacterium marinum str. Europe MMEU_4939 S7S303

Mycobacterium marinum MB2 MMMB2 4134 S7QZY6

Mycobacterium orygis 112400015 MORY_17288 M8DBT2

Mycobacterium tuberculosis NCGM2209 alkB NCGM2209 3538 G2UTS8

Mycobacterium bovis BCG str. Moreau RDJ alkB BCGM_3265c F9UZB9

Mycobacterium tuberculosis W-148 TBPG_00365 F2VCH4

Mycobacterium tuberculosis CDC1551A TMMG_02400 E9ZP16

Mycobacterium tuberculosis SUMu012 TMLG_02024 E2WM40

Mycobacterium tuberculosis SUMuOl l TMKG_02511 E2WA16 Species Origin Gene Name Accession No

Mycobacterium tuberculosis SUMuOlO TMJG_03436 E2VYW3

Mycobacterium tuberculosis SUMu009 TMIG_02769 E2VMD7

Mycobacterium tuberculosis SUMu006 TMFG_00461 E2UQS7

Mycobacterium tuberculosis SUMu005 TMEG_03649 E2UEQ4

Mycobacterium tuberculosis SUMu004 TMDG_02087 E2U2V2

Mycobacterium tuberculosis SUMu003 TMCG_01675 E2TRB4

Mycobacterium tuberculosis SUMu002 TMBG_01947 E2TG69

Mycobacterium tuberculosis SUMuOOl TMAG_02705 E1HE07

Mycobacterium africanum K85 TBOG_03815 D6FRF3

Mycobacterium tuberculosis CPHL A TBNG_02887 D6FLF8

Mycobacterium tuberculosis T46 TBLG_03890 D6F9Q1

Mycobacterium tuberculosis T17 TBJG_02010 D5ZLD1

Mycobacterium tuberculosis GM 1503 TBIG_02964 D5Z897

Mycobacterium tuberculosis 02 1987 TBBG_01719 D5YWK4

Mycobacterium tuberculosis EAS054 TBGG_02463 D5YJM0

Mycobacterium tuberculosis T85 TBEG_02389 D5Y8I4

Mycobacterium tuberculosis T92 TBDG_02114 D5XYS2

Mycobacterium tuberculosis C TBCG_03191 A2VP49

Rhodococcus sp. EsD8 EBESD8 35530 N1M6K3

Amycolatopsis orientalis HCCB 10007 AORI_4274 R4SU00

Mycobacterium tuberculosis SUMu008 TMHG_02473 E2VD73

Mycobacterium tuberculosis SUMu007 TMGG_02800 E2V1Z1

Mycobacterium tuberculosis 94 M4241A TBAG_02148 D7EUC2

Gordonia amarae NBRC 15530 alkB GOAMR 02 00080 G7GIN7

Rhodococcus rhodochrous ATCC 21198 RR21198 2302 W4A7D8

Amycolatopsis decaplanina DSM 44594 H074 07696 M2XNH0

Mycobacterium sp. 012931 MMSP_4721 S7R3L1

Rhodococcus erythropolis (strain PR4 / NBRC 100887) alkB RER 07460 C0ZPX6

Rhodococcus sp. (strain Q 15) alkBl Q93DN3

Rhodococcus erythropolis CCM2595 O5Y 03630 T1VI31

Rhodococcus sp. P27 N806 28900 U0EPX3

Rhodococcus erythropolis (Arthrobacter picolinophilus) alkBl Q9XAU0

Rhodococcus qingshengii BKS 20-40 G418 23516 M2V230

Rhodococcus erythropolis SK121 RHOER0001 0742 C3JUT8

Rhodococcus erythropolis DN1 N601_07180 T5HYU5

Nocardia farcinica (strain IFM 10152) NFA 46180 Q5YQS2

Rhodococcus equi NBRC 101255 = C 7 H849 17115 U5DRE7

Shewanella sp. NJ49 alkBl E3VRS8 Species Origin Gene Name Accession No

Mycobacterium canettii CIPT 140070010 alkB BN42 41302 L0QPN9

Nocardia nova SH22a NONO_c63220 W5TPB1

Rhodococcus equi (strain 103 S) (Corynebacterium equi) alkB REQ 33430 E4WK80

Gordonia terrae C-6 GTC6 09699 R7YA99

Nocardioides sp. (strain BAA-499 / JS614) Noca_0122 A1SCY2

Gordonia sp. TF6 alkB2 Q5WA49

Hydrocarboniphaga effusa AP103 WQQ_18760 I7ZII6

Gordonia terrae NBRC 100016 alkB GOTRE 037 00320 H5UBE8

Nocardia brasiliensis ATCC 700358 O3I 035145 K0FBU4

Amycolatopsis mediterranei RB B737_6308 T1V895

Amycolatopsis mediterranei (strain S699) (Nocardia

AMES_6308 RAM 32810 G0FN68 mediterranei)

Amycolatopsis mediterranei (strain U-32) AMED 6400 D8HXC8

Rhodococcus sp. p52 alkB U5S065

Rhodococcus pyridinivorans AK37 AK37_01067 H0JKW2

Rhodococcus pyridinivorans SB3094 Y013_07620 V9XAS5

Janibacter sp. HTCC2649 JNB 17248 A3TPZ2

Gordonia sp. KTR9 KTR9 2914 J9SIP3

Aero microb ium marinum DSM 15272 HMPREF0063 10220 E2S863

Dietzia cinnamea P4 ES5 02159 E6J5E4

Micromonospora aurantiaca (strain ATCC 27029 / DSM

Micau_3940 D9T1D7 43813 / JCM 10878 / NBRC 16125 / INA 9442)

Dietzia sp. El alkB/rub fusion C0LMW6

Rhodococcus ruber BKS 20-38 G352 24171 Μ2ΥΎΒ5

Mycobacterium gilvum (strain PYR-GCK)

(Mycobacterium flavescens (strain ATCC 700033 / PYR- Mflv_3369 A4TAB7 GCK))

Nocardioidaceae bacterium Broad- 1 NBCG_03866 E9UYJ8

Rhodococcus rhodochrous ATCC 21198 RR21198 2485 W4A610

Salinisphaera shabanensis E1L3A SSPSH 001855 U2E637

Rhodococcus erythropolis (strain PR4 / NBRC 100887) alkB RER 54580 C0ZSH4

Corynebacterium falsenii DSM 44353 CFAL 02965 W5WPK1

Rhodococcus erythropolis CCM2595 05Y 25995 T1WR3 gamma proteobacterium BDW918 DOK 04793 I2JMI2

Rhodococcus sp. P27 N806_02390 U0DZR9

Rhodococcus erythropolis DN1 N601 00885 T5IAL6

Rhodococcus erythropolis SK121 RHOER0001 2104 C3JNE0

Rhodococcus qingshengii BKS 20-40 G418 13569 M2WBK9 [0116] In some embodiments, the invention provides synthetic methods as described above, wherein the enzyme is a long-chain alkane hydroxylase. In some embodiments, the long- chain alkane hydroxylase is selected from Table 8 or a variant thereof having at least 90% identity thereto. Table 8. Exemplary long chain alkane hydroxylase enzymes.

Species Origin Gene names Accession No

Burkholdena pseudomallei MSHR1043 D512 19607 M7EBY4

Burkholderia pseudomallei 1655 BURPS1655 10183 B2H6F2

Burkholdena pseudomallei 305 BURPS305 5546 A4LI20

Segniliparus rugosus ATCC BAA-974 HMPREF9336 02889 E5XTR7

Burkholderia pseudomallei 1026b BP1026B II0759 I1WRX2

Burkholderia pseudomallei 354a BP354A 4019 I2MG65

Burkholderia pseudomallei 354e BP354E 3240 I2M2Q7

Burkholderia pseudomallei 1026a BP1026A 2436 I2L127

Burkholderia pseudomallei 1258b BP1258B 3899 I2KY00

Burkholderia pseudomallei 1258a BP1258A 3523 I2KWT0

Pseudomonas putida (strain DOT-TIE) T1E 2746 I7B0Q5

Pseudomonas putida ND6 YSA 09788 I3V2W3

Pseudomonas putida TROl C206 18269 N9VYA0

Pseudomonas putida LS46 PPUTLS46 018911 M7RI48

Burkholderia graminis C4D1M BgramDRAFT 6182 B1G9Y6

Burkholderia phytofirmans (strain DSM 17436 / PsJN) Bphyt_4538 B2TDZ4

Bhargavaea cecembensis DSE10 moxC 3 C772 02411 M7NEH3

Burkholderia thailandensis MSMB121 BTI 5494 N0APC1

Burkholderia pseudomallei (strain 668) BURPS668 A1016 A3NI44

Burkholderia pseudomallei (strain 1710b) BURPS 1710b_A2257 Q3JG95

Burkholderia pseudomallei 1710a BURPS 1710A A0072 C6U1I8

Planomicrobium glaciei CHR43 G159 14295 W3AA87

Burkholderia thailandensis MSMB43 A33K_16732 I6AD68

Pseudomonas sp. GM50 PMI30 04278 J3GFD6

Pseudomonas fluorescens BBc6R8 MHB 001910 V7EA47

Pseudomonas sp. Agl A462 06954 JOYEGl

Pseudomonas sp. GM102 PMI18 00569 J2VSE5

Pseudomonas fluorescens (strain SBW25) PFLU_3858 C3JYC1

Pseudomonas sp. (strain Ml) PM1 0212365 W5IVB1

Pseudomonas sp. TKP U771 20325 V9R055

Pseudomonas putida (strain Fl / ATCC 700007) Pput_3007 A5W4S5

Pseudomonas putida (strain GB-1) PputGBl 1120 B0KS73

Azotobacter vinelandii CA6 seuA AvCA6 43810 M9YDA5

Azotobacter vinelandii CA seuA AvCA 43810 M9Y6B1

Azotobacter vinelandii (strain DJ / ATCC BAA-1303) seuA Avin_43810 C1DGK6

Pseudomonas brassicacearum (strain NFM421) PSEBR_a2282 F2KFH4

Pseudomonas fluorescens Q8rl-96 PflQ8 2313 I4KKG5

Klebsiella oxytoca E718 A225 4709 16X485

Pseudomonas putida (strain KT2440) PP 2746 Q88JA3

Pseudomonas fluorescens BBc6R8 MHB 002244 V7E7E4

Pseudomonas fluorescens Q2-87 PflQ2 2259 J2EFB8

Pseudomonas sp. Agl A462 04671 J0PSS9

Klebsiella oxytoca MGH 42 L388 04093 V3KYZ2

Klebsiella oxytoca 10-5245 HMPREF9689 03721 H3M9T3

Klebsiella oxytoca 10-5243 HMPREF9687 03258 H3LSS6

Klebsiella oxytoca (strain ATCC 8724 / DSM 4798 /

KOX_01240 G8WD25 JCM 20051 / NBRC 3318 / NRRL B-199 / KCTC 1686)

Streptomyces himastatinicus ATCC 53653 SSOG_01846 D9WSJ3

Klebsiella oxytoca MGH 28 L374 04760 V3PRZ9

Klebsiella oxytoca 10-5250 HMPREF9694 02187 H3N1Z4

Klebsiella sp. OBRC7 HMPREF1144 4230 J8VYP0

Klebsiella oxytoca 10-5242 HMPREF9686 03185 H3LCA0

Pantoea ananatis LMG 5342 soxA PANA5342 1855 G9ARF4

Pantoea ananatis PA13 PAGR gl792 G7UD55

Pantoea ananatis (strain AJ13355) soxA PAJ 1557 F2EW92

Species Origin Gene names Accession No

Klebsiella pneumoniae UHKPC02 H229 0083 S7EFH7

Klebsiella pneumoniae UHKPC17 H225 0083 S7E3F9

Klebsiella pneumoniae UHKPC31 H227 0223 S7E0F6

Klebsiella pneumoniae UHKPC59 H223 2084 S7DJY5

Klebsiella pneumoniae UHKPC18 H226 0627 S7CZN2

Klebsiella pneumoniae UHKPC61 H220 0228 S7CKP4

Klebsiella pneumoniae UHKPC07 H224 0554 S7C1T8

Klebsiella pneumoniae DMC1316 H219 1515 S7C0U0

Klebsiella pneumoniae UHKPC33 H222 0227 S7BH54

Klebsiella pneumoniae DMC1097 H218 2245 S7A1J0

Klebsiella pneumoniae UHKPC96 H215 0710 S6YYA8

Klebsiella pneumoniae UHKPC77 H214 0083 S6YU31

Klebsiella pneumoniae UHKPC28 H209 0679 S6YQS7

Klebsiella pneumoniae UHKPC69 H213 0083 S6YBZ0

Klebsiella pneumoniae UHKPC47 H211 0128 S6XBP3

Klebsiella pneumoniae UHKPC32 H242 0078 S2J6Y7

Klebsiella pneumoniae UHKPC48 H221 0076 S2I2J3

Klebsiella pneumoniae DMC0526 H216 2445 S2I0S2

Klebsiella pneumoniae VAKPC278 H247 0907 S2H7F7

Klebsiella pneumoniae UHKPC29 H241 0227 S2GQ63

Klebsiella pneumoniae UHKPC05 H210 0554 S2G118

Klebsiella pneumoniae UHKPC45 H239 0077 S2FVN7

Klebsiella pneumoniae UHKPC 52 H234 0218 S2FQ55

Klebsiella pneumoniae 646 1568 J054 0227 S2E5R5

Klebsiella pneumoniae 540 1460 J053 0083 S2E2M9

Klebsiella pneumoniae 440 1540 J051 2140 S2CWI6

Klebsiella pneumoniae 500 1420 J052 0542 S2CKG8

Klebsiella pneumoniae VAKPC309 H252 1202 S2C6A5

Klebsiella pneumoniae KP-11 H254 0775 S2BTB1

Klebsiella pneumoniae 361 1301 J050 2658 S2B565

Klebsiella pneumoniae VAKPC297 H251 0083 S2ACA5

Klebsiella pneumoniae VAKPC270 H249 0897 S1ZBB5

Klebsiella pneumoniae VAKPC280 H248 0984 S1Z9L1

Klebsiella pneumoniae VAKPC276 H250 1158 S1Z4C6

Klebsiella pneumoniae VAKPC269 H246 1198 S1YJN2

Klebsiella pneumoniae VAKPC254 H245 0083 S1XZP2

Klebsiella pneumoniae UHKPC22 H240 0083 S1XYX9

Klebsiella pneumoniae UHKPC04 H243 0549 S1X5H6

Klebsiella pneumoniae VAKPC252 H244 3523 S1WWW4

Klebsiella pneumoniae UHKPC26 H236 0227 S1W5H8

Klebsiella pneumoniae UHKPC27 H233 0552 S1VUY3

Klebsiella pneumoniae UHKPC24 H235 0228 S1V9Y4

Klebsiella pneumoniae UHKPCOl H231 1154 S1V1B9

Klebsiella pneumoniae UHKPC81 H232 2378 S1TWU9

Klebsiella pneumoniae UHKPC40 H207 0083 S1TR15

Klebsiella pneumoniae UHKPC09 H230 0227 S1TQU1

Klebsiella pneumoniae KP-7 H253 1042 S1T453

Klebsiella pneumoniae UHKPC23 H208 0755 R9BIA6

Klebsiella pneumoniae subsp. pneumoniae KpMDUl C210 21528 N9SXP2

Klebsiella pneumoniae ATCC BAA-1705 KPBAA1705 02256 M7QWX8

Klebsiella pneumoniae ATCC BAA-2146 G000 17665 Kpn2146_4394 M7PZV3

Klebsiella pneumoniae VA360 MTE2 213 M5T2W9

Klebsiella pneumoniae RYC492 KPRYC492 05065 M5Q5H7

Klebsiella pneumoniae subsp. pneumoniae KpQ3 B819 29014 M5GIZ6

Klebsiella pneumoniae subsp. pneumoniae Ecl8 BN373_37921 K4UK89 Species Origin Gene names Accession No

Klebsiella pneumoniae subsp. pneumoniae WGLW5 HMPREF1308 03340 K1NXD5

Klebsiella pneumoniae subsp. pneumoniae WGLW3 HMPREF1307 01233 K1NCK1

Klebsiella pneumoniae subsp. pneumoniae WGLW1 HMPREF1305 01058 K1MMN7

Klebsiella pneumoniae subsp. pneumoniae KPNIH23 KPNIH23 01714 J2W4N5

Klebsiella pneumoniae subsp. pneumoniae KPNIH21 KPNIH21 18909 J2UUP0

Klebsiella pneumoniae subsp. pneumoniae KPNIH18 KPNIHl 8 04648 J2TP42

Klebsiella pneumoniae subsp. pneumoniae KPNIH17 KPNIH17 07852 J2SZ94

Klebsiella pneumoniae subsp. pneumoniae KPNIH9 KPNIH9 07912 J2PY88

Klebsiella pneumoniae subsp. pneumoniae KPNIH6 KPNIH6 12977 J2NIU0

Klebsiella pneumoniae subsp. pneumoniae KPNIH1 KPNIH1 04615 J2MHH3

Klebsiella pneumoniae subsp. pneumoniae KPNIH22 KPNIH22 01396 J2KA06

Klebsiella pneumoniae subsp. pneumoniae KPNIH19 KPNIHl 9 02887 J2JA47

Klebsiella pneumoniae subsp. pneumoniae KPNIH16 KPNIH16 07898 J2HIQ1

Klebsiella pneumoniae subsp. pneumoniae KPNIH14 KPNIH14 01932 J2GTK1

Klebsiella pneumoniae subsp. pneumoniae KPNIHl 1 KPNIHl 1 05794 J2G1J7

Klebsiella pneumoniae subsp. pneumoniae KPNIH2 KPNIH2 14379 J2BUC4

Klebsiella pneumoniae subsp. pneumoniae KPNIH20 KPNIH20 08348 J2BFJ4

Klebsiella pneumoniae subsp. pneumoniae KPNIH12 KPNIH12 01874 J1YXJ0

Klebsiella pneumoniae subsp. pneumoniae KPNIHIO KPNIH10 07382 J1X9E8

Klebsiella pneumoniae subsp. pneumoniae KPNIH8 KPNIH8 09376 J1WTX7

Klebsiella pneumoniae subsp. pneumoniae KPNIH7 KPNIH7 03054 J1WDZ3

Klebsiella pneumoniae subsp. pneumoniae KPNIH5 KPNIH5 11286 J1V7M9

Klebsiella pneumoniae subsp. pneumoniae KPNIH4 KPNIH4 01334 J1UFY7

Klebsiella sp. 4 1 44FAA HMPREF1024 02306 G9REB7

Klebsiella pneumoniae JM45 N559 1083 S5YDY6

Klebsiella pneumoniae subsp. pneumoniae Kpl3 KP13 02362 V9ZFM9

Klebsiella pneumoniae subsp. rhinoscleromatis ATCC

HMPREF0484 1763 C8T2C2 13884

Klebsiella pneumoniae subsp. pneumoniae ST258-

BN426_1797 K4RX40 K26BO

Klebsiella variicola (strain At-22) Kvar 0908 D3RIP8

Klebsiella pneumoniae (strain 342) KPK 0975 B5XUZ5

Klebsiella pneumoniae MGH 20 L366 04030 V3R3V0

Klebsiella pneumoniae UCICRE 10 L421 04096 V3DSZ3

Klebsiella sp. KTE92 AIWC 04002 R8X357

Klebsiella pneumoniae hvKPl G057 03698 M2A8M6

Mycobacterium hassiacum DSM 44199 C731 0966 K5B980

Klebsiella pneumoniae MGH 48 L394 03318 V3J564

Pantoea vagans (strain C9-1) (Pantoea agglomerans

Pvag_pPagl0056 E1PKF9 (strain C9-1))

Klebsiella pneumoniae IS22 W1BJB8

Klebsiella pneumoniae subsp. pneumoniae NTUH-K2044 KP1 4424 C4XCS7

Burkholderia sp. CCGEIOOI BCIOOI 4137 E8YTA8

Microvirga lotononidis MicloDRAFT 00046760 I4YW6

Burkholderia phenoliruptrix BR3459a BUPH 00719 K0DVZ1

Pseudomonas cichorii JBC1 PCH70 03420 W0H3V5

Burkholderia sp. (strain CCGE1003) BC1003 5279 E1TDZ6

Pseudomonas protegens CHAO soxAl PFLCHAO c02440 R4QZ42

Herbaspirillum sp. CF444 PMI16 04881 J2L7C4

Pseudomonas fluorescens (strain Pf-5 / ATCC B AA-477) PFL 0243 Q4KK44

Bacillus megaterium WSH-002 BMWSH 4371 G2RTT4

Pseudomonas sp. GM30 PMI25 001642 W6W1D9

Pseudomonas sp. GM78 PMI35 05139 J3D9L9

Species Origin Gene names Accession No

Pseudomonas aeraginosa VRFPAOl G039 0203575 V4QMQ4

Pseudomonas aeraginosa HB15 PA15 0330520 V4MN40

Pseudomonas aeraginosa M8A.3 Q082 00075 U9SHI5

Pseudomonas aeraginosa CF27 Q003 00104 U9RU06

Pseudomonas aeraginosa MSH10 Q000 02112 U9RT23

Pseudomonas aeraginosa CF127 Q001 02232 U9RQB8

Pseudomonas aeraginosa CF5 Q004 02036 U9R042

Pseudomonas aeraginosa S54485 Q007 00776 U9QQE4

Pseudomonas aeraginosa BWHPSA007 Q020 00157 U9PK67

Pseudomonas aeraginosa BWHPSA009 Q022 02698 U9NGB4

Pseudomonas aeraginosa BWHPSA008 Q021 00149 U9NF67

Pseudomonas aeraginosa BWHPSAOIO Q023 01638 U9MXZ6

Pseudomonas aeraginosa BWHPSA015 Q028 00447 U9MBW2

Pseudomonas aeraginosa BWHPSA016 Q029 01714 U9LQK4

Pseudomonas aeraginosa BL03 Q057 00105 U9LB58

Pseudomonas aeraginosa BL01 Q055 02736 U9KLQ0

Pseudomonas aeraginosa BL02 Q056 06394 U9JUP8

Pseudomonas aeraginosa BL05 Q059 02100 U9JF28

Pseudomonas aeraginosa BL06 Q060 06378 U9IJ92

Pseudomonas aeraginosa BL21 Q075 03038 U9GQQ1

Pseudomonas aeraginosa BL23 Q077 03073 U9FQH5

Pseudomonas aeraginosa BL24 Q078 06288 U9EQY5

Pseudomonas aeraginosa M8A.4 Q083 01720 U9ECA2

Pseudomonas aeraginosa MSH3 P999 02290 U9D2B6

Pseudomonas aeraginosa X24509 Q005 02076 U9CCX5

Pseudomonas aeraginosa UDL Q006 01725 U9C927

Pseudomonas aeraginosa CF18 Q002 02068 U9BVH8

Pseudomonas aeraginosa 19660 Q010 02159 U9AF43

Pseudomonas aeraginosa X13273 Q013 02044 U8Z334

Pseudomonas aeraginosa S35004 Q012 06204 U8YF61

Pseudomonas aeraginosa BWHPSA001 Q014 02765 U8YAB2

Pseudomonas aeraginosa BWHPSA003 Q016 02194 U8XR83

Pseudomonas aeraginosa BWHPSA002 Q015 02292 U8XP62

Pseudomonas aeraginosa BWHPSA004 Q017 02030 U8X7A0

Pseudomonas aeraginosa BWHPSA005 Q018 03069 U8W6E8

Pseudomonas aeraginosa BWHPSA011 Q024 01957 U8VA48

Pseudomonas aeraginosa BWHPSA013 Q026 03028 U8URW4

Pseudomonas aeraginosa BWHPSA012 Q025 02769 U8UQP2

Pseudomonas aeraginosa BWHPSA014 Q027 01719 U8TK96

Pseudomonas aeraginosa BWHPSA017 Q030 05589 U8SKH8

Pseudomonas aeraginosa BWHPSA020 Q033 02593 U8S609

Pseudomonas aeraginosa BWHPSA019 Q032 03133 U8RPR9

Pseudomonas aeraginosa BWHPSA022 Q035 01895 U8R8U4

Pseudomonas aeraginosa BWHPSA023 Q036 00320 U8R6B4

Pseudomonas aeraginosa BWHPSA021 Q034 02035 U8R1N4

Pseudomonas aeraginosa BWHPSA025 Q038 01757 U8PR31

Pseudomonas aeraginosa BWHPSA024 Q037 02761 U8PP93

Pseudomonas aeraginosa BWHPSA027 Q040 02049 U8N8N1

Pseudomonas aeraginosa BL07 Q061 01439 U8LYS6

Pseudomonas aeraginosa BL04 Q058 06192 U8LL05

Pseudomonas aeraginosa BL11 Q065 03099 U8K8S5

Pseudomonas aeraginosa BL10 Q064 02801 U8JQ84

Pseudomonas aeraginosa BL15 Q069 01997 U8IMR3 Species Origin Gene names Accession No

Pseudomonas aeruginosa BL16 Q070 01957 U8IID0

Pseudomonas aeruginosa BL18 Q072 02105 U8H8J8

Pseudomonas aeruginosa M8A.2 Q081 01961 U8FTG3

Pseudomonas aeruginosa M8A.1 Q080 04721 U8FHJ8

Pseudomonas aeruginosa M9A.1 Q084 05530 U8EPH5

Pseudomonas aeruginosa C20 Q085 03119 U8EML6

Pseudomonas aeruginosa C23 Q086 03122 U8EJ68

Pseudomonas aeruginosa C40 Q087 02201 U8DKJ1

Pseudomonas aeruginosa C48 Q089 02700 U8CPW7

Pseudomonas aeruginosa C51 Q090 05806 U8BVH7

Pseudomonas aeruginosa CF77 Q092 01904 U8BA80

Pseudomonas aeruginosa C52 Q091 05688 U8AZD2

Pseudomonas aeruginosa CF614 Q093 06204 U8ACM4

Pseudomonas aeruginosa VRFPA04 P797 30195 U5AHY5

Pseudomonas aeruginosa HB13 PA13 1029315 U1E3A4

Pseudomonas aeruginosa MSH-10 L346 02111 S0IJJ1

Pseudomonas aeruginosa PA14 CIA 02266 S0I9C6

Pseudomonas aeruginosa PAK PAK 02986 S0I695

Pseudomonas sp. P179 HMPREF1224 05539 N2DDM6

Pseudomonas aeruginosa str. Stone 130 HMPREF1223 07114 N2D7D2

Pseudomonas aeruginosa PA21 ST175 H123 24636 M3AW72

Pseudomonas aeruginosa E2 P998 02032 PAE2 2544 K1DHT6

Pseudomonas aeruginosa ATCC 25324 PABE173 3188 K1DD82

Pseudomonas aeruginosa CI27 PACI27 2786 K1CTB3

Pseudomonas aeruginosa ATCC 700888 PABE177 2660 K1CGR7

Pseudomonas aeruginosa ATCC 14886 PABE171 3115 K1BXJ5

Pseudomonas aeruginosa PADK2 CF510 CF510 22344 I1ACS3

Pseudomonas aeruginosa MPA01 P2 OlQ_15090 H3TFC3

Pseudomonas aeruginosa MPA01 P1 010_28545 H3T6G4

Pseudomonas sp. 2 1 26 HMPREF1030 05556 G5G1F3

Pseudomonas aeruginosa 2192 PA2G 01431 A3LB74

Pseudomonas aeruginosa C3719 PACG 01235 A3KU95

Erwinia billingiae (strain Eb661) EbC 20720 D8MRZ6

Xanthomonas axonopodis pv. citri (strain 306) XAC0855 Q8PP33

Xanthomonas citri subsp. citri Awl2879 XCAW_03724 M4W2T5

Xanthomonas axonopodis Xac29-1 XAC29 04355 M4U7K3

Xanthomonas citri pv. mangiferaeindicae LMG 941 ladA ΧΜΓΝ 2789 H8FHG1

Xanthomonas axonopodis pv. punicae str. LMG 859 ladA XAPC_728 H1XCV7

Leifsonia aquatica ATCC 14665 N136 01626 U2TBF7

Serratia marcescens subsp. marcescens Dbl l SMDB11 2421 V6A0D9

Pseudomonas aeruginosa VRFPA05 T266 33830 V4WJP9

Pseudomonas aeruginosa BL22 Q076 01761 U9GCW5

Xanthomonas axonopodis pv. malvacearam str.

