Development and characterization of Saccharomyces cerevisiae strains genetically modified to over-express the pentose phosphate pathway regulating transcription factor STB5 in the presence of xylose

Abstract

Several enzymes in the pentose phosphate pathway of Saccharomyces cerevisiae have been identified as relating to the constraint of xylose consumption and conversion to ethanol. However, no strategy has been proposed for simultaneous regulation of all contributing enzymes. If multiple enzymes contribute to constraint, over expression of a native transcription factor controlling the entire constraining pathway may provide optimal pathway wide regulation. Further characterization of this strain on both pure sugars and lignocellulosic hydrolysates would provide an opportunity to identify additional bottlenecks not addressed by the modification of the pentose phosphate pathway expression pattern. A series of strains were developed expressing STB5 and PGI1 under the control of a novel xylose inducible promoter. Increased transcription of STB5 and its regulatory targets was verified via qRT-PCR. No statistically significant difference was found in terms of xylose consumption or ethanol yield in these strains versus control strains. Xylose consumption through both the fermentative and respiratory pathways appeared to be related to oxygen availability and culture density with high-density (low oxygen) cultures consuming xylose more slowly than low-density cultures. The maximum specific consumption rate for high-density cultures was 0.21 g xylose/gDCW/h versus 0.41 g xylose/gDCW/h in lower density cultures. Statistically similar ethanol yields at high and low density (approximately 0.25 g ethanol/ g xylose) suggest that the maximum rate of fermentation is linked to the rate of respiration in a stoichiometric fashion. This study did not find evidence supporting the pentose phosphate pathway constraint identified in other works. Instead, NAD + availability mediated by oxygen availability and citric acid cycle flux was suggested to limit fermentation. While increased aeration could provide increased conversion of NAD + to NADH (and a stoichiometric increase in fermentation flux), this increase would not be expected improve ethanol yield beyond 50% of the theoretical maximum. Based on these findings, future work in Saccharomyces cerevisiae development for fermentation of lignocellulosic hydrolysates should focus on balancing NAD + / NADH availability through non-respiratory pathways.