Fig 1.
Expression of different glycosyl hydrolase (GH) genes from a common expression vector backbone and test of their effects on growth of E. coli DH5α in MOPS minimal medium [19] and Z. mobilis ZM4 in Zymomonas minimal medium [20] with glucose or cellobiose as a carbon source.
(A) Expression of GH genes in a pVector backbone and a summary of the constructs and their effects on growth in minimal medium supplemented with cellobiose. (B-D) Growth of E. coli DH5α containing plasmids pVector or pCel3A or pGH3 in MOPS minimal medium supplied with 0.4% glucose or cellobiose. (E-G) Growth of Z. mobilis ZM4 containing plasmids pVector or pCel3A or pGH3 in a Zymomonas minimal medium containing 2% glucose or 2% cellobiose. The growth curves are averages of three replicates. *Gene from Cellvibrio japonicus, #Gene from Caulobacter crescentus.
Table 1.
Localization and activity of glycosyl hydrolase expressed in Z. mobilis ZM4 and E. coli DH5α.
Fig 2.
Growth and physiological changes induced by adaptation to cellobiose medium.
(A) Growth of Z. mobilis ZM4 transformed with control pVector or pGH3 in 2% cellobiose medium with 0.05% glucose (RMCG). Z. mobilis GH3 growth in RMCG can be described in three stages: initial growth on glucose, a long lag phase, and growth on cellobiose. (B) Growth comparison of Z. mobilis GH3 in RMCG. Cells were either adapted to cellobiose, unadapted to cellobiose, or adapted to cellobiose and then regrown in RMG (reRMG). Extracellular (C) and whole-cell (D) GH activity for RMCG-adapted or -unadapted Z. mobilis GH3. (E) Extracellular and whole cell GH activity of RMCG-adapted Z. mobilis GH3 grown in RMG after adaptation before returning to RMCG. GH activity reported as relative fluorescence signal produced per min normalized by input cell number (apparent OD600). Error bars are standard deviations of biological triplicates.
Fig 3.
Growth adaptation by serial passage and its effect on growth in cellobiose and ethanol production.
(A) Schematic representation of adaptation showing serial passages of Z. mobilis GH3 and pVector control. Apparent OD600 was measured for Z. mobilis GH3 at the end of each passage. (B, C, D). Growth of Z. mobilis GH3 and pVector control in RMC after first, second, and third passages. (E, F, G) Cellobiose conversion after first, second, and third passages, respectively. (H, I, J) Ethanol production after first, second, and third passages, respectively. Error bars are standard deviations of triplicate experiments.
Fig 4.
Sucrose adaptation allows Z. mobilis GH3 to grow on cellobiose.
(A) Growth of sucrose-adapted Z. mobilis GH3, pVector control, and unadapted Z. mobilis GH3 in RMC supplemented with 0.1% sucrose (RMCSuc). (B) Growth of sucrose-adapted and unadapted Z. mobilis GH3 and pVector control, in RMC plus sucrose (0.2–0.8%). Gray line, Z. mobilis pVector RMC alone. Black lines, Z. mobilis + pVector in RMC+sucrose. Blue lines, Z. mobilis GH3 in RMC+sucrose. Green lines, sucrose-adapted Z. mobilis GH3 in RMC+sucrose.
Fig 5.
Volcano plots of Gene Ontology (GO) enrichment analysis showing differential expression of GO term proteins.
The upper panels show (A) intracellular and (B) extracellular proteomics for cellobiose-adapted Z. mobilis GH3 compared to unadapted strain. The lower panels show (C) intracellular and (D) extracellular proteomics for sucrose-adapted Z. mobilis GH3 compared to unadapted strain. All enriched GO term proteins are indicated with spheres of distinct colors and β-glucosidase upregulation is shown as a yellow star.
Fig 6.
Heat map displaying hierarchical clustering of control-normalized log2 fold changes of 1199 molecules quantified across 12 replicates.
Both row-wise and column-wise clustering was performed using Euclidean distance and average linkage calculations. 50 distinct hierarchical clusters are represented in the color bar shown alongside the heat map and only the significant protein families are indicated.