MOU_00060 K8GBN4 GSPB1386

Pseudomonas aeruginosa VRFPA07 X778 28580 V8E3G0

Pseudomonas aeruginosa BL20 Q074 02826 U9HSV9

Pseudomonas aeruginosa BL25 Q079 01143 U9F0W8

Pseudomonas aeruginosa BL09 Q063 00187 U8L2Y0

Serratia marcescens WW4 SMWW4 vlc31920 L7ZQQ5

Serratia marcescens VGH107 F518 24469 M3BTM0

Pseudomonas aeruginosa BWHPSA018 Q031 00379 U8TSK3

Pseudomonas aeruginosa M18 PAM18 2715 G2L1H6

Pseudomonas aeruginosa BL12 Q066 03852 U9I855

Pseudomonas aeruginosa BWHPSA028 Q041 02218 U8NES6 Species Origin Gene names Accession No

Pseudomonas aeruginosa WC55 L683 26830 T5KSU5

Pseudomonas aeruginosa NCMG1179 NCGM1179 2739 G2U5R3

Rhodococcus erythropolis SK121 RHOER0001 2299 C3JDL9

Pseudomonas aeruginosa VRFPA03 M770 16185 W1MK34

Pseudomonas aeruginosa BL13 Q067 03184 U9I925

Serratia marcescens EGD-HP20 N040 11055 U1TLQ0

Pseudomonas aeruginosa NCGM2.S1 NCGM2 3338 G4LI50

Pseudomonas aeruginosa 39016 PA39016 002700003 E3A2U8

Pseudomonas aeruginosa MH27 PAMH27 2887 V6AFD9

Pseudomonas aeruginosa JJ692 Q008 02805 U9PMT7

Pseudomonas aeruginosa 6077 Q011 02150 U9ATK4

Pseudomonas aeruginosa U2504 Q009 02593 U9AAM5

Pseudomonas aeruginosa BWHPSA006 Q019 02936 U8VL16

Pseudomonas aeruginosa BL08 Q062 04340 U8KSZ8

Pseudomonas aeruginosa BL14 Q068 02182 U8JUF2

Pseudomonas aeruginosa BL17 Q071 02971 U8H8J5

Pseudomonas aeruginosa PA45 H734 07342 N4W202

Rhodococcus erythropolis CCM2595 05Y 21155 T1VSG7

Rhodococcus sp. P27 N806 09240 U0ED84

Kosakonia radicincitans DSM 16656 Y71 0158 J1QW00

Rhodococcus erythropolis (strain PR4 / NBRC 100887) RER 45000 C0ZMF0

Klebsiella pneumoniae MGH 46 L392 03264 V3LZ98

Klebsiella pneumoniae MGH 44 L390 02205 V3JUR2

Klebsiella pneumoniae UCICRE 4 L415 03363 V3FXF6

Klebsiella pneumoniae 303K N598 24365 U6T101

Klebsiella pneumoniae UHKPC179 H238 2267 S7F9A7

Klebsiella pneumoniae UHKPC57 H237 2247 S2EDB5

Klebsiella pneumoniae JHCK1 MTE1 213 M3U9Q5

Klebsiella pneumoniae subsp. pneumoniae WGLW2 HMPREF1306 03733 K1NBI6

Klebsiella pneumoniae UCICRE 14 L425 03054 V3CJD9

Rhodococcus qingshengii BKS 20-40 G418 04858 M2XMT9

Pantoea sp. Scl S7A 19914 H8DUB8

Klebsiella sp. 1 1 55 HMPREF0485 02899 D6GIG4

Pantoea agglomerans TxlO L584 13665 U4VW62

Escherichia coli 909957 HMPREF1619 02817 V0B421

Klebsiella pneumoniae KP-1 KLP1 1662 U2ABR1

Rhodococcus erythropolis DN1 N601 05680 T5I9L8

Klebsiella pneumoniae UCICRE 8 L419 03300 V3F3T1

Brenneria sp. EniD312 BrE312 1717 G7LVX2

Klebsiella pneumoniae BIDMC 23 L459 03205 V3BAE8

Raoultella ornithinolytica B6 RORB6 23555 M9W8P0

Klebsiella oxytoca 10-5246 HMPREF9690 03902 H3MRJ7

Pantoea agglomerans 299R F385 1445 L7BV82

Pantoea sp. aB PanABDRAFT 3926 E0M3F8

Pseudomonas sp. CFII64 CFII64 23274 S6GXI3

Pseudomonas synxantha BG33R PseBG33 0275 I4KV50

Pseudomonas syringae pv. actinidiae ICMP 18801 A221_07756 S6XYV3

Pseudomonas syringae pv. actinidiae ICMP 19072 A3SO_07400 S6PNP2

Pseudomonas syringae pv. actinidiae ICMP 19073 A262_20054 S6MLA8

Pseudomonas syringae pv. actinidiae ICMP 19071 A264_07551 S6M2E1

Pseudomonas syringae pv. actinidiae ICMP 19104 A258_19792 S6QSB5

Pseudomonas syringae pv. actinidiae ICMP 9855 A252 19596 S6QRN6

Pseudomonas syringae pv. actinidiae ICMP 19102 A253_19857 S6Q6B9

Pseudomonas syringae pv. actinidiae ICMP 19068 A260 20086 S6Q126

Pseudomonas syringae pv. theae ICMP 3923 A584 21008 S6MKD2 Species Origin Gene names Accession No

Pseudomonas syringae pv. actinidiae ICMP 19103 A256 19800 S6M4P1

Rhizobium leguminosaram bv. viciae (strain 3841) pRL90300 Q1M8E2

Pseudomonas sp. GM25 PMI24 01694 J2PHH1

Herbaspirillum sp. YR522 PMI40 00700 J3HY53

Pseudomonas syringae pv. morsprunorum str. M302280 PSYMP 05599 F3DS65

Pseudomonas fluorescens (strain PfO-1) PH01 0238 Q3KJS4

Pseudomonas avellanae BPIC 631 Pav631 4731 K2RRZ8

Pseudomonas fluorescens R124 I1A 000262 K0W8U4

Pseudomonas syringae pv. syringae (strain B728a) Psyr_2869 Q4ZSG7

Pseudomonas syringae CC1557 N018 12850 W0MW63

Pseudomonas sp. GM80 PMI37 03766 J3DKC5

Pseudomonas syringae pv. syringae SM PssSM_2902 S3MKC4

Pseudomonas syringae pv. avellanae str. ISPaVe037 Pav037_2494 K2T3F9

Pseudomonas syringae pv. aceris str. M302273 PSYAR 06142 F3JE47

Pseudomonas syringae pv. maculicola str. ES4326 PMA4326 07981 F3HHE2

Pseudomonas syringae BRIP39023 A988 19986 L7GSY0

Pseudomonas syringae pv. aptata str. DSM 50252 PSYAP 18083 F3J2D2

Pseudomonas savastanoi pv. savastanoi NCPPB 3335 PSA3335_0550 D7HUP0

Pseudomonas syringae pv. aesculi str. 0893 23 PSYAE 00125 F3D7S6

Pseudomonas syringae BRIP34881 A987 17762 L7G2P2

Pseudomonas syringae BRIP34876 A979 21556 L7FTL3

Rhizobium leguminosarum bv. viciae WSM1455 Rleg5DRAFT_0033 J0URT9

Pseudomonas syringae Cit 7 PSYCIT7 07619 F3GWQ5

Acinetobacter baumannii NIPH 410 F910 02332 S3TEC4

Acinetobacter baumannii OIFC110 ACIN5110 2029 K5S1X4

Acinetobacter baumannii WC-692 ACINWC692 1619 K1ER91

Pseudomonas sp. T P U771 01460 V9QPN2

Pseudomonas syringae pv. syringae B64 PssB64_3039 L8NFP3

Pseudomonas syringae pv. actinidiae ICMP 19094 A241 11585 S6VCM5

Pseudomonas syringae pv. actinidiae ICMP 18883 A243 23241 S6TZP7

Pseudomonas syringae pv. actinidiae ICMP 19095 A242 23680 S6TDL4

Pseudomonas syringae pv. actinidiae ICMP 19099 A247 15969 S6S3V9

Pseudomonas syringae pv. actinidiae ICMP 19100 A248_23237 S6R962

Pseudomonas syringae pv. actinidiae ICMP 19098 A246 16023 S6LVQ8

[0117] In some embodiments, the invention provides synthetic methods as described above, wherein the enzyme is a cytochrome P450. In some embodiments, the cytochrome P450 is selected from Table 9 or a variant thereof having at least 90% identity thereto. In some embodiments, the cytochrome P450 is a member of the CYP52 or CYP153 family.

Table 9. Exemplary cytochrome P450 enzymes.

Species Origin Gene names Accession No

(Yeast) orfl9.13150

Candida albicans (strain SC5314 / ATCC MYA-2876) ALK1 Ca019.5728

Q5A8U5 (Yeast) orfl9.5728

Candida maltosa (strain Xu316) (Yeast) G210 4862 M3HRI7

Candida maltosa (Yeast) CYP52A3-A P16496

Candida orthopsilosis (strain 90-125) (Yeast) CORT 0F01930 H8X8E5

Candida parapsilosis (strain CDC 317 / ATCC MYA-

CPAR2 600870 G8B4X9 4646) (Y east) (Monilia parapsilosis)

Lodderomyces elongisporus (strain ATCC 11503 / CBS

2605 / JCM 1781 / NBRC 1676 / NRRL YB-4239) LELG_04957 A5E5R8

(Yeast) (Saccharomyces elongisporus)

Candida maltosa (Yeast) ALK3-B (CYP52A4) B0VX53

Candida maltosa (Yeast) ALK8-B Q12584

Candida tropicalis (Yeast) CYP52A8 P30610

Debaryomyces hansenii (strain ATCC 36239 / CBS 767 /

JCM 1990 / NBRC 0083 / IGC 2968) (Yeast) (Torulaspora DEHA2E18634g Q6BNV8 hansenii)

Candida tropicalis (Yeast) CYP52A17 Q874I9

Candida maltosa (strain Xu316) (Yeast) G210 3820 M3II00

Spathaspora passalidarum (strain NRRL Y-27907 / 11-Yl) SPAPADRAFT 59378 G3AJR6

Scheffersomyces stipitis (strain ATCC 58785 / CBS 6054

CP52M PICST 58031 A3LRT5 / NBRC 10063 / NRRL Y-11545) (Yeast) (Pichia stipitis)

Candida parapsilosis (strain CDC 317 / ATCC MYA-

CPAR2 503950 G8BH23 4646) (Y east) (Monilia parapsilosis)

Candida parapsilosis (strain CDC 317 / ATCC MYA-

CPAR2 800510 G8BBI4 4646) (Y east) (Monilia parapsilosis)

Candida tropicalis (Yeast) CYP52A18 Q874I8

Candida maltosa (strain Xu316) (Yeast) G210 4812 M3K5V3

Debaryomyces hansenii (Yeast) (Torulaspora hansenii) CYP52A13 ALK2 Q9Y758

Meyerozyma guilliermondii (strain ATCC 6260 / CBS 566

/ DSM 6381 / JCM 1539 / NBRC 10279 / NRRL Y-324) PGUG_05855 A5DRF4

(Yeast) (Candida guilliermondii)

Debaryomyces hansenii (strain ATCC 36239 / CBS 767 /

JCM 1990 / NBRC 0083 / IGC 2968) (Yeast) (Torulaspora DEHA2C02596g Q6BVH7 hansenii)

Candida maltosa (Yeast) CYP52A5 Q12581

Meyerozyma guilliermondii (strain ATCC 6260 / CBS 566

/ DSM 6381 / JCM 1539 / NBRC 10279 / NRRL Y-324) PGUG_01238 A5DD87

(Yeast) (Candida guilliermondii)

Debaryomyces hansenii (Yeast) (Torulaspora hansenii) CYP52A12 ALK1 Q9Y757

Candida dubliniensis (strain CD36 / ATCC MYA-646 /

CYP52A14 CD36 25250 B9WKL6 CBS 7987 / NCPF 3949 / NRRL Y-17841) (Yeast)

Meyerozyma guilliermondii (strain ATCC 6260 / CBS 566

/ DSM 6381 / JCM 1539 / NBRC 10279 / NRRL Y-324) PGUG_05670 A5DQW9

(Yeast) (Candida guilliermondii)

Candida albicans (strain SC5314 / ATCC MYA-2876) ALK2 Ca019.7513

Q5AAH6 (Yeast) orfl9.7513

Candida albicans (strain WO-1) (Yeast) CAWG 01382 C4YNC3

Candida tropicalis (Yeast) CYP52A14 CYP14 Q874J3

Candida tropicalis (Yeast) CYP52A13 Q874J4

Pichia sorbitophila (strain ATCC MYA-4447 / BCRC

PisoO 002820 GNLVRSOl

22081 / CBS 7064 / NBRC 10061 / NRRL Y-12695) G8YG24

PISO0I18532g

(Hybrid yeast)

Candida parapsilosis (strain CDC 317 / ATCC MYA-

CPAR2 204220 G8BFZ5 4646) (Y east) (Monilia parapsilosis)

Candida tropicalis (Yeast) CYP52A20 Q874I6

Candida tropicalis (Yeast) CYP52A19 Q874I7 Species Origin Gene names Accession No

Lodderomyces elongisporus (strain ATCC 11503 / CBS

2605 / JCM 1781 / NBRC 1676 / NRRL YB-4239) LELG_00044 A5DRQ8

(Yeast) (Saccharomyces elongisporus)

Candida albicans (strain WO-1) (Yeast) CAWG 02011 C4YMD2

Candida albicans (strain SC5314 / ATCC MYA-2876) ALK8 CaO19.10

Q59K96

(Yeast) Ca019.7683

Candida albicans (Yeast) alk8 074626

Candida maltosa (strain Xu316) (Yeast) G210 4811 M3JDC1

Scheffersomyces stipitis (strain ATCC 58785 / CBS 6054

CP52C PICST 56580 A3LR60 / NBRC 10063 / NRRL Y-11545) (Yeast) (Pichia stipitis)

Lodderomyces elongisporus (strain ATCC 11503 / CBS

2605 / JCM 1781 / NBRC 1676 / NRRL YB-4239) LELG_03506 A5E1L9

(Yeast) (Saccharomyces elongisporus)

Candida tropicalis (strain ATCC MYA-3404 / Tl) (Yeast) CTRG 03115 C5MAM3

Pichia sorbitophila (strain ATCC MYA-4447 / BCRC

PisoO 002820 GNLVRSOl

22081 / CBS 7064 / NBRC 10061 / NRRL Y-12695) G8YDL5

PISO0J20293g

(Hybrid yeast)

Candida parapsilosis (strain CDC 317 / ATCC MYA-

CPAR2 204210 G8BFZ4 4646) (Y east) (Monilia parapsilosis)

Spathaspora passalidarum (strain NRRL Y-27907 / 11-Yl) SPAPADRAFT 134963 G3AJD3

Candida tropicalis (strain ATCC MYA-3404 / Tl) (Yeast) CTRG 01061 C5M4S1

Candida tropicalis (Yeast) CYP52A2 P30607

Candida parapsilosis (strain CDC 317 / ATCC MYA-

CPAR2 800520 G8BBI5 4646) (Y east) (Monilia parapsilosis)

Scheffersomyces stipitis (strain ATCC 58785 / CBS 6054

CP52L PICST 56638 A3LSP0 / NBRC 10063 / NRRL Y-11545) (Yeast) (Pichia stipitis)

Candida parapsilosis (strain CDC 317 / ATCC MYA-

CPAR2 203780 G8BFV1 4646) (Y east) (Monilia parapsilosis)

Candida maltosa (strain Xu316) (Yeast) G210 4902 M3IU34

Candida orthopsilosis (strain 90-125) (Yeast) CORT 0D03890 H8X5Y1

Candida dubliniensis (strain CD36 / ATCC MYA-646 /

CD36 32710 B9WMB3 CBS 7987 / NCPF 3949 / NRRL Y-17841) (Yeast)

Pichia sorbitophila (strain ATCC MYA-4447 / BCRC

22081 / CBS 7064 / NBRC 10061 / NRRL Y-12695) G8YJP0

(Hybrid yeast)

Debaryomyces hansenii (strain ATCC 36239 / CBS 767 /

JCM 1990 / NBRC 0083 / IGC 2968) (Yeast) (Torulaspora DEHA2E18590g Q6BNW0 hansenii)

Candida maltosa (Yeast) CYP52A9 Q12586

Scheffersomyces stipitis (strain ATCC 58785 / CBS 6054

ALK2 PICST 35590 A3LS01 / NBRC 10063 / NRRL Y-11545) (Yeast) (Pichia stipitis)

Spathaspora passalidarum (strain NRRL Y-27907 / 11-Yl) SPAPADRAFT 67265 G3APG2

Candida tropicalis (strain ATCC MYA-3404 / Tl) (Yeast) CTRG 03120 C5MAM8

Candida maltosa (Yeast) CYP52A11 Q12589

Candida albicans (strain WO-1) (Yeast) CAWG 01383 C4YNC4

Candida tropicalis (strain ATCC MYA-3404 / Tl) (Yeast) CTRG 01060 C5M4S0

Candida albicans (strain SC5314 / ATCC MYA-2876) ALK3 Ca019.7512

Q5AAH7 (Yeast) orfl9.7512

Candida tropicalis (Yeast) CYP52A1 P10615

Scheffersomyces stipitis (strain ATCC 58785 / CBS 6054

CYP52 PICST 37142 A3LZV9 / NBRC 10063 / NRRL Y-11545) (Yeast) (Pichia stipitis)

Debaryomyces hansenii (strain ATCC 36239 / CBS 767 /

JCM 1990 / NBRC 0083 / IGC 2968) (Yeast) (Torulaspora DEHA2E18612g Q6BNV9 hansenii)

Candida tenuis (strain ATCC 10573 / BCRC 21748 / CBS

615 / JCM 9827 / NBRC 10315 / NRRL Y-1498 / VKM CANTEDRAFT l 15474 G3BA51

Y-70) (Yeast) Species Origin Gene names Accession No

Lodderomyces elongisporus (strain ATCC 11503 / CBS

2605 / JCM 1781 / NBRC 1676 / NRRL YB-4239) LELG_03309 A5E122

(Yeast) (Saccharomyces elongisporus)

Lodderomyces elongisporus (strain ATCC 11503 / CBS

2605 / JCM 1781 / NBRC 1676 / NRRL YB-4239) LELG_03505 A5E1L8

(Yeast) (Saccharomyces elongisporus)

Candida tropicalis (Yeast) CYP52A16 CYP16 Q874J1

Candida tropicalis (Yeast) CYP52A15 Q874J2

Candida maltosa (Yeast) CYP52A10 Q12588

Candida dubliniensis (strain CD36 / ATCC MYA-646 /

ALK3-A CD36 25260 B9WKL7 CBS 7987 / NCPF 3949 / NRRL Y-17841) (Yeast)

Candida maltosa (Yeast) CYP52A4 P16141

Candida tenuis (strain ATCC 10573 / BCRC 21748 / CBS

615 / JCM 9827 / NBRC 10315 / NRRL Y-1498 / VKM CANTEDRAFT l 13909 G3B3X3

Y-70) (Yeast)

Meyerozyma guilliermondii (Yeast) (Candida

CYP52 I6UGD5 guilliermondii)

Spathaspora passalidamm (strain NRRL Y-27907 / 11-Yl) SPAPADRAFT 153278 G3AMY8

Candida tenuis (strain ATCC 10573 / BCRC 21748 / CBS

615 / JCM 9827 / NBRC 10315 / NRRL Y-1498 / VKM CANTEDRAFT l 16673 G3BEU9

Y-70) (Yeast)

Candida maltosa (strain Xu316) (Yeast) G210 3821 M3J257

Candida tropicalis (Yeast) CYP52A7 P30609

Clavispora lusitaniae (strain ATCC 42720) (Yeast)

CLUG_03984 C4Y750 (Candida lusitaniae)

Debaryomyces hansenii (strain ATCC 36239 / CBS 767 /

JCM 1990 / NBRC 0083 / IGC 2968) (Yeast) (Torulaspora DEHA2C01100g Q6BVP2 hansenii)

Candida tropicalis (Yeast) CYP52D2 Q874J0

Clavispora lusitaniae (strain ATCC 42720) (Yeast)

CLUG_04851 C4Y9G1 (Candida lusitaniae)

Meyerozyma guilliermondii (strain ATCC 6260 / CBS 566

/ DSM 6381 / JCM 1539 / NBRC 10279 / NRRL Y-324) PGUG_04005 A5DL54

(Yeast) (Candida guilliermondii)

Yarrowia lipolytica (Candida lipolytica) ALK6 074132

Yarrowia lipolytica (strain CLIB 122 / E 150) (Yeast)

YALI0_B01848g F2Z623 (Candida lipolytica)

Yarrowia lipolytica (strain CLIB 122 / E 150) (Yeast)

YALI0_E25982g Q6C4K6 (Candida lipolytica)

Yarrowia lipolytica (Candida lipolytica) ALK1 074127

Yarrowia lipolytica (Candida lipolytica) ALK2 074128

Yarrowia lipolytica (strain CLIB 122 / E 150) (Yeast)

YALI0_F01320g F2Z6J3 (Candida lipolytica)

Candida maltosa (Yeast) CYP52D1 Q12585

Yarrowia lipolytica (strain CLIB 122 / E 150) (Yeast)

YALI0_B20702g Q6CDW4 (Candida lipolytica)

Byssochlamys spectabilis (strain No. 5 / NBRC 109023)

PVAR5 4403 V5G4E7 (Paecilomyces variotii)

Byssochlamys spectabilis (strain No. 5 / NBRC 109023)

PVAR5 4403 V5G4E7 (Paecilomyces variotii)

Aspergillus terreus (strain NIH 2624 / FGSC Al 156) ATEG 02198 Q0CVT6

Neosartorya fischeri (strain ATCC 1020 / DSM 3700 /

NFIA 029600 A1D9P7 FGSC Al 164 / NRRL 181) (Aspergillus fischerianus)

Yarrowia lipolytica (Candida lipolytica) ALK4 074130

Yarrowia lipolytica (strain CLIB 122 / E 150) (Yeast)

YALI0_B13816g F2Z6H3 (Candida lipolytica)

Penicillium digitatum (strain PHI26 / CECT 20796)

PDIG_58170 K9G9Y0 (Green mold)

muscar ne sease ungus trac um s otae

Species Origin Gene names Accession No

Cochliobolus heterostrophus (strain C5 / ATCC 48332 /

race 0) (Southern corn leaf blight fungus) (Bipolaris COCHEDRAFT 1208754 M2VA93 maydis)

Aspergillus clavatus (strain ATCC 1007 / CBS 513.65 /

ACLA 032820 A1CSC5 DSM 816 / NCTC 3887 / NRRL 1)

Hypocrea jecorina (strain QM6a) (Trichoderma reesei) TRIREDRAFT 103147 G0R9K0

Trichophyton tonsurans (strain CBS 112818) (Scalp

TESG_02758 F2RVB9 ringworm fungus)

Glarea lozoyensis (strain ATCC 20868 / MF5171) GLAREA 12102 S3D2G7

Trichophyton rubrum (strain ATCC MYA-4607 / CBS

TERG_03231 F2SJM4 118892) (Athlete's foot fungus)

Leptosphaeria maculans (strain JN3 / isolate v23.1.3 / race

LEMA_P073070.1 E5A7X3 Avl-4-5-6-7-8) (Blackleg fungus) (Phoma lingam)

Cyphellophora europaea CBS 101466 HMPREF1541 04444 W2RWL1

Hypocrea jecorina (strain QM6a) (Trichoderma reesei) TRIREDRAFT 65036 G0RNX6

Beauveria bassiana (strain ARSEF 2860) (White

BBA 09022 J5J6F5 muscardine disease fungus) (Tritirachium shiotae)

