Figure 1.
Effect of xylose reductase (XR) activity on the specific xylose consumption rates of various S. cerevisiae D452-2 strains (D10) expressing different copy numbers of XYL1 derived from Sch. stipitis.
Figure 2.
NADPH-specific xylose reductase (XR) activity and NAD-specific xylitol dehydrogenase (XDH) activity of three xylose-assimilating strains expressing different copy numbers of XYL1, XYL2, and XYL3.
D452-2, wild type S. cerevisiae; DX123, D452-2 expressing one copy of XYL1, XYL2, and XYL3; SR6, D452-2 expressing multiple copies of XYL1 and one copy of XYL2 and XYL3; SR7, expressing multiple copies of XYL1 and two copies of XYL2 and XYL3. The figure illustrates the means and standard deviations of triplicate experiments.
Figure 3.
Fermentation profiles of three xylose-assimilating strains in YP media containing 40 g/l xylose (a) and a mixture of 70 g/l glucose and 40 g/l xylose (b).
DX123, D452-2 expressing one copy of XYL1, XYL2, and XYL3; SR6, D452-2 expressing multiple copies of XYL1 and one copy of XYL2 and XYL3; SR7, expressing multiple copies of XYL1 and two copies of XYL2 and XYL3. All fermentations were performed under an oxygen-limited condition (100 rpm). An initial cell density was adjusted to 0.3 g/l. The figure illustrates the means of duplicate fermentations for each strain.
Figure 4.
Effect of xylose concentration on the specific growth rates of engineered S. cerevisiae SR7 expressing a xylose assimilation pathway consisting of XYL1, XYL2, and XYL3.
The figure illustrates the means and standard deviations of duplicate experiments.
Figure 5.
Evolution of the SR7 strain by serial subculturing on xylose.
(a) Changes in the growth rate and ethanol yield during serial subcultures of engineered S. cerevisiae SR7 expressing a xylose assimilation pathway (XYL1, XYL2, and XYL3) in 40 g/l xylose (YPX40). (b and c) The xylose fermentation capability of three single colonies (SR7e1, SR7e2, and SR7e3), isolated from the last subculture, were evaluated in YPX40 as compared to the wild type SR7 (the means of duplicate experiments for each strain). All serial subcultures and fermentations were performed under an oxygen-limited condition (100 rpm). An initial cell density for serial subcultures or fermentations was adjusted to 0.03 g/l and 0.3 g/l, respectively.
Table 1.
SNPs identified in the three evolved SR7 strains as compared with the wild type SR7.
Figure 6.
Comparison of wild type SR7 (a), SR7e3 (b), and SR7 pho13Δ (c) when fermenting 40 g/l xylose (YPX40) under an oxygen-limited condition.
An initial cell density was adjusted to 0.3 g/l. The figure illustrates the means of duplicate experiments for each strain.
Figure 7.
PHO13 deletion affects xylose fermentation differently in various strain backgrounds.
(a-d) Xylose fermentation parameters in three different xylose-assimilating strains and (e and f) growth patterns of the SR7 strain in various xylose concentrations: a, xylose consumption rate (g/h); b, specific xylose consumption rate (g/g cell/h); c, xylitol yield (g/g xylose); d, ethanol yield (g/g xylose); e, cell growth of SR7; f, cell growth of SR7 pho13Δ. All parameters were calculated at 36 h during 40 g/l xylose fermentation. An initial cell density was adjusted to 0.3 g/l (a-d) or 0.03 g/l (e-f). All experiments were duplicated.
Figure 8.
In vitro phosphatase activity of crude cell extracts from SR7 control (pRS423), SR7 pho13Δ, and PHO13-overexpressing SR7 (SR7 pRS423-PHO13) on p-nitrophenyl phosphate (p-NPP, an artificial phosphatase substrate, a positive control) and xylulose-5-phosphate.
The figure illustrates the means and standard deviations of triplicate experiments.
Figure 9.
Fermentation profiles and pH changes of SR7e3 (a), SR7e3 with pH control (b), and SR7e3 ald6Δ (i.e. SR8, c) when fermenting 80 g/l xylose (YPX80) under an oxygen-limited condition.
An initial cell density was adjusted to 0.3 g/l.
Table 2.
Fermentation profiles of SR7e3 and SR8 (SR7e3 ald6Δ) in various sugar conditions1).
Figure 10.
Summary of metabolic engineering strategies used in this study to develop efficient xylose-fermenting S. cerevisiae.
(a) Rational metabolic engineering strategies to optimize a xylose-assimilating pathway consisting of the XYL1, XYL2, and XYL3 genes and to overcome acetate toxicity (ald6Δ), (b) evolutionary engineering to isolate mutants (SR7e1, SR7e2, SR7e3) that grow faster on xylose and to identify genetic changes (pho13Δ) of the mutants through genome sequencing, and (c) targeted analysis to confirm inhibitory effect of Pho13p on xylose metabolism by engineered S. cerevisiae.
Table 3.
Plasmids and strains used in this study.