Brazilian Journal of Microbiology (2007) 38:52-57
ISSN 1517-8283
ISOLATION OF RECOMBINANT STRAINS WITH ENHANCED PECTINASE PRODUCTION BY
PROTOPLAST FUSION BETWEEN PENICILLIUM EXPANSUM AND PENICILLIUM
GRISEOROSEUM
Maurilio Antonio Varavallo1; Marisa Vieira de Queiroz2*; Taís Guimarães Lana2; Admilson Toscano Ribeiro de Brito3;
Daniel Bonoto Gonçalves2; Elza Fernandes de Araújo2
1
Faculdade de Biomedicina, Centro Universitário das Faculdades Metropolitanas Unidas, São Paulo-SP, Brazil; 2Departamento
de Microbiologia/BIOAGRO, Universidade Federal de Viçosa, Viçosa-MG, Brazil; 3CODAI, Universidade Federal Rural de
Pernambuco, Recife-PE
Submitted: February 07, 2006; Returned to authors for corrections: March 30, 2006; Approved: October 13, 2006
ABSTRACT
Protoplast fusion between complementary auxotrophic and morphological mutant strains of Penicillium
griseoroseum and P. expansum was induced by polyethylene glycol and calcium ions (Ca2+). Fusant strains
were obtained in minimal medium and a prototrophic strain, possibly diploid, was chosen for haplodization
with the fungicide benomyl. Different recombinant strains were isolated and characterized for occurrence of
auxotrophic mutations and pectinolytic enzyme production. The fusant prototrophic did not present higher
pectinase production than the parental strains, but among 29 recombinants analyzed, four presented enhanced
enzyme activities. The recombinant RGE27, which possesses the same auxotrophic and morphologic mutations
as the P. griseoroseum parental strain, presented a considerable increase in polygalacturonase (3-fold) and
pectin lyase production (1.2-fold).
Key words: Protoplast fusion, pectinases, Penicillium
INTRODUCTION
Since the parasexual cycle was first discovered by
Pontecorvo and Roper (31), it has been described in important
fungal species (6). From a biotechnological point of view, the
parasexual cycle is of great importance for the improvement of
fungi of industrial interest since most of these fungi do not
have a sexual cycle. The parasexual cycle has successfully been
applied in enzyme producing fungi, antibiotics and has been
used for improvement of biocontrol efficiency in the genus
Beauveria (9,21,32,40,44).
The parasexual cycle initiates with heterokaryose, occurring
through anastomosis of vegetative cells; followed by nuclear
fusion, which gives rise to heterozygous diploids putatives
and terminates with recombinant production by mitotic
recombination and haplodization (6,10,18,19,44). Spontaneous
anastosis between different species can be hindered by the
vegetative incompatibility among different strains. The
incompatibility can be overcome by use of the protoplast fusion
technique, which allows for production of haploid recombinants
with the desired characteristics of the parental species.
Interspecific protoplast fusion has already been described for
several fungi, including Aspergillus nidulans with A. fumigatus
(16), A. nidulans with A. rugulosus (22), A. oryzae with A. sojae
(42), Aspergillus sp. with A. flavipes (38), Volvariella volvacea
with V. bombycina (46) and Beauveria bassiana with B.
sulfurescens (13,44).
The spontaneous parasexual cycle in the Penicillium genus
has been described for the species P. chrysogenum (30), P.
expansum (8), P. italicum and P. digitatum (39), and P. roqueforti
(15), while the induced parasexual cycle in P. griseoroseum was
described by Santos (37). Fusions of interspecific protoplasts
were obtained between P. chrysogenum and P. notatum (3) and
between P. chrysogenum and P. roqueforti (4).
*Corresponding Author. Mailing address: Departamento de Microbiologia/BIOAGRO, Universidade Federal de Viçosa, Viçosa-MG. Cep: 36570-000,
Brazil. Tel.: (31) 3899-2553 ou (31) 3899-2573. E-mail: mvqueiro@ufv.br
52
Isolation of pectinase producing recombinants
Pectinolytic enzymes are of great commercial interest in the
food industry for clarification of fruit juices and wines, for
ripening cacao and coffee, for processing of conserved fruit
(12,25). These enzymes are also important in the textile industry,
since pectic substances contained in the middle lamella and
primary cell wall can be softened with enzymes in order to liberate
the cellulose fibers from the plant cell wall (7,41,45).