Cordyceps militaris (strain CMOl) (Cate illar fungus) CCM 02084 G3JCK3

Trichophyton rubrum (strain ATCC MYA-4607 / CBS

TERG_05441 F2SSI7 118892) (Athlete's foot fungus)

Botryotinia fuckeliana (strain BcDWl) (Noble rot fungus)

BcDWl_9224 M7U6H3 (Botrytis cinerea)

Magnaporthe oryzae (strain P131) (Rice blast fungus)

OOW_P13 Iscaffold01201g5 L7J0M9 (Pyricularia oryzae)

Magnaporthe oryzae (strain Y34) (Rice blast fungus)

OOU_Y34scaffold00145gl3 L7IJZ9 (Pyricularia oryzae)

Magnaporthe oryzae (strain 70-15 / ATCC MYA-4617 /

MGG_09920 G4MR75 FGSC 8958) (Rice blast fungus) (Pyricularia oryzae)

Paracoccidioides lutzii (strain ATCC MYA-826 / PbOl)

PAAG_01378 C1GS83 (Paracoccidioides brasiliensis)

Bipolaris zeicola 26-R-13 COCCADRAFT 9928 W6Y8S8

Verticillium dahliae (strain VdLs.17 / ATCC MYA-4575 /

VDAG_04483 G2X2F9 FGSC 10137) (Verticillium wilt)

Trichophyton verrucosum (strain HKI 0517) TRV 02251 D4D581

Arthroderma benhamiae (strain ATCC MYA-4681 / CBS

ARB 01131 D4AY62 112371) (Trichophyton mentagrophytes)

Chaetomium globosum (strain ATCC 6205 / CBS 148.51 /

CHGG_01610 Q2HDU4 DSM 1962 / NBRC 6347 / NRRL 1970) (Soil fungus)

Magnaporthe poae (strain ATCC 64411 / 73-15)

M4G6C3 (Kentucky bluegrass fungus)

Hypocrea atroviridis (strain ATCC 20476 / IMI 206040)

TRIATDRAFT 45536 G9NQR1 (Trichoderma atroviride)

Colletotrichum orbiculare (strain 104-T / ATCC 96160 /

CBS 514.97 / LARS 414 / MAFF 240422) (Cucumber Cob_03064 N4W651 anthracnose fungus) (Colletotrichum lagenarium)

Penicillium chrysogenum (strain ATCC 28089 / DSM Pc20gl l290

B6HG66 1075 / Wisconsin 54-1255) (Penicillium notatum) PCH Pc20gl l290

Ophiocordyceps sinensis (strain Col8 / CGMCC 3.14243)

OCS_02874 T5AG58 (Yarsagumba caterpillar fungus) (Hirsutella sinensis)

Pyrenophora teres f. teres (strain 0-1) (Barley net blotch

PTT 07245 E3RH76 fungus) (Drechslera teres f. teres)

Baudoinia compniacensis (strain UAMH 10762) (Angels'

B AUCODRAFT 71913 M2MX22 share fungus)

Podospora anserina (strain S / ATCC MYA-4624 / DSM

PODANS_0_160 B2AFV1 980 / FGSC 10383) (Pleurage anserina)

Aspergillus terreus (strain NIH 2624 / FGSC Al 156) ATEG 05807 Q0CKH7

Hypocrea jecorina (strain QM6a) (Trichoderma reesei) TRIREDRAFT 75713 G0RDE9

Claviceps purpurea (strain 20.1) (Ergot fungus) (Sphacelia CPUR 06997 M1WHP2

Species Origin Gene names Accession No

980 / FGSC 10383) (Pleurage anserina)

Sporothrix schenckii (strain ATCC 58251 / de Perez

HMPREF1624 01101 U7Q4H5 2211183) (Rose-picker's disease fungus)

Exophiala dermatitidis (strain ATCC 34100 / CBS 525.76

HMPREF1120 04188 H6BWM7 / NIH/UT8656) (Black yeast) (Wangiella dermatitidis)

Colletotrichum gloeosporioides (strain Cg-14)

CGLO_16096 T0JPF3 (Anthracnose fungus) (Glomerella cingulata)

Arthroderma benhamiae (strain ATCC MYA-4681 / CBS

ARB 05099 D4ALA2 112371) (Trichophyton mentagrophytes)

Macrophomina phaseolina (strain MS6) (Charcoal rot

MPH 10488 K2QR42 fungus)

Trichophyton tonsurans (strain CBS 112818) (Scalp

TESG_05856 F2S4I4 ringworm fungus)

Trichophyton equinum (strain ATCC MYA-4606 / CBS

TEQG_04559 F2PUI2 127.97) (Horse ringworm fungus)

Arthroderma benhamiae (strain ATCC MYA-4681 / CBS

ARB 07892 D4AUH5 112371) (Trichophyton mentagrophytes)

Arthroderma otae (strain ATCC MYA-4605 / CBS

MCYG_08648 C5G126 113480) (Microsporum cards)

Aspergillus flavus (strain ATCC 200026 / FGSC Al 120 /

AFLA l 16530 B8NVG6 NRRL 3357 / JCM 12722 / SRRC 167)

Mycosphaerella graminicola (strain CBS 115943 / CYP-

F9XPH9 IP0323) (Speckled leaf blotch fungus) (Septoria tritici) 28MYCGRDRAFT 111399

Penicillium chrysogenum (strain ATCC 28089 / DSM Pcl8g04990

B6HBW9

1075 / Wisconsin 54-1255) (Penicillium notatum) PCH Pcl8g04990

Alternaria solani alt2 Q5KTN2

Colletotrichum higginsianum (strain IMI 349063)

CH063 05380 H1UYS7 (Crucifer anthracnose fungus)

Thielavia heterothallica (strain ATCC 42464 / BCRC

MYCTH 2060315 G2QDC4 31852 / DSM 1799) (Myceliophthora thermophila)

Togninia minima (strain UCR-PA7) (Esca disease fungus)

UCRPA7 1480 R8BUP2 (Phaeoacremonium aleophilum)

Ophiostoma piceae (strain UAMH 11346) (Sap stain

F503 00556 S3C2T4 fungus)

Cladophialophora carrionii CBS 160.54 G647 02236 V9DGM2

Botryotinia fuckeliana (strain BcDWl) (Noble rot fungus)

BcDWl_141 M7UBZ7 (Botrytis cinerea)

Mycobacterium sp. HXN-1500 cypl53 Q65A64

Gordonia amicalis NBRC 100051 = JCM 11271 GOAMI 64 00090 L7L6P4

Mycobacterium austroafricanum B6UKY3

Mycobacterium sp. ENV421 ahpG I7CD96 uncultured bacterium cypl53 W0UDE1 uncultured bacterium P450 Q33DR8 uncultured bacterium P450 Q33DR9 uncultured bacterium cypl53 W0UDG2 uncultured bacterium cypl53 W0UDM1 uncultured bacterium cypl53 W0UCX8 uncultured bacterium cypl53 W0UAP1 uncultured bacterium cypl53 W0UCW9

Polaromonas sp. (strain JS666 / ATCC BAA-500) Bpro_5301 Q11ZY2 uncultured bacterium cypl53 W0UDK1 uncultured bacterium cypl53 W0UD29 uncultured bacterium cypl53 W0UD32 uncultured bacterium cypl53 W0UD27 uncultured bacterium cypl53 W0UAW2 uncultured bacterium cypl53 W0UAW6 Species Origin Gene names Accession No

Parvibaculum sp. S13-6 CYP153A C7A8P8 uncultured bacterium cypl53 W0UDM5 uncultured bacterium cypl53 W0UD31 uncultured bacterium cypl53 W0UDB6

Parvibaculum sp. S13-5 CYP153A C7A8P2 uncultured bacterium P450 Q33DS1 uncultured bacterium cypl53 W0UDK5 uncultured bacterium cypl53 W0UDU1

Tistrella mobilis CYP153A C7A8Q6 uncultured bacterium cypl53 W0UDS7

Parvibaculum sp. S13-6 CYP153A C7A8P9 uncultured bacterium cypl53 W0UB47

Parvibaculum sp. S13-6 CYP153A C7A8P7 gamma proteobacterium S 10-1 CYP153A C7A8N2 uncultured bacterium cypl53 W0UDS4 uncultured bacterium cypl53 W0UAY8 uncultured bacterium cypl53 W0UDB2 uncultured bacterium cypl53 W0UB02 uncultured bacterium cypl53 W0UDV5 uncultured bacterium cypl53 W0UDM7 uncultured bacterium cypl53 W0UD83 uncultured bacterium cypl53 W0UD50

Parvibaculum sp. S13-5 CYP153A C7A8P4

Parvibaculum sp. S18-4 CYP153A C7A8S8

Parvibaculum sp. S18-4 CYP153A C7A8S9 uncultured bacterium cypl53 W0UB69

Parvibaculum sp. S13-5 CYP153A C7A8P5 uncultured bacterium cypl53 W0UDU6 uncultured bacterium cypl53 W0UDD0 uncultured bacterium cypl53 W0UDA8 uncultured bacterium cypl53 W0UDC3 uncultured bacterium cypl53 W0UDF5 uncultured bacterium cypl53 W0UDD2 uncultured bacterium cypl53 W0UD99 uncultured bacterium cypl53 W0UB78 uncultured bacterium cypl53 W0UDU2 uncultured bacterium cypl53 W0UD95 uncultured bacterium cypl53 W0UDT1 uncultured bacterium cypl53 W0UD70 uncultured bacterium cypl53 W0UAV3 uncultured bacterium cypl53 WOUDJO

Parvibaculum sp. S18-4 CYP153A C7A8S7 uncultured bacterium cypl53 W0UD49 uncultured bacterium cypl53 W0UB74 uncultured bacterium cypl53 W0UDG4 uncultured bacterium cypl53 W0UDJ4 uncultured bacterium cypl53 W0UDL1 uncultured bacterium cypl53 W0UD80 uncultured bacterium cypl53 W0UDP8 uncultured bacterium cypl53 W0UDS6 uncultured bacterium cypl53 W0UDC9 uncultured bacterium cypl53 W0UDE6 uncultured bacterium cypl53 W0UDU9 uncultured bacterium cypl53 WOUDCO Species Origin Gene names Accession No uncultured bacterium cypl53 W0UDW1 uncultured bacterium cypl53 W0UDT4 uncultured bacterium cypl53 W0UDB5 uncultured bacterium cypl53 W0UB64 uncultured bacterium cypl53 W0UDA3 uncultured bacterium cypl53 W0UDR7 uncultured bacterium cypl53 W0UB52 uncultured bacterium cypl53 W0UDA5 uncultured bacterium cypl53 W0UDT6

Caulobacter sp. (strain K31) Caul 0020 B0T154 uncultured bacterium cypl53 W0UCV6 uncultured bacterium cypl53 WOUCUl uncultured bacterium cypl53 W0UDK0 uncultured bacterium cypl53 W0UDI6 uncultured bacterium cypl53 W0UAU9 uncultured bacterium cypl53 W0UAZ2 uncultured bacterium cypl53 W0UD75 uncultured bacterium cypl53 W0UD14 uncultured bacterium cypl53 W0UB97 uncultured bacterium cypl53 W0UD23 uncultured bacterium cypl53 W0UD18 uncultured bacterium cypl53 W0UDQ2 uncultured bacterium cypl53 W0UDH4 uncultured bacterium cypl53 W0UAT6 uncultured bacterium cypl53 W0UD79 uncultured bacterium cypl53 W0UAN4 uncultured bacterium cypl53 W0UDW9 uncultured bacterium cypl53 W0UCZ3 uncultured bacterium cypl53 W0UCZ3

Erythrobacter sp. SI 1-13 CYP153A C7A8R4 uncultured bacterium cypl53 W0UDK7

Parvibaculum sp. S13-5 CYP153A C7A8P3 uncultured bacterium cypl53 W0UDS2 uncultured bacterium cypl53 W0UD84 uncultured bacterium cypl53 W0UD90 uncultured bacterium cypl53 W0UB38 uncultured bacterium cypl53 W0UCW4 uncultured bacterium cypl53 W0UB22 uncultured bacterium cypl53 W0UDQ8 uncultured Rhizobiales bacterium HF4000 48A13 E0XZ55 uncultured Rhizobiales bacterium HF4000 48A13 E0XZ44 uncultured bacterium P450 Q33DS2 uncultured bacterium P450 Q33DS0 uncultured bacterium cypl53 W0UDB4

Erythrobacter flavus C5MKK1 uncultured bacterium cypl53 W0UD08 uncultured bacterium cypl53 W0UCW2

Sphingobium sp. S13-2 CYP153A C7A8P1

Sphingopyxis sp. S16-14 CYP153A C7A8R8 uncultured bacterium cypl53 W0UD46

Parvibaculum sp. S13-6 CYP153A C7A8P6 uncultured bacterium cypl53 W0UDQ1 uncultured bacterium cypl53 W0UB27 uncultured bacterium cypl53 W0UD73 Species Origin Gene names Accession No uncultured bacterium cypl53 W0UDE2 uncultured bacterium cypl53 W0UD17

Erythrobacter sp. S17-1 CYP153A C7A8R9 uncultured bacterium cypl53 W0UD15 uncultured bacterium cypl53 W0UAU6

Erythrobacter flavus CYP153A C7A8N4 uncultured bacterium cypl53 W0UDD6 uncultured bacterium cypl53 W0UDP1 uncultured bacterium cypl53 W0UDF8 uncultured bacterium cypl53 W0UDN8 uncultured bacterium cypl53 W0UDD3 uncultured bacterium cypl53 W0UDN1 uncultured bacterium cypl53 W0UDK3 uncultured bacterium cypl53 W0UD11 uncultured bacterium cypl53 W0UB85 uncultured bacterium cypl53 W0UDI2

Bradyrhizobium sp. CCGE-LAOOl BCCGELAOOl 36078 W1JJD5 uncultured bacterium cypl53 W0UDP5 uncultured bacterium cypl53 W0UB19 uncultured bacterium cypl53 W0UAL6 uncultured bacterium cypl53 W0UDN3 uncultured bacterium cypl53 W0UD72 uncultured bacterium cypl53 W0UCX1 uncultured bacterium cypl53 W0UDF6 uncultured bacterium cypl53 W0UD00 uncultured bacterium cypl53 W0UD65

Caulobacter sp. AP07 PMI01 00728 J2H335

Parvibaculum lavamentivorans (strain DS-1 / DSM 13023

Plav_1765 A7HU01 / NCIMB 13966)

uncultured bacterium P450 Q33DS3 uncultured bacterium cypl53 W0UDH8

Erythrobacter flavus CYP153A C7A8R2

Erythrobacter sp. S2-1 CYP153A C7A8K9

Erythrobacter citreus CYP153A C7A8R1

Erythrobacter citreus CYP153A C7A8R3

Erythrobacter flavus CYP153A C7A8N5 uncultured bacterium cypl53 W0UD37

Erythrobacter sp. S14-1 CYP153A C7A8Q4 uncultured bacterium cypl53 W0UDF2 uncultured bacterium cypl53 W0UDR6 uncultured bacterium cypl53 W0UAN1 uncultured bacterium cypl53 W0UCX5 uncultured bacterium cypl53 W0UD38 uncultured bacterium cypl53 W0UDM9 uncultured bacterium cypl53 W0UCW7 uncultured bacterium cypl53 W0UB12 uncultured bacterium cypl53 W0UD04 uncultured bacterium cypl53 W0UDQ6

Sphingopyxis macrogoltabida (Sphingomonas

ahpGl Q5F4D9 macrogoltabidus)

Afipia broomeae ATCC 49717 HMPREF9695 03199 K8P5Q2 uncultured bacterium cypl53 W0UD96

Parvibaculum sp. S18-4 CYP153A C7A8S5 uncultured bacterium cypl53 W0UAN7 Species Origin Gene names Accession No uncultured bacterium cypl53 W0UCS9 uncultured bacterium cypl53 W0UDX6 uncultured bacterium cypl53 W0UDB7 uncultured bacterium cypl53 W0UD56 uncultured bacterium cypl53 W0UD44

Parvibaculum lavamentivorans (strain DS-1 / DSM 13023

Plav_2128 A7HV09 / NCIMB 13966)

Caulobacter crescentus (strain NA 1000 / CB15N) CCNA 00061 B8GXF2

Caulobacter crescentus (strain ATCC 19089 / CB15) CC 0063 Q9AC06

Parvibaculum lavamentivorans (strain DS-1 / DSM 13023

Plav_0025 A7HP15 / NCIMB 13966)

Caulobacter segnis (strain ATCC 21756 / DSM 7131 /

JCM 7823 / NBRC 15250 / LMG 17158 / TK0059) Cseg_0011 D5VDJ3 (Mycoplana segnis)

Novosphingobium sp. PP1Y PP1Y AT31178 F6IH26 uncultured bacterium cypl53 W0UDC7 uncultured bacterium cypl53 W0UDA2 uncultured bacterium cypl53 W0UDP7

Parvibaculum sp. S18-4 CYP153A C7A8S6 uncultured bacterium cypl53 W0UAK6 uncultured bacterium cypl53 W0UD52 uncultured bacterium cypl53 W0UCU6 uncultured bacterium cypl53 W0UCR4 uncultured bacterium cypl53 W0UCS6 uncultured bacterium cypl53 W0UDV6 uncultured bacterium cypl53 W0UDY0 uncultured bacterium cypl53 W0UDF0 uncultured bacterium cypl53 W0UDF0 uncultured bacterium cypl53 W0UAV7 uncultured bacterium cypl53 W0UDL7

Bradyrhizobium sp. STM 3843 BRAS3843 1530026 H0THQ7

Bradyrhizobium sp. (strain ORS278) BRAD01446 A4YN62

Bradyrhizobium sp. (strain BTAil / ATCC BAA-1182) BBta 6659 A5EQW5

Caulobacter crescentus OR37 OR37 01714 R0EKG8

Afipia broomeae ATCC 49717 HMPREF9695 03200 K8P2K6

Afipia clevelandensis ATCC 49720 HMPREF9696 02236 K8P5K9

Bradyrhizobiaceae bacterium SG-6C CSIRO 4275 F7QRQ2

Novosphingobium pentaromativorans US6-1 ahpG3 NSU_pLA1167 G6EL94 marine gamma proteobacterium HTCC2143 GP2143 12206 A0YHG8

Sphingopyxis macrogoltabida (Sphingomonas

ahpG2 Q5F4D6 macrogoltabidus)

uncultured bacterium cypl53 W0UD98 uncultured bacterium cypl53 W0UAZ7 uncultured bacterium cypl53 WOUCUO uncultured bacterium cypl53 W0UCW6

Bradyrhizobium sp. ORS 375 BRA0375 960079 H0SSR8

Bradyrhizobium sp. ORS 285 BRA0285 1310010 H0RSU1

Bradyrhizobium sp. STM 3809 BRAS3809 1790009 H0SVY3

Rhodopseudomonas palustris (strain BisA53) RPE 4309 Q07IK1

Bradyrhizobium sp. YR681 PMI42 06128 J3CQJ7

Bradyrhizobium sp. STM 3843 BRAS3843 1530027 H0THQ8

Rhodopseudomonas palustris (strain BisB18) RPC 4264 Q20YJ8

Caulobacter sp. (strain K31) Caul 5296 B0T9L7

Sphingopyxis macrogoltabida (Sphingomonas

ahpG3 Q5F4D3 macrogoltabidus)

Species Origin Gene names Accession No

Bradyrhizobium sp. WSM1253 Bral253DRAFT 06024 I2QN59

Bradyrhizobium sp. WSM471 Bra471DRAFT 01541 H5Y7S1 uncultured gamma proteobactenum EB000 65A11 E0XZZ2 marine gamma proteobacterium HTCC2148 GPB2148 1452 B7RXX8 marine gamma proteobacterium HTCC2143 GP2143 15156 A0Y901

Afipia sp. P52-10 X566 17435 W3RGW1 gamma proteobacterium NOR5-3 NOR53 537 B8KPR5

Glaciecola psychrophila 170 C427 3047 GPSY 3092 K7ADG3

Marinobacter lipolyticus SMI 9 MARLIPOL 15764 R8AWZ8 gamma proteobacterium IMCC3088 IMCC3088 2432 F3L451 uncultured bacterium P450 Q33DT3 uncultured bacterium P450 Q33DS9 uncultured bacterium P450 Q33DS8 uncultured bacterium cypl53 W0UD71

Congregibacter litoralis KT71 KT71 02837 A4A779 marine gamma proteobacterium HTCC2080 MGP2080 14441 A0Z7J1

Marinobacter santoriniensis NKSG1 MSNKSG1 10343 M7CRK4

Alcanivorax hongdengensis G1C7P2

Alcanivorax sp. DG881 ADG881 2620 B4WXL2 uncultured bacterium P450 Q33DS6 uncultured bacterium cypl53 W0UCP6 uncultured bacterium cypl53 W0UCQ6

Ochrobactrum anthropi CYP153A C7A8M0 uncultured bacterium cypl53 W0UCN8 uncultured bacterium cypl53 W0UCT1 uncultured bacterium cypl53 W0UCT1 uncultured bacterium cypl53 W0UAI3 gamma proteobacterium HIMB55 OMB55 00002070 H3NWG4

Bradyrhizobium sp. DFCI-1 C207 06143 U1H776 gamma proteobacterium HIMB55 OMB55 00014510 H3NWP3 marine gamma proteobacterium HTCC2080 MGP2080 06587 A0Z166

Burkholderia xenovorans (strain LB400) Bxe A3593 Q143U3

Alcanivorax sp. P2S70 Q670 08165 U7G5C1

Marinobacter hydrocarbonoclasticus ATCC 49840 MARHY3773 H8WA08

Marinobacter sp. EVN1 Q672 10645 U7NYR4 uncultured bacterium P450 Q33DS4 uncultured bacterium cypl53 W0UDA1 uncultured bacterium cypl53 W0UCR5 uncultured bacterium cypl53 W0UD97 uncultured bacterium cypl53 W0UD81 uncultured bacterium cypl53 W0UCN3 uncultured bacterium cypl53 W0UCN5 uncultured bacterium cypl53 W0UCT3 gamma proteobacterium HdNl ahpG HDN1F 17560 E1VKJ7

Marinobacter adhaerens (strain HP 15) HP15_pl87gl48 E4PSB0 uncultured bacterium P450 Q33DT0 uncultured bacterium P450 Q33DS5 uncultured bacterium cypl53 W0UD61 uncultured bacterium P450 Q33DT1

Alcanivorax hongdengensis B3U002 uncultured bacterium P450 Q33DT2 uncultured bacterium P450 Q33DS7 uncultured bacterium cypl53 W0UCL9 uncultured bacterium cypl53 W0UDB3

Species Origin Gene names Accession No

Er throbacter litoralis (strain HTCC2594) ELI 12445 Q2N6W0

Erythrobacter sp. SD-21 ED21 18817 A5P986

Novosphingobium nitrogenifigens DSM 19370 Y88 2850 F1Z4F0

Sphingopyxis macrogoltabida (Sphingomonas

ahpG5 Q5F4D8 macrogoltabidus)

Sphingopyxis alaskensis (strain DSM 13593 / LMG 18877

Sala_2865 Q1GP52 / RB2256) (Sphingomonas alaskensis)

Sphingopyxis macrogoltabida (Sphingomonas

ahpG4 Q5F4D1 macrogoltabidus)

Novosphingobium aromaticivorans (strain DSM 12444) Saro 0220 Q2GBV5

Dickey a dadantii (strain Ech586) Dd586 1369 D2BW78

Sphingopyxis sp. MCI EBMC1 05939 N9UVB0

Dietzia sp. D5 W0C650

Sphingobium indicum B90A SIDU 06697 I5BFE4

Sphingobium chinhatense IP26 M527 09955 W1KG42

Sphingobium sp. HDIP04 L286 21540 T0G3B9

Erythrobacter sp. NAP1 NAP1 13673 A3WFL2

Dickeya dadantii (strain 3937) (Erwinia chrysanthemi

Dda3937_03358 E0SIQ2 (strain 3937))

Sphingomonas sanxanigenens DSM 19645 = NX02 NX02 10200 W0AB84

Sphingopyxis sp. MCI EBMC1 03994 N9WE44

Dickeya sp. D s0432-l A544 2711 U6Z9W7

Novosphingobium aromaticivorans (strain DSM 12444) Saro 1821 Q2G7B2

Erythrobacter litoralis (strain HTCC2594) ELI 09815 Q2N8D6

Parvibaculum lavamentivorans (strain DS-1 / DSM 13023

Plav_0029 A7HP19 / NCIMB 13966)

Novosphingobium pentaromativorans US6-1 NSU 3817 G6EHJ6

[0118] In some embodiments, the invention provide synthetic methods as described above, wherein the enzyme is selected from AlkB, AlkB PI, and AlkBl AB. In some embodiments, the enzyme is selected from CYP153 M. sp.; CYP153A M. aq.; CYP153A M. aq. (G307A); Cypl53A M. aq. (G307A)-CPR_BM3; Cypl53A P.sp.-CPR_BM3; CYP153A13N2; CYP153A13N3; CYP153A13P2; and CYP153A7. In some embodiments, the enzyme is selected from CYP52A13 and CYP52A3.

[0119] In some embodiments, the enzyme is not Mycobacterium marinum CYP153A16. In some embodiments, the enzyme is not Marinobacter aquaelolei CYP153A.

[0120] The hydroxylase reactions can be conducting using a whole cell catalyst comprising an enzyme capable of selectively hydroxylating one terminal carbon of an enzyme substrate. In some embodiments, the cell is a microbial cell. In some embodiments, the enzyme is selected from the group consisting of a non-heme diiron monooxygenase, a long-chain alkane hydroxylase, a cytochrome P450, and combinations thereof. In some embodiments, the enzyme is selected from Table 7, Table 8, Table 9, or a variant thereof having at least 90% identity thereto. Exemplary strains include, but are not limited to, those set forth in Table 10. Table 10. Exemplary strains suitable for the present invention.

