Enhanced expression of genes involved in initial xylose metabolism and the oxidative pentose phosphate pathway in the improved xylose-utilizing Saccharomyces cerevisiae through evolutionary engineering

Abstract

Fermentation of xylose in lignocellulosic hydrolysates by Saccharomyces cerevisiae has been achieved through heterologous expression of the xylose reductase (XR)-xylitol dehydrogenase (XDH) pathway. However, the fermentation efficiency is far from the requirement for industrial application due to high yield of the byproduct xylitol, low ethanol yield, and low xylose consumption rate. Through evolutionary engineering, an improved xylose-utilizing strain SyBE005 was obtained with 78.3 % lower xylitol production and a 2.6-fold higher specific ethanol production rate than those of the parent strain SyBE004, which expressed an engineered NADP(+)-preferring XDH. The transcriptional differences between SyBE005 and SyBE004 were investigated by quantitative RT-PCR. Genes including XYL1, XYL2, and XKS1 in the initial xylose metabolic pathway showed the highest up-regulation in SyBE005. The increased expression of XYL1 and XYL2 correlated with enhanced enzymatic activities of XR and XDH. In addition, the expression level of ZWF1 in the oxidative pentose phosphate pathway increased significantly in SyBE005, indicating an elevated demand for NADPH from XR. Genes involved in the TCA cycle (LAT1, CIT1, CIT2, KGD1, KGD, SDH2) and gluconeogenesis (ICL1, PYC1) were also up-regulated in SyBE005. Genomic analysis revealed that point mutations in transcriptional regulators CYC8 and PHD1 might be responsible for the altered expression. In addition, a mutation (Y89S) in ZWF1 was identified which might improve NADPH production in SyBE005. Our results suggest that increasing the expression of XYL1, XYL2, XKS1, and enhancing NADPH supply are promising strategies to improve xylose fermentation in recombinant S. cerevisiae.

Improved Xylose Metabolism by a CYC8 Mutant of Saccharomyces cerevisiae

“…Further mutagenesis of the Y353 position showed that other substitutions can result in improved growth, but the original cysteine mutant appears to exhibit the strongest phenotype. A previous study also described a point mutation in CYC8 in an evolutionary engineering experiment which optimized D-xylose consumption in a D-xylose-fermenting S. cerevisiae strain (26). Either because of that mutation or because of two other mutations found in the evolved strain (26), the expression levels of the genes involved in xylose metabolism (e.g., XYL1 and XYL2) were increased, causing improved D-xylose consumption but not increased growth on D-xylose in the presence of D-glucose.…”

Enhancement of Simultaneous Xylose and Glucose Utilization by Regulating ZWF1 and PGI1 in Saccharomyces Cerevisiae

“…The increase of glucose consumption in SyBE_Sc122001 was due to the increase of metabolic flux into the PP pathway from glucose, which drove the increase in xylose consumption due to the coupling by RPE1 deletion. Besides, ZWF1 overexpression also offered more NADPH as the co-factor for xylose reductase to accelerate xylose utilization [38,39].…”

Hybridization Improves Inhibitor Tolerance of Xylose-fermenting Saccharomyces cerevisiae

“…6634), we named as E7. (Zha et al 2014). The mating selection medium (YNBX) contained 6.7 g/L yeast nitrogen base (YNB), 20 g/L xylose, 20 g/L agar, and 20 g/L of xylose.…”

“…Recently, engineered strains for efficient xylose utilization, by introducing a xylose pathway, has attracted extensive attention. A xylose fermenting yeast, which expresses the xylose reductase (XR) and a NADP+-preferring xylitol dehydrogenase (XDH) mutant, was genetically constructed (Zha et al 2014). Then an adapted strain (E7) with improved xylose fermentation capability (S. cerevisiae CGMCC NO.…”