Several genetic studies have been carried out in our
laboratories in order to sequence the structural and regulatory
genes of the pectinolytic complex, and to isolate strains with
enhanced pectinolytic enzyme activities. Different methodologies
which have been used to achieve these goals include protoplast
production and regeneration, mutant isolation and
characterization, protoplast transformation, construction of a
genomic library and isolation and characterization of genes coding
polygalacturonases in P. griseoroseum and P. expansum
(14,17,33,34,35).
The fusion of interspecific protoplasts to genetically
enhance pectinase production has only been described for the
Aspergillus genus (38), in which fusion of A. flavipes with
Aspergillus sp. protoplasts, resulted in diploids putatives with
increased pectinase production.
The objective of this study was to carry out protoplast fusion
between P. expansum and P. griseoroseum to obtain recombinant
strains with increased pectinolytic enzyme activity.
MATERIAL AND METHODS
Microrganisms
The wild type strains of Penicillium griseoroseum (PG) and
P. expansum (PE) used in this study were isolated from forest
seeds by Dr. James J. Muchovej, Departamento de Fitopatologia,
Universidade Federal de Viçosa. The P. griseoroseum mutant
PGLBF (leu, bio, fwn) is auxotrophic for leucine and biotin and
produces brown conidia. This mutant was isolated using
nitrosoguanidine (N-methyl-N’-nitro-N-nitrosoguanidine), as
mutagenic agent (11). The mutant P. expansum PELW (lis, whi),
obtained by irradiation of conidia with ultraviolet light, is a
lysine auxotroph and produces white conidia (24). The
reversion frequency was < 1 x 10-6 in all mutants tested.
Culture media
The following media were used in this study: 1) Minimal
Medium (MM) (31): 6.0 g/L NaNO3, 1.5 g/L KH2PO4, 0.5 g/L KCl,
0.5 g/L MgSO4.7H2O, 0.01 g/L FeSO4, 0.01 g/L ZnSO4, 10.0 g/L
glucose, 15.0 g/L agar (SIGMA); 2) Complete Medium (CM) (5,
31): minimal medium supplemented with 2.0 g/L peptone, 1.5 g/
L hydrolyzed casein, 2.0 g/L yeast extract and 1.0 mL of a vitamin
solution (0.2 mg biotin, 10.0 mg p-aminobenzoic acid, 50.0 mg
pyridoxine, 50.0 mg thiamin, 100.0 mg nicotinic acid and 100.0
mg riboflavin in 100 mL distilled water); 3) enriched oatmeal
agar (EOA): 40.0 g/L oatmeal, 1.5 g/L hydrolyzed casein, 2.0 g/
L yeast extract, 2.0 g/L peptone, 15.0 g/L agar and 1.0 mL of the
vitamin solution; 4) buffered mineral medium (BMM): 13.6 g/L
K2HPO4, 7.6 g/L KH2PO4, 2.0 g/L (NH4)2SO4, 3.0 g/L citric pectin,
0.6 g/L yeast extract and 1.1 g/L MgSO4.7H2O; and, 5) nonbuffered mineral medium (NBMM): 4.0 g/L K2HPO4, 1.28 g/L
KH2PO4, 2.0 g/L (NH4)2SO4, 3.0 g/L citric pectin, 0.6 g/L yeast
extract and 1.1 g/L MgSO4.7H2O.
Spontaneous heterokaryons formation
P. griseoroseum PGLBF conidia and P. expansum PELW
conidia were inoculated in sterile test tubes containing 3 mL of
MM enriched with 2% (v/v) CM and incubated at 25ºC for 5 days.