[0121] The methods of the invention allow for the production of hydroxylated products and intermediates with controlled regioselectivity, while disfavoring the formation of unwanted species such as epoxides or elimination products. The stereochemistry of a hydroxylated product will depend on factors including the structure of the particular substrate used in a particular reaction, as well as the identity of the enzyme. The methods of the invention can be conducted with enzymes that are selective for particular substrates (e.g., cis or Z unsaturated acids vs. trans or E unsaturated acids). [0122] In certain instances, a hydroxylase enzyme will exhibit catalytic efficiency with one isomer of an internal unsaturated acid (e.g., the cis or Z isomer of an unsaturated acid) that is greater than the catalytic efficiency exhibited with the other isomer of the same internal alkene (e.g., the trans or E isomer of an unsaturated acid). In some embodiments, the invention provides methods wherein the catalytic efficiency of the hydroxylase enzyme is at least about 2-fold greater with one isomer of an unsaturated acid than with the other isomer of the unsaturated acid. The catalytic efficiency exhibited by a hydroxylase with one isomer of an unsaturated acid can be, for example, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 25-fold, at least about 50-fold, at least about 100-fold, or at least about 500-fold greater than the catalytic efficiency exhibited by the hydroxylase with the other isomer of the unsaturated acid.

[0123] A particular enzyme can therefore produce Z product over E product from a mixture of Z and E isomeric substrates or enrich the Z product over the E product. In certain embodiments, the invention provides methods wherein the Z:E (cis:trans) isomeric ratio of a hydroxylated product or intermediate is different from the Z:E (cis:trans) isomeric ratio of the enzyme substrate. The Z:E isomeric ratio of the hydroxylated product can be, for example, around 2 times greater than the Z:E isomeric ratio of the enzyme substrate. The Z:E isomeric ratio of the hydroxylated product can be, for example, around 1.25 times, 1.5 times, 2 times, 2.5 times, 3 times, 4 times, 5 times, 10 times, 20 times, 30 times, or 40 times greater than the Z:E isomeric ratio of the enzyme substrate.

[0124] In some embodiments, the invention provides methods wherein the E:Z (transxis) isomeric ratio of the hydroxylated product or intermediate is different from the E:Z (transxis) isomeric ratio of the enzyme substrate. The E:Z isomeric ratio of the hydroxylated product can be, for example, around 2 times greater than the E:Z isomeric ratio of the enzyme substrate. The E:Z isomeric ratio of the hydroxylated product can be, for example, around 1.25 times, 1.5 times, 2 times, 2.5 times, 3 times, 4 times, 5 times, 10 times, 20 times, 30 times, or 40 times greater than the E:Z isomeric ratio of the enzyme substrate.

[0125] In some embodiments, the Z:E isomeric ratio of the hydroxylated product is about 1.25 times greater than the Z:E isomeric ratio of the enzyme substrate. In some embodiments, the E:Z isomeric ratio of the hydroxylated product is about 1.25 times greater than the E:Z isomeric ratio of the enzyme substrate.

Synthesis of Terminal Alkenals

[0126] As indicated above, the alcohol moiety generated via hydroxylation can be further modified to generate alkenals or esters such as acetate esters.

Oxidation of Fatty Alcohols [0127] Oxidation of fatty alcohols is often achieved via selective oxidation via pyridinium chlorochromate (PCC) (Scheme 9).

Scheme 9

(Z)-hexadec-l 1 -en-1 -ol

(Z)-hexadec-l 1 -enal

[0128] Alternatively, TEMPO (TEMPO=2,2,6,6-tetramethylpiperidinyl-N-oxyl) and related catalyst systems can be used to selectively oxidize alcohols to aldehydes. These methods are described in Ryland and Stahl (2014), herein incorporated by reference in its entirety.

Bio-oxidation of Terminal Alcohols

[0129] Many insect pheromones are fatty aldehydes or comprise a fatty aldehyde component. As such, the conversion of fatty alcohol intermediates produced via terminal hydroxylation to the fatty aldehyde is required to produce certain products such as aldehyde pheromones. The conversion of a fatty alcohol to a fatty aldehyde is known to be catalyzed by alcohol dehydrogenases (ADH) and alcohol oxidases (AOX). Additionally, the conversion of a length C_n fatty acid to a C_n-i fatty aldehyde is catalyzed by plant oc- di oxygenases (oc-DOX) (Scheme 10).

Scheme 10

NAD(P)+ NAD(P)H

0₂ H₂0₂

0₂ H₂0

[0130] In some embodiments, an alcohol oxidase (AOX) is used to catalyze the conversion of a fatty alcohol to a fatty aldehyde. Alcohol oxidases catalyze the conversion of alcohols into corresponding aldehydes (or ketones) with electron transfer via the use of molecular oxygen to form hydrogen peroxide as a by-product. AOX enzymes utilize flavin adenine dinucleotide (FAD) as an essential cofactor and regenerate with the help of oxygen in the reaction medium. Catalase enzymes may be coupled with the AOX to avoid accumulation of the hydrogen peroxide via catalytic conversion into water and oxygen.

[0131] Based on the substrate specificities, AOXs may be categorized into four groups: (a) short chain alcohol oxidase, (b) long chain alcohol oxidase, (c) aromatic alcohol oxidase, and (d) secondary alcohol oxidase (Goswami et al. 2013). Depending on the chain length of the desired substrate, some member of these four groups are better suited than others as candidates for evaluation.

[0132] Short chain alcohol oxidases (including but not limited to those currently classified as EC 1.1.3.13, Table 11) catalyze the oxidation of lower chain length alcohol substrates in the range of C1-C8 carbons (van der Klei et al. 1991) (Ozimek et al. 2005). Aliphatic alcohol oxidases from methyl otrophic yeasts such as Candida boidinii and Komagataella pastoris (formerly Pichia pastoris) catalyze the oxidation of primary alkanols to the corresponding aldehydes with a preference for unbranched short-chain aliphatic alcohols. The most broad substrate specificity is found for alcohol oxidase from the Pichia pastoris including propargyl alcohol, 2-chloroethanol, 2-cyanoethanol (Dienys et al. 2003). The major challenge encountered in alcohol oxidation is the high reactivity of the aldehyde product. Utilization of a two liquid phase system (water/solvent) can provide in-situ removal of the aldehyde product from the reaction phase before it is further converted to the acid. For example, hexanal production from hexanol using Pichia pastoris alcohol oxidase coupled with bovine liver catalase was achieved in a bi-phasic system by taking advantage of the presence of a stable alcohol oxidase in aqueous phase (Karra-Chaabouni et al. 2003). For example, alcohol oxidase from Pichia pastoris was able to oxidize aliphatic alcohols of C6 to CI 1 when used biphasic organic reaction system (Murray and Duff 1990). Methods for using alcohol oxidases in a biphasic system according to (Karra-Chaabouni et al. 2003) and (Murray and Duff 1990) are incorporated by reference in their entirety.

[0133] Long chain alcohol oxidases (including but not limited to those currently classified as EC 1.1.3.20; Table 12) include fatty alcohol oxidases, long chain fatty acid oxidases, and long chain fatty alcohol oxidases that oxidize alcohol substrates with carbon chain length of greater than six (Goswami et al. 2013). Banthorpe et al. reported a long chain alcohol oxidase purified from the leaves of Tanacetum vulgare that was able to oxidize saturated and unsaturated long chain alcohol substrates including hex-trans-2-en-l-ol and octan-l-ol (Banthorpe 1976) (Cardemil 1978). Other plant species, including Simmondsia chinensis (Moreau, R.A., Huang 1979), Arabidopsis thaliana (Cheng et al. 2004), and Lotus japonicas (Zhao et al. 2008) have also been reported as sources of long chain alcohol oxidases. Fatty alcohol oxidases are mostly reported from yeast species (Hommel and Ratledge 1990) (Vanhanen et al. 2000) (Hommel et al. 1994) (Kemp et al. 1990) and these enzymes play an important role in long chain fatty acid metabolism (Cheng et al. 2005). Fatty alcohol oxidases from yeast species that degrade and grow on long chain alkanes and fatty acid catalyze the oxidation of fatty alcohols. Fatty alcohol oxidase from Candida tropicalis has been isolated as microsomal cell fractions and characterized for a range of substrates (Eirich et al. 2004) (Kemp et al. 1988) (Kemp et al. 1991) (Mauersberger et al. 1992). Significant activity is observed for primary alcohols of length C₈ to C₁₆ with reported K_M in the 10-50 μΜ range (Eirich et al. 2004). Alcohol oxidases described may be used for the conversion of medium chain aliphatic alcohols to aldehydes as described, for example, for whole-cells Candida boidinii (Gabelman and Luzio 1997), and Pichia pastoris (Duff and Murray 1988) (Murray and Duff 1990). Long chain alcohol oxidases from filamentous fungi were produced during growth on hydrocarbon substrates (Kumar and Goswami 2006) (Savitha and Ratledge 1991). The long chain fatty alcohol oxidase (LjFAOl) from Lotus japonicas has been heterologously expressed in E. coli and exhibited broad substrate specificity for alcohol oxidation including 1-dodecanol and 1-hexadecanol (Zhao et al. 2008).

Table 11. Alcohol oxidase enzymes capable of oxidizing short chain alcohols (EC 1.1.3.13)

Organism Gene names Accession No.

Thanatephorus cucumeris (strain AGl-ΓΒ / isolate

MOX BN14 09478 M5C8F8 7/3/14) (Lettuce bottom rot fungus) (Rhizoctonia solani)

Thanatephorus cucumeris (strain AG1-IB / isolate

AOD1 BN14 11356 M5CH40 7/3/14) (Lettuce bottom rot fungus) (Rhizoctonia solani)

Ogataea henricii AOD1 A5LGF0

Candida methanosorbosa AOD1 A5LGE5

Candida methanolovescens AOD1 A5LGE4

Candida succiphila AOD1 A5LGE6

Aspergillus niger (strain CBS 513.88 / FGSC A1513) Anl5g02200 A2R501

Aspergillus niger (strain CBS 513.88 / FGSC A1513) Anl8g05480 A2RB46

Moniliophfhora perniciosa (Witches'-broom disease

I7CMK2 fungus) (Marasmius perniciosus)

Candida cariosilignicola AOD1 A5LGE3

Candida pignaliae AOD1 A5LGE1

Candida pignaliae AOD2 A5LGE2

Candida sonorensis AOD1 A5LGD9

Candida sonorensis AOD2 A5LGE0

Pichia naganishii AOD1 A5LGF2

Ogataea minuta AOD1 A5LGF1

Ogataea philodendri AOD1 A5LGF3

Ogataea wickerhamii AOD1 A5LGE8

Kuraishia capsulata AOD1 A5LGE7

Talaromyces stipitatus (strain ATCC 10500 / CBS

375.48 / QM 6759 / NRRL 1006) (Penicillium TSTA 021940 B8MHF8 stipitatum)

Talaromyces stipitatus (strain ATCC 10500 / CBS

375.48 / QM 6759 / NRRL 1006) (Penicillium TSTA 065150 B8LTH7 stipitatum)

Talaromyces stipitatus (strain ATCC 10500 / CBS

375.48 / QM 6759 / NRRL 1006) (Penicillium TSTA 065150 B8LTH8 stipitatum)

Talaromyces stipitatus (strain ATCC 10500 / CBS

375.48 / QM 6759 / NRRL 1006) (Penicillium TSTA 000410 B8MSB1 stipitatum)

Ogataea glucozyma AOD1 A5LGE9

Ogataea parapolymorpha (strain DL-1 / ATCC 26012 /

HPODL 03886 W1QCJ3 NRRL Y-7560) (Yeast) (Hansenula polymorpha)

Gloeophyllum trabeum (Brown rot fungus) AOX A8DPS4

Pichia angusta (Yeast) (Hansenula polymorpha) moxl A6PZG8

Pichia trehalophila AOD1 A5LGF4 Organism Gene names Accession No.

Pichia angusta (Yeast) (Hansenula polymorpha) moxl A6PZG9

Pichia angusta (Yeast) (Hansenula polymorpha) moxl A6PZG7

Ixodes scapularis (Black-legged tick) (Deer tick) IscWJSCWO 17898 B7PIZ7

Table 12. Alcohol oxidase enzymes capable of oxidizing long chain alcohols including fatty alcohols (EC 1.1.3.20)

Organism Gene names Accession No.

Sordaria macrospora (strain ATCC MYA-333 /

SMAC_06361 F7W6K4 DSM 997 / K(L3346) / K-hell)

Sordaria macrospora (strain ATCC MYA-333 /

SMAC_01933 F7VSA1 DSM 997 / K(L3346) / K-hell)

Meyerozyma guilliermondii (strain ATCC 6260 /

CBS 566 / DSM 6381 / JCM 1539 / NBRC 10279 / PGUG_03467 A5DJL6 NRRL Y-324) (Yeast) (Candida guilliermondii)

Trichophyton rubrum CBS 202.88 H107 00669 A0A023ATC5

Arthrobotrys oligospora (strain ATCC 24927 / CBS

115.81 / DSM 1491) (Nematode-trapping fungus) AOL_s00097g516 G1XJI9 (Didymozoophaga oligospora)

Scheffersomyces stipitis (strain ATCC 58785 / CBS

6054 / NBRC 10063 / NRRL Y-l 1545) (Yeast) FAOl PICST 90828 A3LYX9 (Pichia stipitis)

Scheffersomyces stipitis (strain ATCC 58785 / CBS

6054 / NBRC 10063 / NRRL Y-l 1545) (Yeast) FA02 PICST 32359 A3LW61 (Pichia stipitis)

Aspergillus oryzae (strain 3.042) (Yellow koji mold) Ao3042_09114 I8TL25

Fusarium oxysporum (strain Fo5176) (Fusarium

FOXB 17532 F9GFU8 vascular wilt)

Rhizopus delemar (strain RA 99-880 / ATCC MYA- 4621 / FGSC 9543 / NRRL 43880) (Mucormycosis RO3G_08271 I1C536 agent) (Rhizopus arrhizus var. delemar)

Rhizopus delemar (strain RA 99-880 / ATCC MYA- 4621 / FGSC 9543 / NRRL 43880) (Mucormycosis RO3G_00154 I1BGX0 agent) (Rhizopus arrhizus var. delemar)

Fusarium oxysporum (strain Fo5176) (Fusarium

FOXB 07532 F9FMA2 vascular wilt)

Penicillium roqueforti PROQFM164_S02g001772 W6QPY1

Aspergillus clavatus (strain ATCC 1007 / CBS

ACL A O 18400 A1CNB5 513.65 / DSM 816 / NCTC 3887 / NRRL 1)

Arthroderma otae (strain ATCC MYA-4605 / CBS

MCYG_08732 C5G1B0 113480) (Microsporum cards)

Trichophyton tonsurans (strain CBS 112818) (Scalp

TESG_07214 F2S8I2 ringworm fungus)

Colletotrichum higginsianum (strain IMI 349063)

CH063 13441 H1VUE7 (Crucifer anthracnose fungus)

Ajellomyces capsulatus (strain H143) (Darling's

HCDG_07658 C6HN77 disease fungus) (Histoplasma capsulatum)

Trichophyton rubrum (strain ATCC MYA-4607 /

TERG_08235 F2T096 CBS 118892) (Athlete's foot fungus)

Organism Gene names Accession No.

Podospora anserina (strain S / ATCC MYA-4624 /

PODANS_5_13040 B2AFD8 DSM 980 / FGSC 10383) (Pleurage anserina)

Neosartorya fumigata (strain ATCC MYA-4609 /

Af293 / CBS 101355 / FGSC A1100) (Aspergillus AFUA 1G17110 Q4WR91 fumigatus)

Fusarium oxysporam f. sp. vasinfectum 25433 FOTG_00686 X0MEE6

Fusarium oxysporam f. sp. vasinfectum 25433 FOTG_12485 X0LE98

Trichophyton interdigitale H6 H101 06625 A0A022U717

Beauveria bassiana (strain ARSEF 2860) (White

BBA 04100 J4UNY3 muscardine disease fungus) (Tritirachium shiotae)

Fusarium oxysporam f. sp. radicis-lycopersici 26381 FOCG_00843 X0GQ62

Fusarium oxysporam f. sp. radicis-lycopersici 26381 FOCG_15170 X0F4T1

Neurospora tetrasperma (strain FGSC 2509 / P0656) NEUTE2DRAFT 88670 G4UNN6

Pseudozyma hubeiensis (strain SY62) (Yeast) PHSY_000086 R9NVU1

Lodderomyces elongisporas (strain ATCC 11503 /

CBS 2605 / JCM 1781 / NBRC 1676 / NRRL YB- LELG_03289 A5E102 4239) (Yeast) (Saccharomyces elongisporas)

Malassezia globosa (strain ATCC MYA-4612 / CBS

MGL 3855 A8QAY8 7966) (Dandruff-associated fungus)

Byssochlamys spectabilis (strain No. 5 / NBRC

PVAR5 7014 V5GBL6 109023) (Paecilomyces variotii)

Ajellomyces capsulatus (strain H88) (Darling's

HCEG_03274 F0UF47 disease fungus) (Histoplasma capsulatum)

Trichosporon asahii var. asahii (strain ATCC 90039

/ CBS 2479 / JCM 2466 / KCTC 7840 / NCYC 2677 A1Q1 03669 J6FBP4 / UAMH 7654) (Yeast)

Penicillium oxalicum (strain 114-2 / CGMCC 5302)

PDE 00027 S7Z8U8 (Penicillium decumbens)

Fusarium oxysporam f. sp. conglutinans race 2

FOPG_02304 X0IBE3 54008

Fusarium oxysporam f. sp. conglutinans race 2

FOPG_13066 X0H540 54008

Fusarium oxysporam f. sp. raphani 54005 FOQG_00704 X0D1G8

Fusarium oxysporam f. sp. raphani 54005 FOQG_10402 X0C482

Metarhizium acridum (strain CQMa 102) MAC_03115 E9DZR7

Arthroderma benhamiae (strain ATCC MYA-4681 /

ARB 02250 D4B1C1 CBS 112371) (Trichophyton mentagrophytes)

Fusarium oxysporam f. sp. cubense tropical race 4

FOIG_12161 X0JFI6 54006 Organism Gene names Accession No.

Fusarium oxysporum f. sp. cubense tropical race 4

FOIG_12751 X0JDU5 54006

Cochliobolus heterostrophus (strain C4 / ATCC

48331 / race T) (Southern corn leaf blight fungus) COCC4DRAFT 52836 N4WZZ0 (Bipolaris maydis)

Trichosporon asahii var. asahii (strain CBS 8904)

A1Q2 00631 K1VZW1 (Yeast)

Mycosphaerella graminicola (strain CBS 115943 /

IP0323) (Speckled leaf blotch fungus) (Septoria MYCGRDRAFT 37086 F9X375 tritici)

Botryotinia fuckeliana (strain T4) (Noble rot fungus)

BofuT4_P072020.1 G2XQ18 (Botrytis cinerea)

Metarhizium anisopliae (strain ARSEF 23 / ATCC

MAA_05783 E9F0I4 MYA-3075)

Cladophialophora carrionii CBS 160.54 G647 05801 V9DAR1

Coccidioides posadasii (strain RMSCC 757 /

CPSG_09174 E9DH75 Silveira) (Valley fever fungus)

Rhodosporidium toruloides (strain NP11) (Yeast)

RHTO_06879 M7X159 (Rhodotorula gracilis)

Puccinia graminis f. sp. tritici (strain CRL 75-36-

PGTG_10521 E3KIL8 700-3 / race SCCL) (Black stem rust fungus)

Trichophyton rubrum CBS 288.86 H103 00624 A0A022WG28

Colletotrichum fioriniae PJ7 CFIO01 08202 A0A010RKZ4

Trichophyton rubrum CBS 289.86 H104 00611 A0A022XB46

Cladophialophora yegresii CBS 114405 A1O7 02579 W9WC55

Colletotrichum orbiculare (strain 104-T / ATCC

96160 / CBS 514.97 / LARS 414 / MAFF 240422)

Cob_10151 N4VFP3 (Cucumber anthracnose fungus) (Colletotrichum

lagenarium)

Drechslerella stenobrocha 248 DRE 03459 W7IDL6

Neosartorya fumigata (strain CEAIO / CBS 144.89 /

AFUB 016500 B0XP90 FGSC A1163) (Aspergillus fumigatus)

Thielavia terrestris (strain ATCC 38088 / NRRL

THITE 2117674 G2R8H9 8126) (Acremonium alabamense)

Gibberella fujikuroi (strain CBS 195.34 / IMI 58289

/ NRRL A-6831) (Bakanae and foot rot disease FFUJ_02948 S0DZP7 fungus) (Fusarium fujikuroi)

Gibberella fujikuroi (strain CBS 195.34 / IMI 58289

/ NRRL A-6831) (Bakanae and foot rot disease FFUJ_12030 S0EMC6 fungus) (Fusarium fujikuroi)

Aspergillus flavus (strain ATCC 200026 / FGSC

AFLA 109870 B8N941 Al 120 / NRRL 3357 / JCM 12722 / SRRC 167)

Togninia minima (strain UCR-PA7) (Esca disease

UCRPA7 1719 R8BTZ6 fungus) (Phaeoacremonium aleophilum) Organism Gene names Accession No.

Ajellomyces dermatitidis (strain ATCC 18188 /

BDDG_09783 F2TUC0 CBS 674.68) (Blastomyces dermatitidis)

Macrophomina phaseolina (strain MS6) (Charcoal

MPH 10582 K2RHA5 rot fungus)

Neurospora crassa (strain ATCC 24698 / 74-OR23-

NCU08977 Q7S2Z2 1A / CBS 708.71 / DSM 1257 / FGSC 987)

Neosartorya fischeri (strain ATCC 1020 / DSM

3700 / FGSC Al 164 / NRRL 181) (Aspergillus NFIA_008260 A1D156 fischerianus)

Fusarium pseudograminearum (strain CS3096)

FPSE 11742 K3U9J5 (Wheat and barley crown-rot fungus)

Spathaspora passalidaram (strain NRRL Y-27907 /

SPAPADRAFT 54193 G3AJP0 11-Yl)

Spathaspora passalidaram (strain NRRL Y-27907 /

SPAPADRAFT 67198 G3ANX7 11-Yl)

Trichophyton verrucosum (strain HKI 0517) TRV_07960 D4DL86

Arthroderma gypseum (strain ATCC MYA-4604 /

MGYG_07264 E4V2J0 CBS 118893) (Microsporum gypseum)

Hypocrea jecorina (strain QM6a) (Trichoderma

TRIREDRAFT 43893 G0R7P8 reesei)

Trichophyton rubrum MR1448 H110 00629 A0A022Z1G4

Aspergillus ruber CBS 135680 EURHEDRAFT 512125 A0A017SPR0

Glarea lozoyensis (strain ATCC 20868 / MF5171) GLAREA 04397 S3D6C1

Setosphaeria turcica (strain 28A) (Northern leaf

SETTUDRAFT 20639 R0K6H8 blight fungus) (Exserohilum turcicum)

Paracoccidioides brasiliensis (strain Pbl8) PADG_06552 C1GH16

Fusarium oxysporum Fo47 FOZG_13577 W9JPG9

Fusarium oxysporum Fo47 FOZG_05344 W9KPH3

Trichophyton rubrum MR1459 H113 00628 A0A022ZY09

Penicillium marneffei (strain ATCC 18224 / CBS

PMAA_075740 B6QBY3 334.59 / QM 7333)

Sphaeralina musiva (strain SO2202) (Poplar stem

SEPMUDRAFT 154026 M3DAK6 canker fungus) (Septoria musiva)

Gibberella moniliformis (strain M3125 / FGSC

7600) (Maize ear and stalk rot fungus) (Fusarium FVEG_10526 W7N4P8 verticillioides)

Gibberella moniliformis (strain M3125 / FGSC

7600) (Maize ear and stalk rot fungus) (Fusarium FVEG_08281 W7MVR9 verticillioides)

Pseudozyma antarctica (strain T-34) (Yeast)

PANT_22d00298 M9MGF2 (Candida antarctica)

Paracoccidioides brasiliensis (strain Pb03) PABG_07795 C0SJD4 Organism Gene names Accession No.

Rhizophagus irregularis (strain DAOM 181602 /

DAOM 197198 / MUCL 43194) (Arbuscular GLOINDRAFT 82554 U9TF61 mycorrhizal fungus) (Glomus intraradices)

Penicillium chrysogenum (strain ATCC 28089 /

DSM 1075 / Wisconsin 54-1255) (Penicillium Pc21g23700 PCH_Pc21g23700 B6HJ58 notatum)

Baudoinia compniacensis (strain UAMH 10762)

BAUCODRAFT 274597 M2M6Z5 (Angels' share fungus)

Hypocrea atroviridis (strain ATCC 20476 / IMI

TRIATDRAFT 280929 G9NJ32 206040) (Trichoderma atroviride)

Colletotrichum gloeosporioides (strain Cg-14)

CGLO_06642 T0LPH0 (Anthracnose fungus) (Glomerella cingulata)

Cordyceps militaris (strain CMOl) (Caterpillar

CCM 02665 G3JB34 fungus)

Pyronema omphalodes (strain CBS 100304)

PCON 13062 U4LKE9 (Pyronema confluens)

Colletotrichum graminicola (strain Ml.001 / M2 /

FGSC 10212) (Maize anthracnose fungus) GLRG_08499 E3QR67 (Glomerella graminicola)

Glarea lozoyensis (strain ATCC 74030 / MF5533) M7I_2117 H0EHX4

Fusarium oxysporum f. sp. cubense (strain race 4)

FOC4_gl0002493 N1S969 (Panama disease fungus)

Fusarium oxysporum f. sp. cubense (strain race 4)

FOC4_gl0011461 N1RT80 (Panama disease fungus)

Cochliobolus sativus (strain ND90Pr / ATCC

201652) (Common root rot and spot blotch fungus) COCSADRAFT 295770 M2TBE4 (Bipolaris sorokiniana)

Mixia osmundae (strain CBS 9802 / 1 AM 14324 /

Mo05571 E5Q 05571 G7E7S3 JCM 22182 / KY 12970)

Mycosphaerella pini (strain NZE10 / CBS 128990)

(Red band needle blight fungus) (Dothistroma DOTSEDRAFT 69651 N1PXR0 septosporum)

Grosmannia clavigera (strain kwl407 / UAMH

11150) (Blue stain fungus) (Graphiocladiella CMQ 1113 F0XC64 clavigera)

Fusarium oxysporum FOSC 3-a FOYG_03004 W9IUE5

Fusarium oxysporum FOSC 3-a FOYG_16040 W9HNP0

Fusarium oxysporum FOSC 3-a FOYG_17058 W9HB31

Nectria haematococca (strain 77-13-4 / ATCC

MYA-4622 / FGSC 9596 / MP VI) (Fusarium solani NECHADRAFT 37686 C7YQL1 subsp. pisi)

Nectria haematococca (strain 77-13-4 / ATCC

MYA-4622 / FGSC 9596 / MP VI) (Fusarium solani NECHADRAFT 77262 C7ZJI0 subsp. pisi) Organism Gene names Accession No.

Tuber melanosporum (strain Mel28) (Perigord black

GSTUM 00010376001 D5GLS0 truffle)

Ajellomyces dermatitidis (strain SLH14081)

BDBG_07633 C5JYI9 (Blastomyces dermatitidis)

Chaetomium globosum (strain ATCC 6205 / CBS

148.51 / DSM 1962 / NBRC 6347 / NRRL 1970) CHGG_09885 Q2GQ69 (Soil fungus)

Candida tenuis (strain ATCC 10573 / BCRC 21748 /

CBS 615 / JCM 9827 / NBRC 10315 / NRRL Y- CANTEDRAFT 108652 G3B9Z1 1498 / VKM Y-70) (Yeast)

Trichophyton rubrum CBS 100081 H102 00622 A0A022VKY4

Pyrenophora teres f. teres (strain 0-1) (Barley net

PTT 09421 E3RLZ3 blotch fungus) (Drechslera teres f. teres)

Colletotrichum gloeosporioides (strain Nara gc5)

CGGC5 4608 L2GB29 (Anthracnose fungus) (Glomerella cingulata)

Gibberella zeae (Wheat head blight fungus)

FG05 06918 A0A016PCS4 (Fusarium graminearum)

Trichophyton soudanense CBS 452.61 H105 00612 A0A022Y6A6

Sclerotinia sclerotiorum (strain ATCC 18683 / 1980

SS1G_07437 A7EQ37 / Ss-1) (White mold) (Whetzelinia sclerotiorum)

Fusarium oxysporum f. sp. pisi HD V247 FOVG_14401 W9NWU8

Fusarium oxysporum f. sp. pisi HD V247 FOVG_02874 W9Q5V3

Ustilago hordei (strain Uh4875-4) (Barley covered

UHOR_03009 I2G1Z4 smut fungus)

Sporisorium reilianum (strain SRZ2) (Maize head

srl2985 E6ZYF7 smut fungus)

Bipolaris zeicola 26-R-13 COCCADRAFT 81154 W6YIP8

Melampsora larici-populina (strain 98AG31 /

MELLADRAFT 78490 F4RUZ8 pathotype 3-4-7) (Poplar leaf rust fungus)

Fusarium oxysporum f. sp. lycopersici (strain 4287 /

CBS 123668 / FGSC 9935 / NRRL 34936) FOXG_01901 J9MG95 (Fusarium vascular wilt of tomato)

Fusarium oxysporum f. sp. lycopersici (strain 4287 /

CBS 123668 / FGSC 9935 / NRRL 34936) FOXG_11941 J9N9S4 (Fusarium vascular wilt of tomato)

Bipolaris victoriae FI3 COCVIDRAFT_39053 W7EMJ8

Debaryomyces hansenii (strain ATCC 36239 / CBS

767 / JCM 1990 / NBRC 0083 / IGC 2968) (Yeast) DEHA2E04268g Q6BQL4 (Torulaspora hansenii)

Clavispora lusitaniae (strain ATCC 42720) (Yeast)

CLUG_01505 C4XZX3 (Candida lusitaniae)

Candida albicans (strain WO-1) (Yeast) CAWG_02023 C4YME4

Trichophyton rubrum MR850 H100 00625 A0A022U0Q2 Organism Gene names Accession No.

Candida dubliniensis (strain CD36 / ATCC MYA- 646 / CBS 7987 / NCPF 3949 / NRRL Y-17841) CD36 32890 B9WMC7 (Yeast)

Starmerella bombicola AOX1 A0A024FB95

Thielavia heterothallica (strain ATCC 42464 /

BCRC 31852 / DSM 1799) (Myceliophthora MYCTH 103590 G2QJL7 thermophila)

Claviceps puφurea (strain 20.1) (Ergot fungus)

CPUR_07614 M1WFI4 (Sphacelia segetum)

Aspergillus oryzae (strain ATCC 42149 / RIB 40)

AO090023000571 Q2UH61 (Yellow koji mold)

DDB 0184181

Dictyostelium discoideum (Slime mold) Q54DT6

DDB G0292042

Triticum urartu (Red wild einkorn) (Crithodium

TRIUR3 22733 M7YME5 urartu)

Solanum tuberosum (Potato) PGSC0003DMG400017211 M1BG07

OSJNBb0044B19.5

Oryza sativa subsp. japonica (Rice) Q8W5P8

LOC_Osl0g33540

Oryza sativa subsp. japonica (Rice) OJ1234 B11.20 Os02g0621800 Q6K9N5

OSJNBa0001K12.5

Oryza sativa subsp. japonica (Rice) Q8W5P3

LOC_Osl0g33520

Zea mays (Maize) ZEAMMB73 809149 C0P3J6

Citrus Clementina CICLE vlOOl l ll lmg V4S9P4

Citrus Clementina CICLE_vl0018992mg V4U4C9

Citrus Clementina CICLE_vl0004405mg V4S9D3

Citrus Clementina CICLE_vl0004403mg V4RZZ6

Moras notabilis L484 011703 W9RIK0

Moras notabilis L484 005930 W9RET7

Medicago trancatula (Barrel medic) (Medicago

MTR_lg075650 G7I4U3 tribuloides)

Arabidopsis thaliana (Mouse-ear cress) Q8LDP0

Medicago trancatula (Barrel medic) (Medicago

MTR_4g081080 G7JF07 tribuloides)

Simmondsia chinensis (Jojoba) (Buxus chinensis) L7VFV2

Pranus persica (Peach) (Amygdalus persica) PRUPE_ppaO 18458mg M5VXL1

Aphanomyces astaci H257 07411 W4GI89

Aphanomyces astaci H257_07412 W4GI44

Aphanomyces astaci H257 07411 W4GKE3

Aphanomyces astaci H257 07411 W4GK29

Aphanomyces astaci H257 07411 W4GJ79

Aphanomyces astaci H257 07411 W4GI38

Phaeodactylum tricornutum (strain CCAP 1055/1) PHATRDRAFT 48204 B7G6C1

Hordeum vulgare var. distichum (Two-rowed

F2E4R4 barley) Organism Gene names Accession No.

Hordeum vulgare var. distichum (Two-rowed

F2DZG1 barley)

Hordeum vulgare var. distichum (Two-rowed

M0YPG7 barley)

Hordeum vulgare var. distichum (Two-rowed

M0YPG6 barley)

Hordeum vulgare var. distichum (Two-rowed

F2CUY4 barley)

Ricinus communis (Castor bean) RCOM 0867830 B9S1S3

Brassica rapa subsp. pekinensis (Chinese cabbage)

BRAO 14947 M4DEM5 (Brassica pekinensis)

Ricinus communis (Castor bean) RCOM 0258730 B9SV13

Brassica rapa subsp. pekinensis (Chinese cabbage)

BRA001912 M4CCI2 (Brassica pekinensis)

Brassica rapa subsp. pekinensis (Chinese cabbage)

BRAO 12548 M4D7T8 (Brassica pekinensis)

Brassica rapa subsp. pekinensis (Chinese cabbage)

BRA024190 M4E5Y6 (Brassica pekinensis)

Brassica rapa subsp. pekinensis (Chinese cabbage)

BRAO 15283 M4DFL0 (Brassica pekinensis)

Ricinus communis (Castor bean) RCOM l 168730 B9SS54

Zea mays (Maize) C4J691

Oryza glaberrima (African rice) I1P2B7

Zea mays (Maize) B6SXM3

Zea mays (Maize) C0HFU4

Aegilops tauschii (Tausch's goatgrass) (Aegilops

F775_19577 R7W4J3 squarrosa)

Solanum habrochaites (Wild tomato) (Lycopersicon

R9R6T0 hirsutum)

Physcomitrella patens subsp. patens (Moss) PHYPADRAFT 124285 A9S535

Physcomitrella patens subsp. patens (Moss) PHYPADRAFT l 13581 A9RG13

Physcomitrella patens subsp. patens (Moss) PHYPADRAFT 182504 A9S9A5

Solanum pennellii (Tomato) (Lycopersicon

R9R6Q1 pennellii)

Vitis vinifera (Grape) VIT_02s0087g00630 F6HJ27

Vitis vinifera (Grape) VIT_07s0005g03780 F6HZM3

Vitis vinifera (Grape) VIT_05s0049g01400 F6H8T4

Vitis vinifera (Grape) VmSV_019349 A5AH38

Capsella rubella CARUB_vl0013046mg R0HIT3

Capsella rubella CARUB_vl0004212mg R0GUX4

Capsella rubella CARUB_vl0004208mg R0F3X6

Capsella rubella CARUB_vl0012453mg ROILDO

Capsella rubella CARUB_vl0004208mg R0GUX1 Organism Gene names Accession No.

Eutrema salsugineum (Saltwater cress) (Sisymbrium

EUTSA_vl0024496mg V4MD54 salsugineum)

Eutrema salsugineum (Saltwater cress) (Sisymbrium

EUTSA_vl0020141mg V4NM59 salsugineum)

Eutrema salsugineum (Saltwater cress) (Sisymbrium

EUTSA_vl0024496mg V4LUR9 salsugineum)

Eutrema salsugineum (Saltwater cress) (Sisymbrium

EUTSA_vl0024528mg V4P767 salsugineum)

Eutrema salsugineum (Saltwater cress) (Sisymbrium

EUTSA_vl0006882mg V4L2P6 salsugineum)

Selaginella moellendorffii (Spikemoss) SELMODRAFT 87684 D8R6Z6

Selaginella moellendorffii (Spikemoss) SELMODRAFT 87621 D8R6Z5

Selaginella moellendorffii (Spikemoss) SELMODRAFT 74601 D8QN81

Selaginella moellendorffii (Spikemoss) SELMODRAFT 73531 D8QN82

Sb04g026390

Sorghum bicolor (Sorghum) (Sorghum vulgare) C5XXS4

SORBIDRAFT_04g026390

Sb04g026370

Sorghum bicolor (Sorghum) (Sorghum vulgare) C5XXS1

SORBIDRAFT_04g026370

SbOlgO 19470

Sorghum bicolor (Sorghum) (Sorghum vulgare) C5WYH6

SORBIDRAFT 0 IgO 19470

SbOlgO 19480

Sorghum bicolor (Sorghum) (Sorghum vulgare) C5WYH7

SORBIDRAFT 0 IgO 19480

SbOlgO 19460

Sorghum bicolor (Sorghum) (Sorghum vulgare) C5WYH5

SORBIDRAFT 0 IgO 19460

Solanum pimpinellifolium (Currant tomato)

R9R6J2 (Lycopersicon pimpinellifolium)

Phaseolus vulgaris (Kidney bean) (French bean) PHAVU_007G124200g V7BGM7

Phaseolus vulgaris (Kidney bean) (French bean) PHAVU_011G136600g V7AI35

Phaseolus vulgaris (Kidney bean) (French bean) PHAVU_001G162800g V7D063

Solanum tuberosum (Potato) PGSC0003DMG400024294 M1C923

Solanum tuberosum (Potato) PGSC0003DMG400018458 M1BKV4

Solanum tuberosum (Potato) PGSC0003DMG400018458 M1BKV3

Glycine max (Soybean) (Glycine hispida) K7LK61

Glycine max (Soybean) (Glycine hispida) K7KXQ9

Populus trichocarpa (Western balsam poplar)

POPTR_0008sl6920g B9HKS3 (Populus balsamifera subsp. trichocarpa)

Picea sitchensis (Sitka spruce) (Pinus sitchensis) B8LQ84

Populus trichocarpa (Western balsam poplar)

POPTR_0004s24310g U5GKQ5 (Populus balsamifera subsp. trichocarpa)

Populus trichocarpa (Western balsam poplar)

POPTR_0010s07980g B9HSG9 (Populus balsamifera subsp. trichocarpa)

Glycine max (Soybean) (Glycine hispida) I1N9S7

Glycine max (Soybean) (Glycine hispida) I1LSK5 Organism Gene names Accession No.

Setaria italica (Foxtail millet) (Panicum italicum) Si034362m.g K4A658

Solarium lycopersicum (Tomato) (Lycopersicon

Solyc09g072610.2 K4CUT7 esculentum)

Setaria italica (Foxtail millet) (Panicum italicum) Si016380m.g K3YQ38

Solanum lycopersicum (Tomato) (Lycopersicon

R9R6I9 esculentum)

Solanum lycopersicum (Tomato) (Lycopersicon

Solyc09g090350.2 K4CW61 esculentum)

Solanum lycopersicum (Tomato) (Lycopersicon

Solyc08g005630.2 K4CI54 esculentum)

Solanum lycopersicum (Tomato) (Lycopersicon

Solyc08g075240.2 K4CMP1 esculentum)

Setaria italica (Foxtail millet) (Panicum italicum) Si034359m.g K4A655

Setaria italica (Foxtail millet) (Panicum italicum) Si034354m.g K4A650

Mimulus guttatus (Spotted monkey flower) (Yellow

MIMGU mgv 1 aOO 1896mg A0A022PU07 monkey flower)

Mimulus guttatus (Spotted monkey flower) (Yellow

MIMGU mgv 1 a022390mg A0A022RAV4 monkey flower)

Mimulus guttatus (Spotted monkey flower) (Yellow

MIMGU mgv 1 aOO 1868mg A0A022S2E6 monkey flower)

Mimulus guttatus (Spotted monkey flower) (Yellow

MIMGU_mgvla001883mg A0A022S275 monkey flower)

Mimulus guttatus (Spotted monkey flower) (Yellow

MIMGU_mgvla00176 lmg A0A022QNF0 monkey flower)

Musa acuminata subsp. malaccensis (Wild banana)

M0SNA8 (Musa malaccensis)

Musa acuminata subsp. malaccensis (Wild banana)

M0RUT7 (Musa malaccensis)

Musa acuminata subsp. malaccensis (Wild banana)

M0RUK3 (Musa malaccensis)

Saprolegnia diclina VS20 SDRG_10901 T0RG89

Brachypodium distachyon (Purple false brome)

BRADI3G49085 I1IBP7 (Trachynia distachya)

Brachypodium distachyon (Purple false brome)

BRADI3G28677 I1I4N2 (Trachynia distachya)

Brachypodium distachyon (Purple false brome)

BRADI3G28657 I1I4N0 (Trachynia distachya)

Oryza sativa subsp. indica (Rice) Osl_34012 B8BHG0

Oryza sativa subsp. indica (Rice) Osl_08118 B8AFT8

Oryza sativa subsp. indica (Rice) Osl_34008 A2Z8H1

Oryza sativa subsp. indica (Rice) Osl_34014 B8BHG1

Oryza sativa subsp. japonica (Rice) LOC_Osl0g33460 Q7XDG3

Oryza sativa subsp. japonica (Rice) Osl0g0474800 Q0IX12 Organism Gene names Accession No.

Oryza sativa subsp. japonica (Rice) Osl0g0474966 C7J7R1

Oryza sativa subsp. japonica (Rice) OSJNBa0001K12.13 Q8W5N7

Oryza sativa subsp. japonica (Rice) OsJ_31873 B9G683

Oryza sativa subsp. japonica (Rice) OsJ_31875 B9G684

Oryza sativa subsp. japonica (Rice) OSJNBa0001K12.3 Q8W5P5

Arabidopsis lyrata subsp. lyrata (Lyre-leaved rock-

ARALYDRAFT 470376 D7KDA3 cress)

Arabidopsis lyrata subsp. lyrata (Lyre-leaved rock-

ARALYDRAFT 479855 D7L3B6 cress)

Arabidopsis lyrata subsp. lyrata (Lyre-leaved rock-

ARALYDRAFT 491906 D7MDA9 cress)

Arabidopsis lyrata subsp. lyrata (Lyre-leaved rock-

ARAL YDRAFT 914728 D7MGS9 cress)

[0134] In some embodiments, an alcohol dehydrogenase (ADH, Table 13) is used to catalyze the conversion of a fatty alcohol intermediate to a fatty aldehyde. A number of ADHs identified from alkanotrophic organisms, Pseudomonas fluorescens NRRL B-1244 (Hou et al. 1983), Pseudomonas butanovora ATCC 43655 (Vangnai and Arp 2001), and Acinetobacter sp. strain M-l (Tani et al. 2000), have shown to be active on short to medium- chain alkyl alcohols (C₂ to Ci₄). Additionally, commercially available ADHs from Sigma, Horse liver ADH and Baker's yeast ADH have detectable activity for substrates with length Cio and greater. The reported activities for the longer fatty alcohols may be impacted by the difficulties in solubilizing the substrates. For the yeast ADH from Sigma, little to no activity is observed for Ci₂ to Ci₄ aldehydes by (Tani et al. 2000), however, activity for Ci₂ and Ci₆ hydroxy-co-fatty acids has been observed (Lu et al. 2010). Recently, two ADHs were characterized from Geobacillus thermodenitrificans NG80-2, an organism that degrades C15 to C₃6 alkanes using the LadA hydroxylase. Activity was detected from methanol to 1- triacontanol (C₃₀) for both ADHs, with 1-octanol being the preferred substrate for ADH2 and ethanol for ADH1 (Liu et al. 2009).

[0135] The use of ADHs in whole-cell bioconversions has been mostly focused on the production of chiral alcohols from ketones (Ernst et al. 2005) (Schroer et al. 2007). Using the ADH from Lactobacillus brevis and coupled cofactor regeneration with isopropanol, Schroer et al. reported the production of 797 g of (R)-methyl-3 hydroxybutanoate from methyl acetoacetate, with a space time yield of 29 g/L/h (Schroer et al. 2007). Examples of aliphatic alcohol oxidation in whole-cell transformations have been reported with commercially obtained S. cerevisiae for the conversion of hexanol to hexanal (Presecki et al. 2012) and 2-heptanol to 2-heptanone (Cappaert and Larroche 2004).

Table 13. Exemplary alcohol dehydrogenase enzymes.

Organisms Gene Name Accession No.

Zaprionus tuberculatus (Vinegar fly) Adh P51552

GeobaciUus stearothermophilus (Bacillus stearothermophilus) adh P42327

Drosophila mayaguana (Fruit fly) Adh, Adh2 P25721

Drosophila melanogaster (Fruit fly) Adh, CG3481 P00334

Drosophila pseudoobscura pseudoobscura (Fruit fly) Adh, GA17214 Q6LCE4

Drosophila simulans (Fruit fly) Adh, GD23968 Q24641

Drosophila yakuba (Fruit fly) Adh, GE19037 P26719

Drosophila ananassae (Fruit fly) Adh, GF14888 Q50L96

Drosophila erecta (Fruit fly) Adh, GG25120 P28483

Drosophila grimshawi (Fruit fly) (Idiomyia grimshawi) Adh, GH13025 P51551

Drosophila willistoni (Fruit fly) Adh, GK18290 Q05114

Drosophila persimilis (Fruit fly) Adh, GL25993 P37473

Drosophila sechellia (Fruit fly) Adh, GM15656 Q9GN94

Cupriavidus necator (strain ATCC 17699 / H16 / DSM 428 /

adh, H16 A0757 Q0KDL6 Stanier 337) (Ralstonia eutropha)

Mycobacterium tuberculosis (strain CDC 1551 / Oshkosh) adh, MT1581 P9WQC2

Staphylococcus aureus (strain MW2) adh, MW0568 Q8NXU1

Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv) adh, Rvl530 P9WQC3

Staphylococcus aureus (strain N315) adh, SA0562 Q7A742

Staphylococcus aureus (strain bovine RF122 / ET3-1) adh, SAB0557 Q2YSX0

Sulfolobus acidocaldarius (strain ATCC 33909 / DSM 639 /

adh, Saci_2057 Q4J781 JCM 8929 / NBRC 15157 / NCIMB 11770)

Staphylococcus aureus (strain COL) adh, SACOL0660 Q5HI63 adh,

Staphylococcus aureus (strain NCTC 8325) Q2G0G1

SAOUHSC 00608

Staphylococcus aureus (strain MRSA252) adh, SAR0613 Q6GJ63

Staphylococcus aureus (strain MS SA476) adh, SAS0573 Q6GBM4

Staphylococcus aureus (strain USA300) adh, SAUSA300 0594 Q2FJ31

Staphylococcus aureus (strain Mu50 / ATCC 700699) adh, SAV0605 Q99W07

Staphylococcus epidermidis (strain ATCC 12228) adh, SE 0375 Q8CQ56

Staphylococcus epidermidis (strain ATCC 35984 / RP62A) adh, SERP0257 Q5HRD6

Sulfolobus solfataricus (strain ATCC 35092 / DSM 1617 /

adh, SS02536 P39462 JCM 11322 / P2)

Sulfolobus tokodaii (strain DSM 16993 / JCM 10545 / NBRC

adh, STK 25770 Q96XE0 100140 / 7) Organisms Gene Name Accession No.

Anas platyrhynchos (Domestic duck) (Anas boschas) ADH1 P30350

Apteryx australis (Brown kiwi) ADH1 P49645

Ceratitis capitata (Mediterranean fruit fly) (Tephritis capitata) ADH1 P48814

Ceratitis cosyra (Mango fruit fly) (Trypeta cosyra) ADH1 Q70UN9

Gallus gallus (Chicken) ADH1 P23991

Columba livia (Domestic pigeon) ADH1 P86883

Coturnix coturnix japonica (Japanese quail) (Coturnix

ADH1 P19631 japonica)

Drosophila hydei (Fruit fly) Adhl P23236

Drosophila montana (Fruit fly) Adhl P48586

Drosophila mettleri (Fruit fly) Adhl P22246

Drosophila mulleri (Fruit fly) Adhl P07161

Drosophila navojoa (Fruit fly) Adhl P12854

Geomys attwateri (Attwater's pocket gopher) (Geomys

ADH1 Q9Z2M2 bursarius attwateri)

Geomys bursarius (Plains pocket gopher) ADH1 Q64413

Geomys knoxjonesi (Knox Jones's pocket gopher) ADH1 Q64415

Hordeum vulgare (Barley) ADH1 P05336

Kluyveromyces marxianus (Yeast) (Candida kefyr) ADH1 Q07288

Zea mays (Maize) ADH1 P00333

Mesocricetus auratus (Golden hamster) ADH1 P86885

Pennisetum americanum (Pearl millet) (Pennisetum glaucum) ADH1 P14219

Petunia hybrida (Petunia) ADH1 P25141

Oryctolagus cuniculus (Rabbit) ADH1 Q03505

Solanum tuberosum (Potato) ADH1 P14673

Struthio camelus (Ostrich) ADH1 P80338

Trifolium repens (Creeping white clover) ADH1 P13603

Zea luxurians (Guatemalan teosinte) (Euchlaena luxurians) ADH1 Q07264

Saccharomyces cerevisiae (strain ATCC 204508 / S288c) ADH1, ADC1,

P00330 (Baker's yeast) YOL086C, O0947

ADH1, ADH,

Arabidopsis thaliana (Mouse-ear cress) Atlg77120, P06525

F22K20.19

Schizosaccharomyces pombe (strain 972 / ATCC 24843) adhl, adh,

P00332 (Fission yeast) SPCC13B11.01

Drosophila lacicola (Fruit fly) Adhl, Adh-1 Q27404

Mus musculus (Mouse) Adhl, Adh-1 P00329

Peromyscus maniculatus (North American deer mouse) ADH1, ADH-1 P41680

Rattus norvegicus (Rat) Adhl, Adh-1 P06757 Organisms Gene Name Accession No.

Adhl, Adh-1,

Drosophila virilis (Fruit fly) B4M8Y0

GJ18208

Scheffersomyces stipitis (strain ATCC 58785 / CBS 6054 / ADHl, ADH2,

000097 NBRC 10063 / NRRL Y-11545) (Yeast) (Pichia stipitis) PICST 68558

Aspergillus flavus (strain ATCC 200026 / FGSC Al 120 /

adhl, AFLA 048690 P41747 NRRL 3357 / JCM 12722 / SRRC 167)

Neurospora crassa (strain ATCC 24698 / 74-OR23-1A / CBS adh-1, B17C10.210,

Q9P6C8 708.71 / DSM 1257 / FGSC 987) NCU01754

Candida albicans (Yeast) ADHl, CAD P43067

ADHl, DUPR11.3,

Osl lg0210300,

Oryza sativa subsp. japonica (Rice) Q2R8Z5

LOC Osl lgl0480,

OsJ 032001

Drosophila mojavensis (Fruit fly) Adhl, GI 17644 P09370

Kluyveromyces lactis (strain ATCC 8585 / CBS 2359 / DSM

ADHl,

70799 / NBRC 1267 / NRRL Y-l 140 / WM37) (Yeast) P20369

KLLA0F21010g

(Candida sphaerica)

Oryza sativa subsp. indica (Rice) ADHl, Osl_034290 Q75ZX4

Pongo abelii (Sumatran orangutan) (Pongo pygmaeus abelii) ADH1A Q5RBP7

Homo sapiens (Human) ADHl A, ADHl P07327

Macaca mulatta (Rhesus macaque) ADHl A, ADHl P28469

Pan troglodytes (Chimpanzee) ADH1B Q5R1W2

Papio hamadryas (Hamadryas baboon) ADH1B P14139

Homo sapiens (Human) ADH1B, ADH2 P00325

Homo sapiens (Human) ADH1C, ADH3 P00326

Papio hamadryas (Hamadryas baboon) ADH1C, ADH3 097959

Ceratitis capitata (Mediterranean fruit fly) (Tephritis capitata) ADH2 P48815

Ceratitis cosyra (Mango fruit fly) (Trypeta cosyra) ADH2 Q70UP5

Ceratitis rosa (Natal fruit fly) (Pterandrus rosa) ADH2 Q70UP6

Drosophila arizonae (Fruit fly) Adh2 P27581

Drosophila buzzatii (Fruit fly) Adh2 P25720

Drosophila hydei (Fruit fly) Adh2 P23237

Drosophila montana (Fruit fly) Adh2 P48587

Drosophila mulleri (Fruit fly) Adh2 P07160

Drosophila wheeleri (Fruit fly) Adh2 P24267

Entamoeba histolytica ADH2 Q24803

Hordeum vulgare (Barley) ADH2 P10847

Kluyveromyces marxianus (Yeast) (Candida kefyr) ADH2 Q9P4C2

Zea mays (Maize) ADH2 P04707

Oryza sativa subsp. indica (Rice) ADH2 Q4R1E8

Solanum lycopersicum (Tomato) (Lycopersicon esculentum) ADH2 P28032

Organisms Gene Name Accession No.