Protoplast isolation and fusion
About 106 conidia of the P. griseoroseum (PGLBF) and P.
expansum (PELW) mutant strains were inoculated into 50 mL
CM in 250 mL Erlenmeyer flasks and incubated for 18 hours at
150 rpm and 25ºC. Cultures were filtered in gauze and the retained
mycelia washed twice in distilled water. Approximately 300 mg
of fresh mycelia were incubated in 5.0 mL of osmotic stabilizer
(0.6 M KCl in 10 mM sodium and potassium phosphate buffer,
pH 5.8) containing 5.0 mg/mL of Trichoderma harzianum lytic
enzyme (SIGMA) for 3 hours at 80 rpm and 30ºC to produce
protoplasts. The protoplasts were filtered through gauze,
washed twice in osmotic stabilizer and centrifuged for 10 minutes
at 3000 g and 4ºC. Protoplast fusion was carried out as follows:
protoplast suspensions (106 protoplasts/mL) of each strain were
centrifuged at 3000 g for 10 minutes at 4ºC, protoplasts were
resuspended in 1.0 mL of a solution containing 30% (w/v)
polyethylene glycol (PEG 6000 – SYNTH) and 0.05 mM CaCl2 in
0.05 mM glycine-NaOH buffer, pH 8.0. After 10 minutes of
incubation at 30ºC, the suspension was washed twice with 10
mL of osmotic stabilizer and centrifuged at 3000 g for 10 minutes
at 4ºC. The sedimented protoplasts were resuspended in the
same buffer and serial dilutions were prepared and plated for
regeneration in MM and CM containing 1.0 M sucrose as
osmotic stabilizer, for the calculation of fusion frequency and
incubated at 25ºC for 5 days. Diploids putatives were identified
by growth in MM and by the green conidial color.
Haplodization
To induce haplodization, diploids putatives were transferred
by punctual inoculation to Petri dishes containing CM
supplemented with 1.0 µg/mL of benomyl (methyl-1
(butylcarbamoyl)-2-benzimidazole carbamate) and incubated at
25ºC for 10 days. The sectors presenting color similar to the
parental strains were purified in CM and analyzed for genetic
markers and pectinase activity.
Conditions for enzyme production
The fungi were grown for 9 days on plates containing EOA
and 5.107 conidia were harvested and inoculated in 125 mL
53
Queiroz, M.V. et al.
Erlenmeyer flasks containing 50 mL culture medium (pH 6.3) and
incubated under agitation of 150 rpm at 25ºC. Polygalacturonase
activity was determined after 96 hours of growth in NBMM,
while pectin lyase activity was determined after 72 hours growth
in BMM. After the growth period, mycelia were separated by
filtration through sieves with pore diameters of 37 µm and dried
at 105ºC to constant weight for biomass determination. Enzyme
activity was measured in the culture filtrate. The experiment was
installed in a completely randomized design with three replicates.
Enzyme activity data were submitted to variance analysis and
means were grouped by the Scott-Knott test at 1% probability.
The experiment was repeated three times.
Enzyme assay
Pectin lyase activity was determined as described by
Albersheim & Killias (1). The reaction mixture, containing 1.0 mL
of a 2.5% (w/v) pectin solution in 0.1 M phosphate and potassium
buffer, pH 6.8 and 1.5 mL of the culture filtrate, was incubated at
40ºC for 30 minutes. The reaction was interrupted by transferring
0.5 mL of the reaction mixture to a tube containing 4.5 mL of 0.01
N HCl. The product formed was detected in a Micronal B-390
spectrophotometer by monitoring the absorption increase at
235 nm. Control tubes were prepared at zero reaction time. One
unit of pectin lyase activity (U) was defined as ηmoles of
unsaturated product formed per mL of culture mixture per minute
per gram of dry mycelium (Upectina lyase = ηmoles of unsaturated
product . mL–1 . min–1 . g dry mycelium–1). The molar extinction
coefficient of the unsaturated product (5.500 mM.cm–1) was used
for this calculation (2). Polygalacturonase activity was
determined in a reaction mixture containing 0.5 mL of the culture
filtrate, 0.5 mL of 1.2% polygalacturonic acid in 100 mM sodium
acetate buffer, pH 4.8 and 0.1 M NaCl, which was incubated at
40ºC for 20 minutes. The formation of reducing sugar was
measured by the 3.5 dinitrosalicylic acid method according to
Miller (27) using galacturonic acid as standard. One unit of
polygalacturonase activity (U) was defined as µmoles of
galacturonic acid liberated per mL of culture filtrate per minute
per gram of dry mycelium (Upoligalacturonase = µmoles of galacturonic
acid mL–1 . min–1 . g dry mycelium–1).