Mus musculus (Mouse) Adh5, Adh-2, Adh2 P28474

Rattus norvegicus (Rat) Adh5, Adh-2, Adh2 P12711

Oryctolagus cuniculus (Rabbit) ADH5, ADH3 019053

Homo sapiens (Human) ADH5, ADHX, FDH PI 1766

adh5,

Dictyostelium discoideum (Slime mold) Q54TC2

DDB G0281865

Saccharomyces cerevisiae (strain ATCC 204508 / S288c) ADH5, YBR145W,

P38113 (Baker's yeast) YBR1122

Homo sapiens (Human) ADH6 P28332

Peromyscus maniculatus (North American deer mouse) ADH6 P41681

Pongo abelii (Sumatran orangutan) (Pongo pygmaeus abelii) ADH6 Q5R7Z8

Rattus norvegicus (Rat) Adh6 Q5XI95

Homo sapiens (Human) ADH7 P40394

Rattus norvegicus (Rat) Adh7 P41682

Mus musculus (Mouse) Adh7, Adh-3, Adh3 Q64437

Mycobacterium tuberculosis (strain CDC 1551 / Oshkosh) adhA, MT1911 P9WQC0

Rhizobium meliloti (strain 1021) (Ensifer meliloti) adhA, RA0704,

031186 (Sinorhizobium meliloti) SMal296

Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv) adhA, Rvl862 P9WQC1

Zymomonas mobilis subsp. mobilis (strain ATCC 31821 /

adhA, ZM01236 P20368 ZM4 / CP4)

Mycobacterium bovis (strain ATCC BAA-935 / AF2122/97) adhB, Mb0784c Q7U1B9

Mycobacterium tuberculosis (strain CDC 1551 / Oshkosh) adhB, MT0786 P9WQC6 adhB, Rv0761c,

Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv) P9WQC7

MTCY369.06c

Zymomonas mobilis subsp. mobilis (strain ATCC 31821 /

adhB, ZM01596 P0DJA2 ZM4 / CP4)

Zymomonas mobilis subsp. mobilis (strain ATCC 10988 /

adhB, Zmob_1541 F8DVL8 DSM 424 / LMG 404 / NCIMB 8938 / NRRL B-806 / ZMl)

Mycobacterium tuberculosis (strain CDC 1551 / Oshkosh) adhD, MT3171 P9WQB8

Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv) adhD, Rv3086 P9WQB9

Clostridium acetobutylicum (strain ATCC 824 / DSM 792 /

adhE, aad, CA P0162 P33744 JCM 1419 / LMG 5710 / VKM B-1787)

adhE, ana, bl241,

Escherichia coli (strain K12) P0A9Q7

JW1228

Escherichia coli 0157:H7 adhE, Z2016, P0A9Q8 Organisms Gene Name Accession No.

ECsl741

Rhodobacter sphaeroides (strain ATCC 17023 / 2.4.1 / NCIB adhI, RHOS4 11650,

P72324 8253 / DSM 158) RSP_2576

Oryza sativa subsp. indica (Rice) ADHIIL Osl_009236 A2XAZ3 adhP, yddN, bl478,

Escherichia coli (strain K12) P39451

JW1474

GeobaciUus stearothermophilus (Bacillus stearothermophilus) adhT P12311

Emericella nidulans (strain FGSC A4 / ATCC 38163 / CBS

alcA, AN8979 P08843 112.46 / NRRL 194 / M139) (Aspergillus nidulans)

Emericella nidulans (strain FGSC A4 / ATCC 38163 / CBS

ale, AN3741 P54202 112.46 / NRRL 194 / M139) (Aspergillus nidulans)

Emericella nidulans (strain FGSC A4 / ATCC 38163 / CBS

alcC, adh3, AN2286 P07754 112.46 / NRRL 194 / M139) (Aspergillus nidulans)

Arabidopsis thaliana (Mouse-ear cress) Atlg22430, F12K8.22 Q9SK86

Arabidopsis thaliana (Mouse-ear cress) Atlg22440, F12K8.21 Q9SK87

Arabidopsis thaliana (Mouse-ear cress) Atlg32780, F6N18.16 A1L4Y2

Arabidopsis thaliana (Mouse-ear cress) Atlg64710, F13O11.3 Q8VZ49

At4g22110,

Arabidopsis thaliana (Mouse-ear cress) Q0V7W6

F1N20.210

At5g24760,

Arabidopsis thaliana (Mouse-ear cress) Q8LEB2

T4C12 30

Arabidopsis thaliana (Mouse-ear cress) At5g42250, K5J14.5 Q9FH04

Zea mays (Maize) FDH P93629

Fdh, gfd, ODH,

Drosophila melanogaster (Fruit fly) P46415

CG6598

Bacillus subtilis (strain 168) gbsB, BSU31050 P71017

Caenorhabditis elegans H24K24.3 Q17335

Os02g0815500,

LOC Os02g57040,

Oryza sativa subsp. japonica (Rice) Q0DWH1

OsJ 008550,

P0643F09.4

Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv) Rvl895 007737

Caenorhabditis elegans sodh-l, K12G11.3 Q17334

Caenorhabditis elegans sodh-2, K12G11.4 045687

Pseudomonas sp. terPD P33010

Escherichia coli (strain K12) yiaY, b3589, JW5648 P37686

Moraxella sp. (strain TAE123) P81786

Alligator mississippiensis (American alligator) P80222

Catharanthus roseus (Madagascar periwinkle) (Vinca rosea) P85440 Organisms Gene Name Accession No.

Gadus morhua subsp. callarias (Baltic cod) (Gadus callarias) P26325

Naja naja (Indian cobra) P80512

Pisum sativum (Garden pea) P12886

Pelophylax perezi (Perez's frog) (Rana perezi) P22797

Saara hardwickii (Indian spiny -tailed lizard) (Uromastyx

P25405 hardwickii)

Saara hardwickii (Indian spiny -tailed lizard) (Uromastyx

P25406 hardwickii)

Equus caballus (Horse) P00327

Equus caballus (Horse) P00328

Geobacillus stearothermophilus (Bacillus stearothermophilus) P42328

Gadus morhua (Atlantic cod) P81600

Gadus morhua (Atlantic cod) P81601

Myxine glutinosa (Atlantic hagfish) P80360

Octopus vulgaris (Common octopus) P81431

Pisum sativum (Garden pea) P80572

Saara hardwickii (Indian spiny -tailed lizard) (Uromastyx

P80467 hardwickii)

Scyliorhinus canicula (Small-spotted catshark) (Squalus

P86884 canicula)

Spams aurata (Gilthead sea bream) P79896

[0136] In some embodiments, an a-dioxygenase is used to catalyze the conversion of a fatty acid to a fatty aldehyde (Hamberg et al. 2005). Alpha-dioxygenases catalyze the conversion of a C_n fatty acid to a C_n-i aldehyde and may serve as an alternative to both ADH and AOX for fatty aldehyde production if a fatty acid is used as a biotransformation substrate. Due to the chain shortening of the dioxygenase reaction, this route requires a different synthesis pathway compared to the ADH and AOX routes. Biotransformations of E. coli cells expressing a rice a-dioxygenase exhibited conversion of CIO, C12, C14 and C16 fatty acids to the corresponding C_n-i aldehydes. With the addition of the detergent Triton X 100, 3.7 mM of pentadecanal (0.8 g/L) was obtained after 3 hours from hexadecanoic acid with 74% conversion (Kaehne et al. 2011). Exemplary oc-dioxygenases are shown in Table 14.

Table 14. Exemplary alpha-dioxygenases

P14550 Homo sapiens (Human) AKR1A1 ALDR1 ALR

Solanum lycopersicum (Tomato) (Lycopersicon

Q69EZ9 esculentum) LOC543896

Solanum lycopersicum (Tomato) (Lycopersicon

Q5WM33 esculentum) alpha-DOX2

Solanum lycopersicum (Tomato) (Lycopersicon

Q69F00 esculentum)

Arabidopsis lyrata subsp. lyrata (Lyre-leaved

D7LAG3 rock-cress) ALPHA-DOX1 ARALYDRAFT 317048

D8LJL3 Ectocarpus siliculosus (Brown alga) DOX Esi 0026 0091

E3U9P5 Nicotiana attenuata (Coyote tobacco) adox2

[0137] An enzyme's total turnover number (or TTN) refers to the maximum number of molecules of a substrate that the enzyme can convert before becoming inactivated. In general, the TTN for the hydroxylases and other enzymes used in the methods of the invention range from about 1 to about 100,000 or higher. For example, the TTN can be from about 1 to about 1,000, or from about 1,000 to about 10,000, or from about 10,000 to about 100,000, or from about 50,000 to about 100,000, or at least about 100,000. In particular embodiments, the TTN can be from about 100 to about 10,000, or from about 10,000 to about 50,000, or from about 5,000 to about 10,000, or from about 1,000 to about 5,000, or from about 100 to about 1,000, or from about 250 to about 1,000, or from about 100 to about 500, or at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 , 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 15,000, 20,000, 25,000, 30,000, 35,000, 40,000, 45,000, 50,000, 55,000, 60,000, 65,000, 70,000, 75,000, 80,000, 85,000, 90,000, 95,000, 100,000, or more.

[0138] When whole cells expressing a hydroxylase are used to carry out a hydroxylation reaction, the turnover can be expressed as the amount of substrate that is converted to product by a given amount of cellular material. In general, in vivo hydroxylation reactions exhibit turnovers from at least about 0.01 to at least about 10 mmol -gcdw^"1, wherein g_Cd_w is the mass of cell dry weight in grams. When whole cells expressing a hydroxylase are used to carry out a hydroxylation reaction, the activity can further be expressed as a specific productivity, e.g., concentration of product formed by a given concentration of cellular material per unit time, e.g., in g/L of product per g/L of cellular material per hour (g gcdw^"1 h^"1). In general, in vivo hydroxylation reactions exhibit specific productivities from at least about 0.01 to at least about 0.5 g -gcdw^"1 h^"1, wherein g_cdw is the mass of cell dry weight in grams. [0139] The TTN for heme enzymes, in particular, typically ranges from about 1 to about 100,000 or higher. For example, the TTN can be from about 1 to about 1,000, or from about 1,000 to about 10,000, or from about 10,000 to about 100,000, or from about 50,000 to about 100,000, or at least about 100,000. In particular embodiments, the TTN can be from about 100 to about 10,000, or from about 10,000 to about 50,000, or from about 5,000 to about 10,000, or from about 1,000 to about 5,000, or from about 100 to about 1,000, or from about 250 to about 1,000, or from about 100 to about 500, or at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 , 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 15,000, 20,000, 25,000, 30,000, 35,000, 40,000, 45,000, 50,000, 55,000, 60,000, 65,000, 70,000, 75,000, 80,000, 85,000, 90,000, 95,000, 100,000, or more. In certain embodiments, the variant or chimeric heme enzymes of the present invention have higher TTNs compared to the wild- type sequences. In some instances, the variant or chimeric heme enzymes have TTNs greater than about 100 (e.g., at least about 100, 150, 200, 250, 300, 325, 350, 400, 450, 500, or more) in carrying out in vitro hydroxylation reactions. In other instances, the variant or chimeric heme enzymes have TTNs greater than about 1000 (e.g., at least about 1000, 2500, 5000, 10,000, 25,000, 50,000, 75,000, 100,000, or more) in carrying out in vivo whole cell hydroxylation reactions. [0140] When whole cells expressing a heme enzyme are used to carry out a hydroxylation reaction, the turnover can be expressed as the amount of substrate that is converted to product by a given amount of cellular material. In general, in vivo hydroxylation reactions exhibit turnovers from at least about 0.01 to at least about 10 mmol -gcdw^"1, wherein g_Cd_w is the mass of cell dry weight in grams. For example, the turnover can be from about 0.1 to about 10 mmol gcdw^"1, or from about 1 to about 10 mmol gcdw^"1, or from about 5 to about 10 mmol gcdw^"1, or from about 0.01 to about 1 mmol gcdw^"1, or from about 0.01 to about 0.1 mmol gcdw^"1, or from about 0.1 to about 1 mmol gcdw^"1, or greater than 1 mmol gcdw^"1- The turnover can be about 0.01, 0.015, 0.02, 0.025, 0.03, 0.035, 0.04, 0.045, 0.05, 0.055, 0.06, 0.065, 0.07, 0.075, 0.08, 0.085, 0.09, 0.095, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or about 10 mmol -gcdw^"1. [0141] When whole cells expressing a heme enzyme are used to carry out a hydroxylation reaction, the activity can further be expressed as a specific productivity, e.g., concentration of product formed by a given concentration of cellular material per unit time, e.g., in g/L of product per g/L of cellular material per hour (g -gcdw^"1 h^"1). In general, in vivo hydroxylation reactions exhibit specific productivities from at least about 0.01 to at least about 0.5 g gcdw^"1 h^"1, wherein g_Cd_w is the mass of cell dry weight in grams. For example, the specific productivity can be from about 0.01 to about 0.1 g gcdw^"1 h^"1, or from about 0.1 to about 0.5 g -gcdw^"1 h^"1, or greater than 0.5 g -gcdw^"1 h^"1. The specific productivity can be about 0.01, 0.015, 0.02, 0.025, 0.03, 0.035, 0.04, 0.045, 0.05, 0.055, 0.06, 0.065, 0.07, 0.075, 0.08, 0.085, 0.09, 0.095, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, or about 0.5 g-gcdw^"1 h^"1.

[0142] In certain embodiments, mutations can be introduced into the target gene using standard cloning techniques {e.g., site-directed mutagenesis) or by gene synthesis to produce the hydroxylases {e.g., cytochrome P450 variants) of the present invention. The mutated gene can be expressed in a host cell {e.g., bacterial cell) using an expression vector under the control of an inducible promoter or by means of chromosomal integration under the control of a constitutive promoter. Hydroxylation activity can be screened in vivo or in vitro by following product formation by GC or HPLC as described herein.

[0143] The expression vector comprising a nucleic acid sequence that encodes a heme enzyme of the invention can be a viral vector, a plasmid, a phage, a phagemid, a cosmid, a fosmid, a bacteriophage {e.g., a bacteriophage PI -derived vector (PAC)), a baculovirus vector, a yeast plasmid, or an artificial chromosome {e.g., bacterial artificial chromosome (BAC), a yeast artificial chromosome (YAC), a mammalian artificial chromosome (MAC), and human artificial chromosome (HAC)). Expression vectors can include chromosomal, non-chromosomal, and synthetic DNA sequences. Equivalent expression vectors to those described herein are known in the art and will be apparent to the ordinarily skilled artisan.

[0144] The expression vector can include a nucleic acid sequence encoding a heme enzyme that is operably linked to a promoter, wherein the promoter comprises a viral, bacterial, archaeal, fungal, insect, or mammalian promoter. In certain embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is an inducible promoter. In other embodiments, the promoter is a tissue-specific promoter or an environmentally regulated or a developmentally regulated promoter. [0145] It is to be understood that affinity tags may be added to the N- and/or C-terminus of a heme enzyme expressed using an expression vector to facilitate protein purification. Non- limiting examples of affinity tags include metal binding tags such as His6-tags and other tags such as glutathione S-transferase (GST). [0146] Non-limiting expression vectors for use in bacterial host cells include pCWori, pET vectors such as pET22 (EMD Millipore), pBR322 (ATCC37017), pQE™ vectors (Qiagen), pBluescript™ vectors (Stratagene), pNH vectors, lambda-ZAP vectors (Stratagene); ptrc99a, pKK223-3, pDR540, pRIT2T (Pharmacia), pRSET, pCR-TOPO vectors, pET vectors, pSyn l vectors, pChlamy l vectors (Life Technologies, Carlsbad, CA), pGEMl (Promega, Madison, WI), and pMAL (New England Biolabs, Ipswich, MA). Non-limiting examples of expression vectors for use in eukaryotic host cells include pXTl, pSG5 (Stratagene), pSVK3, pBPV, pMSG, pSVLSV40 (Pharmacia), pcDNA3.3, pcDNA4/TO, pcDNA6/TR, pLenti6/TR, pMT vectors (Life Technologies), pKLACl vectors, pKLAC2 vectors (New England Biolabs), pQE™ vectors (Qiagen), BacPak baculoviral vectors, pAdeno-X™ adenoviral vectors (Clontech), and pBABE retroviral vectors. Any other vector may be used as long as it is replicable and viable in the host cell.

[0147] The host cell can be a bacterial cell, an archaeal cell, a fungal cell, a yeast cell, an insect cell, or a mammalian cell.

[0148] Suitable bacterial host cells include, but are not limited to, BL21 E. coli, DE3 strain E. coli, E. coli Ml 5, DH5a, DH10β, HB 101 , T7 Express Competent E. coli (NEB), B. subtilis cells, Pseudomonas fluorescens cells, and cyanobacterial cells such as Chlamydomonas reinhardtii cells and Synechococcus elongates cells. Non-limiting examples of archaeal host cells include Pyrococcus furiosus, Metallosphera sedula, Thermococcus litoralis, Methanobacterium thermoautotrophicum, Methanococcus jannaschii, Pyrococcus abyssi, Sulfolobus solfataricus, Pyrococcus woesei, Sulfolobus shibatae, and variants thereof. Fungal host cells include, but are not limited to, yeast cells from the genera Saccharomyces (e.g., S. cerevisiae), Pichia (P. Pastoris), Kluyveromyces (e.g., K. lactis), Hansenula and Yarrowia, and filamentous fungal cells from the genera Aspergillus, Trichoderma, and Myceliophthora. Suitable insect host cells include, but are not limited to, Sf9 cells from Spodoptera frugiperda, Sf21 cells from Spodoptera frugiperda, Hi-Five cells, BTI-TN-5B1-4 Trichophusia ni cells, and Schneider 2 (S2) cells and Schneider 3 (S3) cells from Drosophila melanogaster. Non-limiting examples of mammalian host cells include HEK293 cells, HeLa cells, CHO cells, COS cells, Jurkat cells, NSO hybridoma cells, baby hamster kidney (BHK) cells, MDCK cells, NIH-3T3 fibroblast cells, and any other immortalized cell line derived from a mammalian cell.

[0149] In certain embodiments, the present invention provides heme enzymes such as the P450 variants described herein that are active hydroxylation catalysts inside living cells. As a non-limiting example, bacterial cells (e.g., E. coli) can be used as whole cell catalysts for the in vivo hydroxylation reactions of the present invention. In some embodiments, whole cell catalysts containing P450 enzymes with the equivalent C400X mutation are found to significantly enhance the total turnover number (TTN) compared to in vitro reactions using i sol ated P450 enzyme s .

Biohydroxylation Reaction Conditions

[0150] The methods of the invention include forming reaction mixtures that contain the hydroxylases described herein. The hydroxylases can be, for example, purified prior to addition to a reaction mixture or secreted by a cell present in the reaction mixture. The reaction mixture can contain a cell lysate including the enzyme, as well as other proteins and other cellular materials. Alternatively, a hydroxylase can catalyze the reaction within a cell expressing the hydroxylase. Any suitable amount of hydroxylase can be used in the methods of the invention. In general, hydroxylation reaction mixtures contain from about 0.01 weight % (wt%) to about 100 wt% hydroxylase with respect to the enzyme substrate. The reaction mixtures can contain, for example, from about 0.01 wt% to about 0.1 wt% hydroxylase, or from about 0.1 wt% to about 1 wt% hydroxylase, or from about 1 wt% to about 10 wt% hydroxylase, or from about 10 wt% to about 100 wt% hydroxylase. The reaction mixtures can contain from about 0.05 wt% to about 5 wt% hydroxylase, or from about 0.05 wt% to about 0.5 wt% hydroxylase. The reaction mixtures can contain about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.2, 1.4, 1.6, 1.8, 2, 2.2, 2.4, 2.6, 2.8, or about 3 wt% hydroxylase. One of skill in the art will understand how to convert wt% values to mol% values with respect to the hydroxylase and/or substrate concentrations set forth herein.

[0151] If the hydroxylase catalyzes the reaction within a cell expressing the hydroxylase then any suitable amount of cells can be used in the methods of the invention. In general, hydroxylation whole-cell reaction mixtures contain from about 1 weight% to about 10,000 wt% of cells on a cell dry weight basis with respect to the enzyme substrate. The whole-cell reaction mixtures can contain, for example, from about 1 wt% to about 10 wt% cells, or from about 10 wt% to about 100 wt% cells, or from about 100 wt% to about 1000 wt% cells, or from about 1000 wt% cells to about 2500 wt% cells, or from about 2500 wt% cells to about 5000 wt% cells, or from about 5000 wt% cells to about 7500 wt% cells, or from about 7500 wt% cells to about 10000 wt% cells with respect to the enzyme substrate. The whole-cell reaction mixtures can contain from about 2 wt% to about 1000 wt% cells, or from about 5 wt% to about 500 wt% cells with respect to the enzyme substrate. The whole-cell reaction mixtures can contain about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or about 1000 wt% cells with respect to the enzyme substrate.

[0152] The concentration of an enzyme substrate is typically in the range of from about 100 μΜ to about 1 M. The concentration can be, for example, from about 100 μΜ to about 1 mM, or about from 1 mM to about 100 mM, or from about 100 mM to about 500 mM, or from about 500 mM to 1 M. The concentration can be from about 500 μΜ to about 500 mM, 500 μΜ to about 50 mM, or from about 1 mM to about 50 mM, or from about 15 mM to about 45 mM, or from about 15 mM to about 30 mM. The concentration of the enzyme substrate can be, for example, about 100, 200, 300, 400, 500, 600, 700, 800, or 900 μΜ. The concentration of the enzyme substrate can be about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, or 500 mM.

[0153] Reaction mixtures can contain additional reagents. As non-limiting examples, the reaction mixtures can contain buffers (e.g., 2-(N-morpholino)ethanesulfonic acid (MES), 2- [4-(2-hydroxyethyl)piperazin-l-yl]ethanesulfonic acid (HEPES), 3-morpholinopropane-l- sulfonic acid (MOPS), 2-amino-2-hydroxymethyl-propane-l,3-diol (TRIS), potassium phosphate, sodium phosphate, phosphate-buffered saline, sodium citrate, sodium acetate, and sodium borate), cosolvents (e.g., dimethylsulfoxide, dimethylformamide, ethanol, methanol, isopropanol, glycerol, tetrahydrofuran, acetone, acetonitrile, and acetic acid), salts (e.g., NaCl, KC1, CaCl₂, and salts of Mn²⁺ and Mg²⁺), denaturants (e.g., urea and guanidinium hydrochloride), detergents (e.g., sodium dodecyl sulfate and Triton-X 100), chelators (e.g., ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA), 2-({2- [Bis(carboxymethyl)amino]ethyl} (carboxymethyl)amino)acetic acid (EDTA), and l,2-bis(o- aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)), sugars (e.g., glucose, sucrose, and the like), and reducing agents (e.g., sodium dithionite, NADPH, dithiothreitol (DTT), β- mercaptoethanol (BME), and tris(2-carboxyethyl)phosphine (TCEP)). Buffers, cosolvents, salts, denaturants, detergents, chelators, sugars, and reducing agents can be used at any suitable concentration, which can be readily determined by one of skill in the art. In general, buffers, cosolvents, salts, denaturants, detergents, chelators, sugars, and reducing agents, if present, are included in reaction mixtures at concentrations ranging from about 1 μΜ to about 1 M. For example, a buffer, a cosolvent, a salt, a denaturant, a detergent, a chelator, a sugar, or a reducing agent can be included in a reaction mixture at a concentration of about 1 μΜ, or about 10 μΜ, or about 100 μΜ, or about 1 mM, or about 10 mM, or about 25 mM, or about 50 mM, or about 100 mM, or about 250 mM, or about 500 mM, or about 1 M. Cosolvents, in particular, can be included in the reaction mixtures in amounts ranging from about 1% v/v to about 75% v/v, or higher. A cosolvent can be included in the reaction mixture, for example, in an amount of about 5, 10, 20, 30, 40, or 50% (v/v). [0154] Reactions are conducted under conditions sufficient to catalyze the formation of a hydroxylation product. The reactions can be conducted at any suitable temperature. In general, the reactions are conducted at a temperature of from about 4°C to about 40°C. The reactions can be conducted, for example, at about 25°C or about 37°C. The reactions can be conducted at any suitable pH. In general, the reactions are conducted at a pH of from about 3 to about 10. The reactions can be conducted, for example, at a pH of from about 6.5 to about 9. The reactions can be conducted for any suitable length of time. In general, the reaction mixtures are incubated under suitable conditions for anywhere between about 1 minute and several hours. The reactions can be conducted, for example, for about 1 minute, or about 5 minutes, or about 10 minutes, or about 30 minutes, or about 1 hour, or about 2 hours, or about 4 hours, or about 8 hours, or about 12 hours, or about 24 hours, or about 48 hours, or about 72 hours, or about 96 hours, or about 120 hours, or about 144 hours, or about 168 hours, or about 192 hours. In general, reactions are conducted under aerobic conditions. In some embodiments, the solvent forms a second phase, and the hydroxylation occurs in the aqueous phase. In some embodiments, the hydroxylases is located in the aqueous layer whereas the substrates and/or products occur in an organic layer. Other reaction conditions may be employed in the methods of the invention, depending on the identity of a particular hydroxylase, or olefinic substrate.

[0155] Reactions can be conducted in vivo with intact cells expressing a hydroxylase of the invention. The in vivo reactions can be conducted with any of the host cells used for expression of the hydroxylases, as described herein. A suspension of cells can be formed in a suitable medium supplemented with nutrients (such as mineral micronutrients, glucose and other fuel sources, and the like). Hydroxylation yields from reactions in vivo can be controlled, in part, by controlling the cell density in the reaction mixtures. Cellular suspensions exhibiting optical densities ranging from about 0.1 to about 50 at 600 nm can be used for hydroxylation reactions. Other densities can be useful, depending on the cell type, specific hydroxylases, or other factors.

Metathesis Catalysts [0156] In general, any metathesis catalyst stable under the reaction conditions and nonreactive with the functional groups present on the reactant shown in Schemes 3 through 6 may be used with the present invention. Such catalysts are, for example, those described by Grubbs (Grubbs, R.H., "Synthesis of large and small molecules using olefin metathesis catalysts." PMSE Prepr., 2012), herein incorporated by reference in its entirety. Depending on the desired isomer of the olefin, as cis-selective (or Z-selective) metathesis catalyst can be used, for example one of those described by Shahane et al. (Shahane, S., et al. ChemCatChem, 2013. 5(12): p. 3436-3459), herein incorporated by reference in its entirety. Specific catalysts 1-5 exhibiting cis-selectivity are shown below (Scheme 11) and have been described previously (Khan, R.K., et al. J. Am. Chem. Soc, 2013. 135(28): p. 10258-61; Hartung, J. et al. J. Am. Chem. Soc, 2013. 135(28): p. 10183-5.; Rosebrugh, L.E., et al. J. Am. Chem. Soc, 2013. 135(4): p. 1276-9.; Marx, V.M., et al. J. Am. Chem. Soc, 2013. 135(1): p. 94-7.; Herbert, M.B., et al. Angew. Chem. Int. Ed. Engl, 2013. 52(1): p. 310-4; Keitz, B.K., et al. J. Am. Chem. Soc, 2012. 134(4): p. 2040-3.; Keitz, B.K., et al. J. Am. Chem. Soc, 2012. 134(1): p. 693-9.; Endo, K. et al. J. Am. Chem. Soc, 2011. 133(22): p. 8525-7).

Scheme 11.

4 [0157] Additional Z-selective catalysts are described in (Cannon and Grubbs 2013; Bronner et al. 2014; Hartung et al. 2014; Pribisko et al. 2014; Quigley and Grubbs 2014) and are herein incorporated by reference in their entirety. Due to their excellent stability and functional group tolerance, preferred metathesis catalysts include, but are not limited to, neutral ruthenium or osmium metal carbene complexes that possess metal centers that are formally in the +2 oxidation state, have an electron count of 16, are penta-coordinated, and are of the general formula LL'AA'M=CRbRc or LL'AA'M=(C=)nCRbRc (Pederson and Grubbs 2002); wherein

M is ruthenium or osmium;

L and L' are each independently any neutral electron donor ligand and preferably selected from phosphine, sulfonated phosphine, phosphite, phosphinite, phosphonite, arsine, stibnite, ether, amine, amide, imine, sulfoxide, carboxyl, nitrosyl, pyridine, thioether, or heterocyclic carbenes; and

A and A' are anionic ligands independently selected from halogen, hydrogen, C1-C20 alkyl, aryl, C1-C20 alkoxide, aryloxide, C2-C20 alkoxycarbonyl, aryl carboxyl ate, Ci- C₂o carboxylate, arylsulfonyl, C1-C20 alkylsulfonyl, C1-C20 alkyl sulfinyl; each ligand optionally being substituted with C1-C5 alkyl, halogen, C1-C5 alkoxy; or with a phenyl group that is optionally substituted with halogen, C1-C5 alkyl, or C1-C5 alkoxy; and A and A' together may optionally comprise a bidentate ligand; and

Rb and Re are independently selected from hydrogen, C1-C20 alkyl, aryl, C1-C20 carboxylate, C1-C20 alkoxy, aryloxy, C1-C20 alkoxycarbonyl, C1-C20 alkylthio, C1-C20 alkylsulfonyl and C1-C20 alkylsulfinyl, each of R and R_c optionally substituted with C1-C5 alkyl, halogen, C1-C5 alkoxy or with a phenyl group that is optionally substituted with halogen, C1-C5 alkyl, or C1-C5 alkoxy.

[0158] Other metathesis catalysts such as "well defined catalysts" can also be used. Such catalysts include, but are not limited to, Schrock's molybdenum metathesis catalyst, 2,6- diisopropylphenylimido neophylidenemolybdenum (VI) bis(hexafluoro-t-butoxide), described by Grubbs et al. {Tetrahedron 1998, 54: 4413-4450) and Basset's tungsten metathesis catalyst described by Couturier, J. L. et al. (Angew. Chem. Int. Ed. Engl. 1992, 31 : 628).

[0159] Catalysts useful in the methods of the invention also include those described by Peryshkov, et al. J. Am. Chem. Soc. 2011, 133 : 20754-20757; Wang, et al. Angewandte Chemie, 2013, 52: 1939-1943; Yu, et al. J. Am. Chem. Soc, 2012, 134: 2788-2799; Halford. Chem. Eng. News, 2011, 89 (45): 11; Yu, et al. Nature, 2011, 479: 88-93; Lee. Nature, 2011, 471 : 452-453; Meek, et al. Nature, 2011 : 471, 461-466; Flook, et al. J. Am. Chem. Soc. 2011, 133 : 1784-1786; Zhao, et al. Org Lett., 2011, 13(4): 784-787; Ondi, et al. "High activity, stabilized formulations, efficient synthesis and industrial use of Mo- and W-based metathesis catalysts" XiMo Technology Updates, 2015: http://www.ximo-inc.com/files/ximo/uploads/ download/Summary _3.11.15.pdf; Schrock, et al. Macromolecules, 2010: 43, 7515-7522; Peryshkov, et al. Organometallics 2013 : 32, 5256-5259; Gerber, et al. Organometallics 2013 : 32, 5573-5580; Marinescu, et al. Organometallics 2012: 31, 6336-6343; Wang, et al. Angew. Chem. Int. Ed. 2013 : 52, 1939 - 1943; Wang, et al. Chem. Eur. J. 2013 : 19, 2726- 2740; and Townsend et al. J. Am. Chem. Soc. 2012: 134, 11334-11337.

[0160] Catalysts useful in the methods of the invention also include those described in International Pub. No. WO 2014/155185; International Pub. No. WO 2014/172534; U.S. Pat. Appl. Pub. No. 2014/0330018; International Pub. No. WO 2015/003815; and International Pub. No. WO 2015/003814. [0161] Catalysts useful in the methods of the invention also include those described in U.S.

Pat. No. 4,231,947; U.S. Pat. No. 4,245, 131; U.S. Pat. No. 4,427,595; U.S. Pat. No.

4,681,956; U.S. Pat. No. 4,727,215; International Pub. No. WO 1991/009825; U.S. Pat. No.

5,0877,10; U.S. Pat. No. 5,142,073; U.S. Pat. No. 5,146,033; International Pub. No. WO

1992/019631; U.S. Pat. No. 6,121,473; U.S. Pat. No. 6,346,652; U.S. Pat. No. 8,987,531; U.S. Pat. Appl. Pub. No. 2008/0119678; International Pub. No. WO 2008/066754;

International Pub. No. WO 2009/094201; U.S. Pat. Appl. Pub. No. 2011/0015430; U.S. Pat.

Appl. Pub. No. 2011/0065915; U.S. Pat. Appl. Pub. No. 2011/0077421; International Pub.

No. WO 2011/040963; International Pub. No. WO 2011/097642; U.S. Pat. Appl. Pub. No.

2011/0237815; U.S. Pat. Appl. Pub. No. 2012/0302710; International Pub. No. WO 2012/167171; U.S. Pat. Appl. Pub. No. 2012/0323000; U.S. Pat. Appl. Pub. No.

2013/0116434; International Pub. No. WO 2013/070725; U.S. Pat. Appl. Pub. No.

2013/0274482; U.S. Pat. Appl. Pub. No. 2013/0281706; International Pub. No. WO

2014/139679; International Pub. No. WO 2014/169014; U.S. Pat. Appl. Pub. No.

2014/0330018; and U.S. Pat. Appl. Pub. No. 2014/0378637. [0162] Catalysts useful in the methods of the invention also include those described in International Pub. No. WO 2007/075427; U.S. Pat. Appl. Pub. No. 2007/0282148; International Pub. No. WO 2009/126831; International Pub. No. WO 2011/069134; U.S. Pat. Appl. Pub. No. 2012/0123133; U.S. Pat. Appl. Pub. No. 2013/0261312; U.S. Pat. Appl. Pub. No. 2013/0296511; International Pub. No. WO 2014/134333; and U.S. Pat. Appl. Pub. No. 2015/0018557.

[0163] Catalysts useful in the methods of the invention also include those set forth in Table 15. In some embodiments, the metathesis catalyst is a Z-selective metathesis catalyst.

Table 15. Exemplary Metathesis Catalysts.

Structure Name

dichloro[l,3-bis(2,6-isopropylphenyl)-2- imidazolidinylidene](benzylidene)(tricyclohex ylphosphine)ruthenium(II)

dichloro[l,3-bis(2,6-isopropylphenyl)-2- imidazolidinylidene](2- isopropoxyphenylmethylene)ruthenium(II)

dichloro[ 1 ,3 -Bis(2-methylphenyl)-2- imidazolidinylidene](benzylidene)(tricyclohex ylphosphine)ruthenium(II)

Structure Name

dichloro[ 1 ,3 -bis(2-methylphenyl)-2- imidazolidinylidene](2- isopropoxyphenylmethylene)ruthenium(II)

dichloro[l,3-bis(2,4,6-trimethylphenyl)-2- imidazolidinylidene](benzylidene)bis(3- bromopyridine)ruthenium(II)

dichloro[l,3-bis(2,4,6-trimethylphenyl)-2- imidazolidinylidene](3-methyl-2- butenylidene) (tricyclohexylphosphine) ruthenium(II)

dichloro[l,3-bis(2,4,6-trimethylphenyl)-2- imidazolidinylidene][3-(2-pyridinyl) propy li dene] ruthenium(II)

Structure Name

dichloro[l,3-bis(2,4,6-trimethylphenyl)-2- imidazolidinylidene][(tricyclohexylphosphora nyl)methylidene]mthenium(II)

tetrafluorob orate

di chl oro(3 -methyl -2-buteny li dene) bis(tricyclohexylphosphine)ruthenium(II)

di chl oro(3 -methyl -2-buteny li dene) bis(tricyclopentylphosphine)ruthenium(II)

Structure Name

dichloro(tricyclohexylphosphine)[(tricyclohex ylphosphoranyl)methylidene]ruthenium(II) tetrafluorob orate

bis(tricyclohexylphosphine) benzylidine ruthenium(IV) di chloride

[l,3-bis-(2,4,6-trimethylphenyl)-2- imidazolidinylidene]dichloro(phenylmethylen e)(tricyclohexylphosphine)ruthenium

Structure Name

(l,3-bis-(2,4,6-trimethylphenyl)-2- imidazolidinylidene)dichloro(o- isopropoxyphenylmethylene)ruthenium

dichloro(o- isopropoxyphenylmethylene)(tricyclohexylph osphine)nithenium(II)

[2-(l-methylethoxy-O)phenylmethyl- ](nitrato-O,O'){/-e/-(2R,5R,7R)-adamantane- 2, 1 -diyl [3 -(2,4,6-trimethylphenyl)- 1 - imidazolidinyl-2-y lidene]} ruthenium

[0164] Catalysts useful in the methods of the invention also include those described in U.S. Pat. Appl. Pub. No. 2008/0009598; U.S. Pat. Appl. Pub. No. 2008/0207911; U.S. Pat. Appl. Pub. No. 2008/0275247; U.S. Pat. Appl. Pub. No. 2011/0040099; U.S. Pat. Appl. Pub. No. 2011/0282068; and U.S. Pat. Appl. Pub. No. 2015/0038723.

[0165] Catalysts useful in the methods of the invention include those described in International Pub. No. WO 2007/140954; U.S. Pat. Appl. Pub. No. 2008/0221345; International Pub. No. WO 2010/037550; U.S. Pat. Appl. Pub. No. 2010/0087644; U.S. Pat. Appl. Pub. No. 2010/01 13795; U. S. Pat. Appl. Pub. No. 2010/0174068; International Pub. No. WO 201 1/091980; International Pub. No. WO 2012/168183; U.S. Pat. Appl. Pub. No. 2013/0079515; U. S. Pat. Appl. Pub. No. 2013/0144060; U.S. Pat. Appl. Pub. No. 2013/021 1096; International Pub. No. WO 2013/135776; International Pub. No. WO 2014/001291 ; International Pub. No. WO 2014/067767; U.S. Pat. Appl. Pub. No. 2014/0171607; and U. S. Pat. Appl. Pub. No. 2015/0045558.

[0166] The catalyst is typically provided in the reaction mixture in a sub-stoichiometric amount (e.g., catalytic amount). In certain embodiments, that amount is in the range of about 0.001 to about 50 mol % with respect to the limiting reagent of the chemical reaction, depending upon which reagent is in stoichiometric excess. In some embodiments, the catalyst is present in less than or equal to about 40 mol % relative to the limiting reagent. In some embodiments, the catalyst is present in less than or equal to about 30 mol % relative to the limiting reagent. In some embodiments, the catalyst is present in less than about 20 mol %, less than about 10 mol %, less than about 5 mol %, less than about 2.5 mol %, less than about 1 mol %, less than about 0.5 mol %, less than about 0.1 mol %, less than about 0.015 mol %, less than about 0.01 mol %, less than about 0.0015 mol %, or less, relative to the limiting reagent. In some embodiments, the catalyst is present in the range of about 2.5 mol % to about 5 mol %, relative to the limiting reagent. In some embodiments, the reaction mixture contains about 0.5 mol% catalyst. In the case where the molecular formula of the catalyst complex includes more than one metal, the amount of the catalyst complex used in the reaction may be adjusted accordingly.

[0167] In some cases, the methods described herein can be performed in the absence of solvent (e.g., neat). In some cases, the methods can include the use of one or more solvents. Examples of solvents that may be suitable for use in the invention include, but are not limited to, benzene, p-cresol, toluene, xylene, diethyl ether, glycol, diethyl ether, petroleum ether, hexane, cyclohexane, pentane, methylene chloride, chloroform, carbon tetrachloride, dioxane, tetrahydrofuran (THF), dimethyl sulfoxide, dimethylformamide, hexamethyl-phosphoric triamide, ethyl acetate, pyridine, triethylamine, picoline, and the like, as well as mixtures thereof. In some embodiments, the solvent is selected from benzene, toluene, pentane, methylene chloride, and THF. In certain embodiments, the solvent is benzene.

[0168] In some embodiments, the method is performed under reduced pressure. This may be advantageous in cases where a volatile byproduct, such as ethylene, may be produced during the course of the metathesis reaction. For example, removal of the ethylene byproduct from the reaction vessel may advantageously shift the equilibrium of the metathesis reaction towards formation of the desired product. In some embodiments, the method is performed at a pressure of about less than 760 torr. In some embodiments, the method is performed at a pressure of about less than 700 torr. In some embodiments, the method is performed at a pressure of about less than 650 torr. In some embodiments, the method is performed at a pressure of about less than 600 torr. In some embodiments, the method is performed at a pressure of about less than 550 torr. In some embodiments, the method is performed at a pressure of about less than 500 torr. In some embodiments, the method is performed at a pressure of about less than 450 torr. In some embodiments, the method is performed at a pressure of about less than 400 torr. In some embodiments, the method is performed at a pressure of about less than 350 torr. In some embodiments, the method is performed at a pressure of about less than 300 torr. In some embodiments, the method is performed at a pressure of about less than 250 torr. In some embodiments, the method is performed at a pressure of about less than 200 torr. In some embodiments, the method is performed at a pressure of about less than 150 torr. In some embodiments, the method is performed at a pressure of about less than 100 torr. In some embodiments, the method is performed at a pressure of about less than 90 torr. In some embodiments, the method is performed at a pressure of about less than 80 torr. In some embodiments, the method is performed at a pressure of about less than 70 torr. In some embodiments, the method is performed at a pressure of about less than 60 torr. In some embodiments, the method is performed at a pressure of about less than 50 torr. In some embodiments, the method is performed at a pressure of about less than 40 torr. In some embodiments, the method is performed at a pressure of about less than 30 torr. In some embodiments, the method is performed at a pressure of about less than 20 torr. In some embodiments, the method is performed at a pressure of about 20 torr.

[0169] In some embodiments, the method is performed at a pressure of about 19 torr. In some embodiments, the method is performed at a pressure of about 18 torr. In some embodiments, the method is performed at a pressure of about 17 torr. In some embodiments, the method is performed at a pressure of about 16 torr. In some embodiments, the method is performed at a pressure of about 15 torr. In some embodiments, the method is performed at a pressure of about 14 torr. In some embodiments, the method is performed at a pressure of about 13 torr. In some embodiments, the method is performed at a pressure of about 12 torr. In some embodiments, the method is performed at a pressure of about 11 torr. In some embodiments, the method is performed at a pressure of about 10 torr. In some embodiments, the method is performed at a pressure of about 10 torr. In some embodiments, the method is performed at a pressure of about 9 torr. In some embodiments, the method is performed at a pressure of about 8 torr. In some embodiments, the method is performed at a pressure of about 7 torr. In some embodiments, the method is performed at a pressure of about 6 torr. In some embodiments, the method is performed at a pressure of about 5 torr. In some embodiments, the method is performed at a pressure of about 4 torr. In some embodiments, the method is performed at a pressure of about 3 torr. In some embodiments, the method is performed at a pressure of about 2 torr. In some embodiments, the method is performed at a pressure of about 1 torr. In some embodiments, the method is performed at a pressure of less than about 1 torr.

[0170] In some embodiments, the two metathesis reactants are present in equimolar amounts. In some embodiments, the two metathesis reactants are not present in equimolar amounts. In certain embodiments, the two reactants are present in a molar ratio of about 20: 1, 19:1, 18:1, 17:1, 16:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, or 1:20. In certain embodiments, the two reactants are present in a molar ratio of about 10:1. In certain embodiments, the two reactants are present in a molar ratio of about 7:1. In certain embodiments, the two reactants are present in a molar ratio of about 5:1. In certain embodiments, the two reactants are present in a molar ratio of about 2:1. In certain embodiments, the two reactants are present in a molar ratio of about 1:10. In certain embodiments, the two reactants are present in a molar ratio of about 1:7. In certain embodiments, the two reactants are present in a molar ratio of about 1:5. In certain embodiments, the two reactants are present in a molar ratio of about 1 :2.

[0171] In general, the reactions with many of the metathesis catalysts disclosed herein provide yields better than 15%, preferably better than 50%, more preferably better than 75%, and most preferably better than 90%. In addition, the reactants and products are chosen to provide at least a 5°C difference, preferably a greater than 20°C difference, and most preferably a greater than 40°C difference in boiling points. Additionally, the use of metathesis catalysts allows for much faster product formation than byproduct, it is desirable to run these reactions as quickly as practical. In particular, the reactions are performed in less than about 24 hours, preferably less than 12 hours, more preferably less than 8 hours, and most preferably less than 4 hours.

[0172] One of skill in the art will appreciate that the time, temperature and solvent can depend on each other, and that changing one can require changing the others to prepare the pyrethroid products and intermediates in the methods of the invention. The metathesis steps can proceed at a variety of temperatures and times. In general, reactions in the methods of the invention are conducted using reaction times of several minutes to several days. For example, reaction times of from about 12 hours to about 7 days can be used. In some embodiments, reaction times of 1-5 days can be used. In some embodiments, reaction times of from about 10 minutes to about 10 hours can be used. In general, reactions in the methods of the invention are conducted at a temperature of from about 0 °C to about 200 °C. For example, reactions can be conducted at 15-100 °C. In some embodiments, reaction can be conducted at 20-80 °C. In some embodiments, reactions can be conducted at 100-150 °C.

Pheromone Compositions and Uses Thereof [0173] As described above, many of the oxy-functionalized products made via the methods described herein are pheromones. Pheromones prepared according to the methods of the invention can be formulated for use as insect control compositions. The pheromone compositions can include a carrier, and/or be contained in a dispenser. The carrier can be, but is not limited to, an inert liquid or solid. [0174] Examples of solid carriers include but are not limited to fillers such as kaolin, bentonite, dolomite, calcium carbonate, talc, powdered magnesia, Fuller's earth, wax, gypsum, diatomaceous earth, rubber, plastic, silica and China clay. Examples of liquid carriers include, but are not limited to, water; alcohols, such as ethanol, butanol or glycol, as well as their ethers or esters, such as methylglycol acetate; ketones, such as acetone, cyclohexanone, methylethyl ketone, methylisobutylketone, or isophorone; alkanes such as hexane, pentane, or heptanes; aromatic hydrocarbons, such as xylenes or alkyl naphthalenes; mineral or vegetable oils; aliphatic chlorinated hydrocarbons, such as trichloroethane or methylene chloride; aromatic chlorinated hydrocarbons, such as chlorobenzenes; water- soluble or strongly polar solvents such as dimethylformamide, dimethyl sulfoxide, or N- methylpyrrolidone; liquefied gases; and mixtures thereof. Baits or feeding stimulants can also be added to the carrier. [0175] Pheromone compositions can be formulated so as to provide slow release into the atmosphere, and/or so as to be protected from degradation following release. For example, the pheromone compositions can be included in carriers such as microcapsules, biodegradable flakes and paraffin wax-based matrices. [0176] Pheromone compositions can contain other pheromones or attractants provided that the other compounds do not substantially interfere with the activity of the composition. The pheromone compositions can also include insecticides. Examples of suitable insecticides include, but are not limited to, buprofezin, pyriproxyfen, flonicamid, acetamiprid, dinotefuran, clothianidin, acephate, malathion, quinolphos, chloropyriphos, profenophos, bendiocarb, bifenthrin, chlorpyrifos, cyfluthrin, diazinon, pyrethrum, fenpropathrin, kinoprene, insecticidal soap or oil, and mixtures thereof.

[0177] Pheromone compositions can be used in conjunction with a dispenser for release of the composition in a particular environment. Any suitable dispenser known in the art can be used. Examples of such dispensers include but are not limited to bubble caps comprising a reservoir with a permeable barrier through which pheromones are slowly released, pads, beads, tubes rods, spirals or balls composed of rubber, plastic, leather, cotton, cotton wool, wood or wood products that are impregnated with the pheromone composition. For example, polyvinyl chloride laminates, pellets, granules, ropes or spirals from which the pheromone composition evaporates, or rubber septa. One of skill in the art will be able to select suitable carriers and/or dispensers for the desired mode of application, storage, transport or handling.

[0178] A variety of pheromones, including those set forth in Table 1 can be prepared according to the methods of the invention and formulated as described above. For example, the methods of the invention can be used to prepare peach twig borer (PTB) sex pheromone, which is a mixture of (E)-dec-5-en-l-ol (17%) and (E)-dec-5-en-l-yl acetate (83%). The PTB sex pheromone can be used in conjunction with a sustained pheromone release device having a polymer container containing a mixture of the PTB sex pheromone and a fatty acid ester (such as a sebacate, laurate, palmitate, stearate or arachidate ester) or a fatty alcohol (such as undecanol, dodecanol, tridecanol, tridecenol, tetradecanol, tetradecenol, tetradecadienol, pentadecanol, pentadecenol, hexadecanol, hexadecenol, hexadecadienol, octadecenol and octadecadienol). The polymer container can be a tube, an ampule, or a bag made of a polyolefin or an olefin component-containing copolymer. Sex pheromones of other pest insects such the cotton bollworm (Helicoverpa armigera), fall army worm (Spodoptera frugiperda), oriental fruit moth (Grapholita molesta) and leaf roller (Tortricidae) can be used in this type of sustained pheromone release device. The sex pheromones typically include one or more aliphatic acetate compounds having from 10 to 16 carbon atoms (e.g., decyl acetate, decenyl acetate, decadienyl acetate, undecyl acetate, undecenyl acetate, dodecyl acetate, dodecenyl acetate, dodecadienyl acetate, tridecyl acetate, tridecenyl acetate, tridecadienyl acetate, tetradecyl acetate, tetradecenyl acetate, tetradecadienyl acetate, and the like) and/or one or more aliphatic aldehyde compounds having from 10 to 16 carbon atoms (e.g., 7-hexadecenal, 11-hexadecenal, 13-octadecenal, and the like). [0179] Pheromones prepared according to the methods of the invention, as well as compositions containing the pheromones, can be used to control the behavior and/or growth of insects in various environments. The pheromones can be used, for example, to attract or repel male or female insects to or from a particular target area. The pheromones can be used to attract insects away from vulnerable crop areas. The pheromones can also be used example to attract insects as part of a strategy for insect monitoring, mass trapping, lure/attract-and-kill or mating disruption.

[0180] Mass trapping involves placing a high density of traps in a crop to be protected so that a high proportion of the insects are removed before the crop is damaged. Lure/attract- and-kill techniques are similar except once the insect is attracted to a lure, it is subjected to a killing agent. Where the killing agent is an insecticide, a dispenser can also contain a bait or feeding stimulant that will entice the insects to ingest an effective amount of the insecticide.

[0181] It will be appreciated by a person skilled in the art that a variety of different traps are possible. Suitable examples of such traps include water traps, sticky traps, and one-way traps. Sticky traps come in many varieties. One example of a sticky trap is of cardboard construction, triangular or wedge-shaped in cross-section, where the interior surfaces are coated with a non-drying sticky substance. The insects contact the sticky surface and are caught. Water traps include pans of water and detergent that are used to trap insects. The detergent destroys the surface tension of the water, causing insects that are attracted to the pan, to drown in the water. One-way traps allow an insect to enter the trap but prevent it from exiting. The traps of the invention can be colored brightly, to provide additional attraction for the insects. [0182] The trap is positioned in an area infested (or potentially infested) with insects. Generally, the trap is placed on or close to a tree or large plant. The aroma of the pheromone attracts the insects to the trap. The insects can then be caught, immobilized and/or killed within the trap, for example, by the killing agent present in the trap. [0183] Pheromones prepared according to the methods of the invention can also be used to disrupt mating. Strategies of mating disruption include confusion, trail-masking and false- trail following. Constant exposure of insects to a high concentration of a pheromone can prevent male insects from responding to normal levels of the pheromone released by female insects. Trail-masking uses a pheromone to destroy the trail of pheromones released by females. False-trail following is carried out by laying numerous spots of a pheromone in high concentration to present the male with many false trails to follow. When released in sufficiently high quantities, the male insects are unable to find the natural source of the sex pheromones (the female insects) so that mating cannot occur.