RESULTS AND DISCUSSION
Heterokaryons and diploid formation
No heterokaryon film formation could be observed when
conidia of P. griseoroseum and P. expansum were mixed in
minimal medium containing 2% CM, indicating incompatibility
between these two species for the formation of heterokaryons
by this technique. Heterokaryons were easily obtained when
conidia of complementary mutants of each species were mixed
(11,24,37).
Anastomosis between hyphae does not always occur when
the fungi belong to different species, possibly because of the
54
presence of a determinant factor in the cell wall that blocks cell
fusion thereby causing incompatibility (28). One way to
overcome the incompatibility between strains is to apply the
technique of protoplast fusion.
Protoplast fusion
Protoplast fusion of P. griseoroseum with P. expansum
produced stable prototrophic fusant products with green
conidia, possible diploids, even when grown in CM, a result
that differs from that obtained by Lana (24), who observed
spontaneous haploidization in putative diploid strains of P.
expansum. Prototrophic fusants of P. griseoroseum were
extremely stable, even when grown in CM with addition of 0.75
µg/mL of benomyl (37).
Conidia of a putative diploid strain (DGE) were transferred
to plates containing CM supplemented with benomyl in order
to obtain recombinant strains with enhanced pectinase
production. Colony formation with white and fawn-colored
sectors similar to the parental strains was observed. Twentynine recombinants were obtained and analyzed for auxotrophic
and morphologic markers. These strains were designated RGEs
(P. griseoroseum and P. expansum recombinants). Recombinant
strains with different auxotrophic markers were isolated, and
although none of them presented markers of both parents,
prototrophic white strains were obtained (Table 1).
Table 1. Characterization of recombinant strains obtained by
haploidization of the putative diploid strain (DGE), after
protoplast fusion between P. expansum PELW and P.
griseoroseum PGLB.
Strain
Genotype
Strain
Genotype
PELW
PGLBF
DGE
RGE01
RGE02
RGE03
RGE04
RGE05
RGE06
RGE07
RGE08
RGE09
RGE10
RGE11
RGE12
RGE13
lys whi
leu bio fwn
prototrophic
leu fwn
leu fwn
leu fwn
leu fwn
leu bio fwn
leu bio fwn
leu bio fwn
leu bio fwn
leu fwn
leu bio fwn
leu fwn
leu fwn
leu fwn
RGE14
RGE15
RGE16
RGE17
RGE18
RGE19
RGE20
RGE21
RGE22
RGE23
RGE24
RGE25
RGE26
RGE27
RGE28
RGE29
leu fwn
leu fwn
prototrophic whi
prototrophic whi
prototrophic whi
leu fwn
leu bio fwn
leu fwn
leu fwn
leu fwn
leu fwn
leu bio fwn
prototrophic whi
leu bio fwn
leu fwn
leu fwn
lys = lysine auxotroph; leu = leucine auxotroph; bio= biotin
auxotroph; whi= white conidia; fwn= fawn-colored conidia.
Isolation of pectinase producing recombinants
When the spontaneous parasexual cycle of P. expansum
was analyzed (24), it was noted that diploids putative were rather
unstable and that recombinant sectors could easily be isolated.
Nevertheless, Santos (37) observed quite different results with
P. griseoroseum, in which the parasexual cycle was not obtained
spontaneously, and the prototrophic products of fusion were
Figure 1. Polygalacturonase (A) and pectin lyase (B) activities of wild type strains of P. expansum (PE) and P. griseoroseum (PG),
of the mutant parental strains (PELW and PGLBF), of the diploid obtained by protoplast fusion (DGE) and of the haploid recombinants
(RGE1 to RGE29) grown in minimal medium at pH 6.3 with pectin as sole carbon source. Segments followed by the same letter are
not significantly different by the Scott-Knott test at a level of 1%.
55
Queiroz, M.V. et al.
stable and produced few discrete sectors even when placed in
CM supplemented with benomyl. The diploid putative resulting
from the fusion between P. expansum and P. griseoroseum
protoplasts presented behavior similar to that observed in P.
expansum, with regard to haploidization, presenting readily
observable sectors.