[0184] Insect populations can be surveyed or monitored by counting the number of insects in a target area (e.g., the number of insects caught in a trap). Inspection by a horticulturist can provide information about the life stage of a population. Knowing where insects are, how many of them there are, and their life stage enables informed decisions to be made as to where and when insecticides or other treatments are warranted. For example, a discovery of a high insect population can necessitate the use of methods for removal of the insect. Early warning of an infestation in a new habitat can allow action to be taken before the population becomes unmanageable. Conversely, a discovery of a low insect population can lead to a decision that it is sufficient to continue monitoring the population. Insect populations can be monitored regularly so that the insects are only controlled when they reach a certain threshold. This provides cost-effective control of the insects and reduces the environmental impact of the use of insecticides.

[0185] As will be apparent to one of skill in the art, the amount of a pheromone or pheromone composition used for a particular application can vary depending on several factors such as the type and level of infestation; the type of composition used; the concentration of the active components; how the composition is provided, for example, the type of dispenser used; the type of location to be treated; the length of time the method is to be used for; and environmental factors such as temperature, wind speed and direction, rainfall and humidity. Those of skill in the art will be able to determine an effective amount of a pheromone or pheromone composition for use in a given application.

EXAMPLES

[0186] The present invention will be described in greater detail by way of specific examples. The following examples are offered for illustrative purposes, and are not intended to limit the invention in any manner. Those of skill in the art will readily recognize a variety of noncritical parameters which can be changed or modified to yield essentially the same results.

Example 1. Utilization of oleic acid for synthesis of pheromones

[0187] Scheme 12 shows an example of how oleic acid (R = H) can be used for synthesis of a Yellow Stem Borer's pheromone component.

Scheme 12. Synthesis of heromones via the use of oleic acid as starting material

Pheromones:Yellow stem borer

[0188] In this process, oleic acid or its ester derivative is first terminally oxygenated through the use of hydroxylase enzyme systems, such as P450s, to provide the desired co- hydroxy oleic acid, which upon esterification and protection of the hydroxyl functional group, can be subjected to cross-metathesis with alkenes of different hydrocarbon chain lengths for synthesis of various pheromones. For instance, ω-hydroxy-protected methyl oleate can be coupled with Z-9-octadecene for the synthesis of Z-9-octadecen-l-al, a Yellow Stem Borer's pheromone component (Insect pheromones and their use in pest management by Howse, P. et al. 1998, Publisher: Chapman & Hall, p. 145).

[0189] Similarly, co-hydroxy-protected methyl oleate can be coupled with Z-7-tetradecene to provide Z-9-hexadecen-l-ol that can be readily oxidize to Z-9-hexadecenal, a component of Corn Earworm's pheromone.

[0190] Other natural fatty acids, such as Z-l l-octadecenoic, Z-13-octadecenoic, Z-l l- hexadecenoic, as well as unnatural fatty acids that can be synthesized through chemical means can also be used for the synthesis of other pheromones.

[0191] Metathesis of methyl acrylate with terminal alkenes is a practical approach for making unsaturated fatty esters that can be biohydroxylated to generate desirable hydroxyl acids that can be used for synthesis of insect pheromones as illustrated in Scheme 13. One of the advantages of this process is that shorter biohydroxylation substrates can be made quickly and inexpensively.

Scheme 13. Utilization of methyl acrylate for synthesis of insect pheromones

Example 2. Utilization of methyl decanoate for synthesis of pheromones

[0192] Scheme 14 shows an example of a saturated methyl ester, methyl decanoate, can be used for synthesis of pheromone Z9-tetradecenyl acetate.

Scheme 10. Synthesis of pheromones via the use of a saturated methyl ester as starting material

Z-Selective

Metathesis

- e ra eceny ace a e

[0193] In this process, methyl decanoate is first terminally oxygenated through the use of hydroxylase enzyme systems, such as P450s, to provide the desired product, methyl 10- hydroxydecanoate, that can be easily converted to its corresponding methyl ester by subjecting the said acid to an acid-catalyzed methanolysis process. Dehydration of methyl 10- hydroxydecanoate can be carried out by first treating the ester with phosphorous tribromide to generate terminally brominated ester, which is then treated with sodium iodide in hexamethylphosphoramide (HMPA) at 170 °C to give the corresponding terminal alkene ester, methyl 9-decenoate. Coupling of methyl 9-decenoate with l-hexene via a Z-selective metathesis process provides methyl Z9-tetradecenoate, which is then converted to its corresponding alcohol utilizing sodium bis(2-methoxyethoxy)aluminum dihydride (Red-Al) as a reducing agent. The resulting Z9-tetradecen-l-ol is then esterified with acetic anhydride to afford the desired product, Z9-tetradecenyl acetate.

[0194] Although the foregoing has been described in some detail by way of illustration and example for purposes of clarity and understanding, one of skill in the art will appreciate that certain changes and modifications can be practiced within the scope of the appended claims. All publications, patents, patent applications, and sequence accession numbers cited herein are hereby incorporated by reference in their entirety for all purposes.

Claims

WHAT IS CLAIMED IS: 1. A method for synthesizing an oxy-functionalized product, the method comprising:

incubating an enzyme substrate with an enzyme capable of hydroxylating one terminal carbon of the enzyme substrate to form a hydroxylated product; and

converting at least a portion of the hydroxylated product to the oxy- functionalized product; wherein

the enzyme substrate is a carboxylic acid or an ester thereof, and the hydroxylated product is a terminal hydroxy-substituted carboxylic acid or an ester thereof.

2. The method of claim 1, wherein the enzyme substrate is an unsaturated carboxylic acid or an ester thereof, and the hydroxylated product is a terminal hydroxy- substituted unsaturated carboxylic acid or an ester thereof.

3. The method of claim 2, wherein the unsaturated carboxylic acid comprises from about 4 to about 22 carbon atoms.

4. The method of claim 3, wherein the unsaturated carboxylic acid comprises from about 8 to about 20 carbon atoms.

5. The method of claim 2, comprising:

incubating an enzyme substrate according to formula I

(I),

with an enzyme capable of hydroxylating the terminal carbon of the enzyme substrate to form a hydroxylation product according to formula II

(ii); and

converting at least a portion of the hydroxylation product to the oxy- functionalized product.

6. The method of claim 5, wherein converting the hydroxylation product to the oxy-functionalized product comprises:

metathesizing the hydroxylation product and a terminal olefin according to formula III ^^e (HI),

wherein e is an integer ranging from 0 to 17,

7. The method of claim 5, further comprising:

protecting the hydroxylation product to form a protected hydroxylation product according to formula Ila

(Ha),

wherein R² is an alcohol protecting group;

metathesizing the protected hydroxylation product and a terminal olefin according to formula III ^^e (HI),

wherein e is an integer ranging from 0 to 17,

8. The method of claim 5, wherein converting the hydroxylation product to the oxy-functionalized product comprises:

cyclizing the hydroxylation product to form a lactone according to formula lib

wherein e is an integer ranging from 0 to 17,

in the presence of a metathesis catalyst to form an ester according to formula IVb:

hydrolyzing the ester to form an olefinic alcohol according to formula IV:

(IV).

9. The method of claim 5, wherein converting the hydroxylation product to the oxy-functionalized product comprises:

cyclizing the hydroxylation product to form a lactone according to formula lib

metathesizing the lactone and a terminal olefin according to formula III

(in),

wherein e is an integer ranging from 0 to 17,

reducing the ester to form an olefinic alcohol according to formula IV:

10. The method of any one of claims 6-9, wherein the olefinic alcohol is the oxy-functionalized product.

11. The method of any one of claims 6-9, further comprising oxidizing the olefinic alcohol to form the oxy-functionalized product, wherein the oxy-functionalized product has a structure according to formula V:

12. The method of any one of claims 6-9, further comprising acylating the olefinic alcohol to form the oxy-functionalized product, wherein the oxy-functionalized product has a s ula VI:

(VI),

wherein R³ is selected from the group consisting of H, Ci_-6 alkyl, and C_6-io aryl.

13. The method of claim 12, wherein R³ is selected from the group consisting of H and methyl.

14. The method of claim 12, wherein R³ is methyl.

15. The method of claim 7, wherein metathesizing the protected hydroxylation product according to formula Ila and the terminal olefin according to formula III comprises formin an ester according to formula IVc:

16. The method of claim 15, further comprising reducing the ester according to formula IVc to form the oxy-functionalized product, wherein the oxy- functionalized product has a structure according to formula V:

17. The method of claim 15, further comprising reducing the ester according to formula IVc to form an olefinic alcohol according to formula IV:

18. The method of claim 17, further comprising acylating the olefinic alcohol to form the oxy-functionalized product, wherein the oxy-functionalized product has a structure according to formula VI:

(VI),

wherein R³ is selected from the group consisting of H, Ci_-6 alkyl, and C₆-io aryl.

19. The method of claim 18, wherein R³ is selected from the group consisting of H and methyl.

20. The method of claim 18, wherein R³ is methyl.

21. The method of claim 8, wherein hydrolyzing the ester according to formula IVb comprises forming an acid according to formula IVd:

O b^e (IVd).

22. The method of claim 21, further comprising reducing the acid according to formula IVd to form the oxy-functionalized product, wherein the oxy- functionalized product has a structure according to formula V: '^¾/¾^{^}=^=/¾H (v).

23. The method of claim 21, further comprising reducing the acid according to formula IVd to form an olefinic alcohol according to formula IV:

^^^H _(IV)

24. The method of claim 23, further comprising acylating the olefinic alcohol to form the oxy-functionalized product, wherein the oxy-functionalized product has a structure according to formula VI:

wherein R³ is selected from the group consisting of H, Ci_-6 alkyl, and

C₆-io aryl.

25. The method of claim 24, wherein R³ is selected from the group consisting of H and methyl.

26. The method of claim 24, wherein R³ is methyl.

27. The method of claim 9, wherein reducing the ester according to formula IVb comprises forming an aldehyde according to formula IVe:

(IVe).

28. The method of claim 27, further comprising reducing the aldehyde according to formula IVe to form an olefinic alcohol according to formula Va:

29. The method of claim 28, further comprising acylating the olefinic alcohol accordin to formula Va to form an ester according to formula Via:

wherein R³ is selected from the group consisting of H, Ci_-6 alkyl, and

C₆-io aryl.

30. The method of claim 29, wherein R³ is selected from the group consisting of H and methyl.

31. The method of claim 29, wherein R³ is methyl.

32. The method of claim 5, wherein converting the hydroxylation product to the oxy-functionalized product comprises:

protecting the hydroxylation product according to formula II to form a protected hydroxylation product according to formula Ila O (Ha),

wherein R² is an alcohol protecting group;

metathesizing the protected hydroxylation product and an internal olefin according to formula VII "c ^C H (VII)

wherein each c is independently selected from integers ranging from 0 to 15, in the presence of a metathesis catalyst to form a protected olefinic alcohol according to formula LIVa: ^^"^^H (LIVa); and

33. The method of claim 5, wherein converting the hydroxylation product to the oxy-functionalized product comprises:

(lib),

metathesizing the lactone and an internal olefin according to formula VII "c ^C H ₍vi_l)

wherein each c is independently selected from integers ranging from 0 to 15, in the presence of a metathesis catalyst to form an ester according to formula LIVb:

hydrolyzing the ester to form an olefinic alcohol according to formula IV:

34. The method of claim 5, wherein converting the hydroxylation product to the oxy-functionalized product comprises:

(lib),

metathesizing the lactone and an internal olefin according to formula VII C H (VII)

reducing the ester to form an olefinic alcohol according to formula IV:

35. The method of any one of claims 32-34, wherein the olefinic alcohol according to formula LIV is the oxy-functionalized product.

36. The method of any one of claims 32-34, further comprising oxidizing the olefinic alcohol according to formula LIV to form the oxy-functionalized product, wherein the ox -functionalized product is an aldehyde according to formula LV:

37. The method of any one of claims 32-34, further comprising oxidizing the olefinic alcohol according to formula LIV to form the oxy-functionalized product, wherein the oxy-functionalized product is an ester according to formula LVI:

wherein R³ is selected from the group consisting of H, Ci_-6 alkyl, and

C_6-io aryl.

38. The method of claim 37, wherein R³ is selected from the group consisting of H and methyl.

39. The method of claim 37, wherein R³ is methyl.

40. The method of claim 32, wherein metathesizing the protected hydroxylation product according to formula Ila and the internal olefin according to formula VII comprises formin an ester according to formula LIVc:

41. The method of claim 40, further comprising reducing the ester according to formula LIVc to form the oxy-functionalized product, wherein the oxy- functionalized product has a structure according to formula LV:

°^ = ^_{H (LV)}

42. The method of claim 40, further comprising reducing the ester according to formula LIVc to form an olefinic alcohol according to formula LIV:

43. The method of claim 42, further comprising acylating the olefinic alcohol to form the oxy-functionalized product, wherein the oxy-functionalized product has a structure accordin to formula LVI:

wherein R is selected from the group consisting of H, Ci_-6 alkyl, and C₆-io aryl.

44. The method of claim 43, wherein R³ is selected from the group consisting of H and methyl.

45. The method of claim 43, wherein R³ is methyl.

46. The method of claim 33, wherein hydrolyzing the ester according to formula LIVb comprises forming an acid according to formula LlVd:

47. The method of claim 46, further comprising reducing the acid according to formula LlVd to form the oxy-functionalized product, wherein the oxy- functionalized product has a structure according to formula LV: ^^=^ H (LV).

48. The method of claim 46, further comprising reducing the acid according to formula LlVd to form an olefinic alcohol according to formula LIV:

49. The method of claim 48, further comprising acylating the olefinic alcohol to form the oxy-functionalized product, wherein the oxy-functionalized product has a structure according to formula LVI:

(LVI),

50. The method of claim 49, wherein R³ is selected from the group consisting of H and methyl.

51. The method of claim 49, wherein R³ is methyl.

52. The method of claim 34, wherein reducing the ester according to formula IVb c mprises forming an aldehyde according to formula LV:

53. The method of claim 52, further comprising reducing the aldehyde according to formula LV to form an olefinic alcohol according to formula LIV:

54. The method of claim 53, further comprising acylating the olefinic alcohol accordin to formula LIV to form an ester according to formula VI:

wherein R³ is selected from the group consisting of H, Ci_-6 alkyl, and

C₆-io aryl.

55. The method of claim 54, wherein R³ is selected from the group consisting of H and methyl.

56. The method of claim 54, wherein R³ is methyl.

57. The method of claim 5, wherein converting the hydroxylation product to the oxy-functionalized product comprises:

metathesizing the hydroxylation product and an olefin according to formula 3 9 h i J ₍₃

HO

a h i ^J ₍₄

58. The method of claim 5, wherein converting the hydroxylation product to the oxy-functionalized product comprises:

(Ha),

wherein R² is an alcohol protecting group;

metathesizing the protected hydroxylation product and an olefin according to formula 3 9 h i J ₍₃

(4a); and

HO

a h i ^J ₍₄₎ 59. The method of claim 5, wherein converting the hydroxylation product to the oxy-functionalized product comprises:

cyclizing the hydroxylation product to form a lactone to formula lib

(lib),

metathesizing the lactone and an olefin according to formula 3 9 h i J ₍₃₎ wherein i is an integer ranging from 1 to 10 and g, h, and j are independently- selected integers ranging from 0 to 15,

in the presence of a metathesis catalyst to form an ester according to formula 4b:

HO

a h i ^J ₍₄₎ 60. The method of any one of claims 57-59, wherein the olefinic alcohol is the oxy-functionalized product.

61. The method of any one of claims 57-59, further comprising oxidizing the olefinic alcohol to form the oxy-functionalized product, wherein the oxy-functionalized product has a structure according to formula 5 : a h i ^J ₍₅₎

62. The method of any one of claims 57-59, further comprising acylating the olefinic alcohol to form the oxy-functionalized product, wherein the oxy-functionalized product has a structure according to formula 6:

63. The method of claim 62, wherein R³ is selected from the group consisting of H and methyl.

64. The method of claim 2, comprising:

metathesizing an enzyme substrate precursor according to formula I

(I),

and an olefin according to formula VII: "c C H _(VII)

incubating the enzyme substrate with an enzyme capable of hydroxylating the terminal carbon of the enzyme substrate to form a hydroxylation product according to formula IX

65. The method of claim 2, comprising:

metathesizing an enzyme substrate precursor according to formula I

(I),

and an olefin according to formula X: d (X)

wherein d is an integer ranging from 0 to 17,

(xi); and incubating the enzyme substrate with an enzyme capable of hydroxylating the terminal carbon of the enzyme substrate to form a hydroxylation product according to formula XII

66. The method of claim 64, wherein converting the hydroxylation product to the oxy-functionalized product comprises:

metathesizing the hydroxylation product according to formula IX and a terminal olefin according to formula III ^^e (HI),

wherein e is an integer ranging from 0 to 17,

67. The method of claim 64, further comprising:

protecting the hydroxylation product according to formula IX to form a protected hydroxylation product according to formula IXa

(IXa),

wherein R² is an alcohol protecting group;

wherein e is an integer ranging from 0 to 17,

in the presence of a metathesis catalyst to form a protected olefinic alcohol according to formula XHIa: K ° ^{c e H} (XHIa); and

68. The method of claim 64, further comprising:

cyclizing the hydroxylation product according to formula IX to form a lactone according to formula IXb

metathesizing the lactone and a terminal olefin according to formula III ^^e (HI),

wherein e is an integer ranging from 0 to 17,

in the presence of a metathesis catalyst to form an ester according to formula

Xlllb:

(XIIIb); and

hydrolyzing the ester to form an olefinic alcohol according to formula XIII:

I I

H_\nO^ c~ Ή _E--.. H _(XM)

69. The method of claim 65, wherein converting the hydroxylation product to the oxy-functionalized product comprises:

metathesizing the hydroxylation product according to formula XII and a terminal olefin according to formula III ^^e (HI),

wherein e is an integer ranging from 0 to 17,

70. The method of claim 65, wherein converting the hydroxylation product to the oxy-functionalized product comprises: protecting the hydroxylation product according to formula XII to form a protected hydroxylation product according to formula Xlla

(Xlla),

wherein R² is an alcohol protecting group;

wherein e is an integer ranging from 0 to 17,

in the presence of a metathesis catalyst to form a protected olefinic alcohol according to formula XlVa: R ° ^{d e H} (XlVa); and

71. The method of claim 65, wherein converting the hydroxylation product to the oxy-functionalized product comprises:

(Xllb),

wherein e is an integer ranging from 0 to 17,

in the presence of a metathesis catalyst to form an ester according to formula XlVb:

(xivb); and hydrolyzing the ester to form an olefmic alcohol according to formula IV:

72. The method of any one of claims 66-71, wherein the olefmic alcohol is the oxy-functionalized product. 73. The method of any one of claims 66-68, further comprising oxidizing the olefmic alcohol according to formula XIII to form the oxy-functionalized product, wherein the oxy-functionalized product has a structure according to formula XV: * ^=^ H _(xv) 74. The method of any one of claims 66-68, further comprising acylating the olefmic alcohol according to Formula XIII to form the oxy-functionalized product, wherein the oxy-functionalized product has a structure according to formula XVI:

O R O c e _{H (X}vi

75. The method of claim 74, wherein R³ is selected from the group consisting of H and methyl.

76. The method of any one of claims 69-71, further comprising oxidizing the olefmic alcohol according to formula XIV to form the oxy-functionalized product, wherein the oxy-functionalized product has a structure according to formula XVII: cT e H ₍xvi_l).

77. The method of any one of claims 69-71, further comprising acylating the olefmic alcohol according to formula IV to form the oxy-functionalized product, wherein the oxy-functionalized product has a structure according to formula XVIII:

O R O _d e _{H (}xvi_ll), wherein R is selected from the group consisting of H, Ci_-6 alkyl, and

C₆-io aryl.

78. The method of claim 77, wherein R³ is selected from the group consisting of H and methyl.

79. The method of any one of claims 2-78, wherein the unsaturated fatty acid or ester thereof is selected from the group consisting of E-5-decenoic acid; Z-5-decenoic acid; Z-l 1-octadecenoic acid; Z-13-octadecenoic acid; E-l 1 -hexadecenoic acid; Z-l 1- hexadecenoic acid; Z-8-dodecanoic acid; (Z,Z)-11,13-hexadecadienoic acid; 2E,4Z,7Z- decatrienoic acid; E8,E10-dodecanoic acid; (E,Z)-3,13-octadecadienoic acid; (E,Z)-2,13- octadecadienoic acid; and E/Z-l 1-tetradecenoic acid.

80. The method of claim 79, wherein the unsaturated fatty acid or ester thereof is selected from Z-l 1-octadecenoic acid, Z-13-octadecenoic acid, and Z-l 1- hexadecenoic acid.

81. The method of claim 1, wherein the enzyme substrate is a saturated carboxylic acid or an ester thereof, and the hydroxylated product is a terminal hydroxy- substituted saturated carboxylic acid or an ester thereof.

82. The method of claim 81, wherein the saturated carboxylic acid comprises from about 4 to about 22 carbon atoms.

83. The method of claim 82, wherein the saturated carboxylic acid comprises from about 8 to about 20 carbon atoms.

84. The method of claim 81, comprising:

incubating an enzyme substrate according to formula XIX

Π ^x

0 (XIX),

wherein x is an integer ranging from 0 to 22, and R⁴ is H, Ci_-6 alkyl, or C₆-io aryl,

85. The method of claim 84, wherein converting the hydroxylation product to the oxy-functionalized product comprises:

Π ^x

° (XXI);

metathesizing the terminal olefin and an alkene according to formula XXII ^ (XXII),

wherein y and z are each independently integers ranging from 0 to 18, and R⁵ is H, Ci-₁₆ alkyl, Ci.i₆ alkenyl, or Ci.i₆ alkynyl,

in the presence of a metathesis catalyst to form an olefinic acid according to formula XXIII x— ^{y R}

0 (XXIII); and

reducing the olefinic acid to form an olefinic alcohol according to formula XXIV x y K (_XXIV).

86. The method of claim 84, wherein converting the hydroxylation product to the oxy-functionalized product comprises:

esterifying the hydroxylation product to form an ester according to formula XXV

^R6 H

0 (XXV),

wherein R⁶ is H, Ci_-6 alkyl, or C₆-₁₀ aryl;

dehydrating the esterification product to form a terminal olefin according to formula XXVI: ^υ (XXVI);

metathesizin the terminal olefin and an alkene according to formula XXII

Y x y ^K

0 (XXVII); and

reducing the compound of formula XXVII to form an olefinic alcohol according to formula XXIV x y K (_XXIV)

87. The method of claim 86, wherein R⁶ is selected from the group consisting of H and methyl.

88. The method of any of claims 85-87, wherein the olefinic alcohol is the oxy-functionalized product.

89. The method of any of claims 85-87, further comprising oxidizing the olefinic alcohol to form the oxy-functionalized product, wherein the oxy-functionalized product has a structure according to formula XXVIII: x^y (XXVIII).

90. The method of any of claims 85-87, further comprising acylating the olefinic alcohol to form the oxy-functionalized product, wherein the oxy-functionalized product has a s ula XXIX:

(XXIX),

wherein R⁷ is selected from the group consisting of H, Ci_-6 alkyl, and C_6-io aryl.

91. The method of any one of claims 81-90, wherein the saturated fatty acid or ester thereof is selected from the group consisting of decanoic acid, tetradecanoic acid, hexadecanoic acid, and octadecanoic acid.

92. The method of claim 91, wherein the saturated fatty acid or ester thereof is decanoic acid.

93. The method of any one of claims 5-78 or 84-90, wherein R¹ is H.

94. The method of any one of claims 5-78 and 84-90, wherein R¹ is

Ci-6 alkyl.

95. The method of claim 94, wherein R¹ is methyl.

96. The method of claim 2 or 81, further comprising converting at least a portion of the hydroxylated product to reconstituted enzyme substrate.

97. The method of claim 96, further comprising recycling the reconstituted enzyme substrate.

98. The method of any one of claims 6-8, 57-59, 64-71, 85, and 86 wherein the metathesis catalyst is a Z-selective metathesis catalyst.

99. The method of any one of claims 2-80, wherein the unsaturated fatty acid or ester thereof is obtained from a source material selected from the group consisting of soybean oil, modified soybean oil, cottonseed oil, peanut oil, sunflower seed oil, canola oil, modified canola oil, rapeseed oil, sesame seed oil, corn oil, olive oil, palm oil, palm kernel oil, coconut oil, butter, lard, fish oil, linseed oil, castor seed oil, and tallow.

100. The method of claim 99, wherein the source material is selected from the group consisting of coconut oil, canola oil, cottonseed oil, linseed oil, olive oil, palm oil, palm kernel oil, peanut oil, sesame seed oil, soybean oil, and sunflower seed oil.

101. The method of any one of claims 2-100, wherein the enzyme is a non- heme diiron monooxygenase.

102. The method of claim 101, wherein the non-heme diiron monooxygenase is selected from Table 7 or a variant thereof having at least 90% identity thereto.

103. The method of any one of claims 2-100, wherein the enzyme is a long- chain alkane hydroxylase.

104. The method of claim 103, wherein the long-chain alkane hydroxylase is selected from Table 8 or a variant thereof having at least 90% identity thereto.

105. The method of any one of claims 2-100, wherein the enzyme is a cytochrome P450.

106. The method of claim 105, wherein the cytochrome P450 is selected from Table 9 or a variant thereof having at least 90% identity thereto.

107. The method of claim 105 or claim 106, wherein the cytochrome P450 is a member of the CYP52 or CYP153 family.

108. The method of any one of claims 2-107, wherein the oxy- functionalized product is selected from the compounds set forth in Table 1.