Enzymatic activity
Polygalacturonase and pectin lyase activities of the wild type
(P. expansum and P. griseoroseum), parental (PGLBF e PELW),
putative diploid strain (DGE) and recombinant strains (RGEs)
were measured. The DGE strain did not present higher
polygalacturonase or pectin lyase activity than the parental strains
(Figure 1A e B). This result is similar to that found for diploids of
A. niger var. awamori, which did not present a higher quantity of
chymosin than the parental strains (9). Hoh et al. (20) obtained
five diploids from A. niger, among which only one presented a
higher β-glucosidase activity than the parental strains. However,
Kirimura et al. (23) obtained one A. niger diploid strain that
produced 1.2 times more citric acid than the parental strain in
solid culture. Martinková et al. (26) also obtained A. niger diploids
with a 15% higher citric acid production than the parental strains.
Several recombinants presented higher polygalacturonase
activities than the wild type strains. Recombinants RGE 14, 20
and 27 had higher activities than the parental mutants (Figure
1A). In relation to pectin lyase production, most recombinants
presented activity similar to that of the parental mutants, and
inferior to the activity of the wild type strains. However, two
recombinants, RGE26 and RGE27, presented increased PL activity,
corresponding to 163.82% and 146.52%, respectively, of the
activity found in the P. griseoroseum wild type strain (Figure
1B). The recombinant RGE27 presented polygalacturonase
activity of 121.58% in relation to PELW and of 340.58% in relation
to the PG. Recombinant RGE26 did not present higher
polygalacturonase activity than the parental mutants, and likewise
for its pectin lyase activity. Among 29 recombinants obtained,
RGE27, which presented the same auxotrophic and morphologic
mutations as the P. griseoroseum parental mutant, was the most
outstanding since it presented a considerable increase in both
polygalacturonase and pectin lyase activities.
Hoh et al. (20) obtained A. niger recombinants by protoplast
fusion which presented glycoamylase activity 2.5 times higher
than in the parental strain. A similar result was obtained by Rubinder
et al. (36) who was able to obtain a Thermomyces lanuginosus
recombinant, which presented five-fold higher α-amylase and aglycoamylase activities than the wild type strain. A. niger var.
awamori recombinants were obtained which presented 15%
greater chymosin production than the parental strains (9).
Tahoun (40) obtained P. chrysogenum segregants with 290 to
390% greater penicillin production than the parental strains.
Our results confirm that protoplast fusion is a promising
technique for obtaining strains with increased enzyme
56
production. Regulatory mechanisms, organization and
localization of the genes responsible for the enzymes of the
pectinolytic complex of the Penicillium genus are not yet
thoroughly understood but, based on the results presented in
this paper, we can affirm that these genes were recombined
during the parasexual cycle initiated by protoplast fusion. The
recombinant RGE27, with enhanced polygalacturonase and
pectin lyase activities, demonstrates that our principal objective,
obtaining a recombinant with the desired characteristics, was
achieved. The production of pectinases by RGE27 strain will be
studied in a submerged fermentation system where different
carbon sources will be tested with the objective of reducing
production costs.
ACKNOWLEDGMENTS
The authors gratefully acknowledge the financial support
of the Brazilian Agencies CNPq, FINEP, FAPEMIG and CAPES.
RESUMO
Isolamento de linhagens recombinantes com maior
produção de pectinases por meio de fusão de
protoplastos entre Penicillium expansum e
Penicillium griseoroseum
Fusões de protoplastos entre linhagens mutantes auxotróficas
e morfológicas complementares de Penicillium griseoroseum e
P. expansum foram induzidas por polietilenoglicol e íons cálcio
(Ca2+). Fusionantes foram obtidos em meio mínimo e uma
linhagem prototrófica, possivelmente diplóide, foi selecionada
para a haploidização com o fungicida benomil. Diferentes
linhagens recombinantes foram isoladas e caracterizadas quanto
à presença de mutações auxotróficas e a produção de enzimas
pectinolíticas. O fusionante prototrófico não apresentou maior
atividade de pectinases em relação às linhagens parentais,
entretanto, entre 29 recombinantes analisados, quatro
apresentaram maiores atividades enzimáticas. O recombinante
RGE27, o qual possui as mesmas mutações auxotróficas e
morfológicas que a linhagem parental de P. griseoroseum,
apresentou um aumento considerável na produção de
poligalacturonase (3 vezes) e de pectina liase (1,2 vezes).
Palavras-chave: Fusão de protoplastos, pectinases, Penicillium
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