Rational promoter elements and evolutionary engineering approaches for efficient xylose fermentation in Saccharomyces cerevisiae

Abstract

We screened and identified a set of efficient promoters in Saccharomyces cerevisiae that maintained their relatively strong strengths to regulate the heterologous xylose-assimilating pathway genes XYL1 and XYL2, and native XKS1 and pentose phosphate pathway four genes, irrespective of glucose or xylose fermentation medium. In this study, we developed a rapid and efficient xylose-fermenting S. cerevisiae strain 7-1 based on balanced pathway expression levels driven by our proposed strong promoters. Next, 7-1 was used to initialize the evolutionary engineering, through first aerobic and anaerobic sequential batch cultivation. The finally evolved strain of 7-1E1 displayed a high ethanol yield (0.45 g/g) and low xylitol accumulation (0.13 g/g). Moreover, the evolved strain of 7-1E1 displays great potential for ethanol production from lignocellulosic biomass. This work reveals that efficient xylose assimilation is attributed to the elevated expression levels of xylose utilization genes, which was accomplished through the strong promoter rational regulation in the chromosome of the evolved strain.

Minimize the Xylitol Production in Saccharomyces cerevisiae by Balancing the Xylose Redox Metabolic Pathway

“…Previous reports employed protein engineering technology to modify the coenzyme specificity of XR and XDH to increase ethanol and reduce xylitol production. Some Xyl1 derivatives, such as K270R, K270M, and R276H, have positive effects on increasing ethanol production and reducing xylitol yield (Kostrzynska et al, 1998;Watanabe et al, 2007a,b;Hyun and Yeong, 2016); however, significant amount of xylitol still remained in our previously reported xylose fermentation systems even after introducing the above XYL1 mutants (Sang et al, 2016).…”

“…In order to further improve xylose utilization toward ethanol production, we asked whether proposed rational promoters could replace the original constitutive ADH1 promoter to effectively drive the XYL1-K270R gene. We previously developed a set of S. cerevisiae promoters to regulate the xylose metabolic pathway including HXT7, HXT4, TPI1, FBA1, CCW12, ACO1, HSP26, HSP70, PGK1, and ADH1, resulting in some rational promoter elements like HXT7 and FBA1 (Sang et al, 2016). We replaced the ADH1 promoter with two previously proposed inducible promoters, HXT7 (Friedrich and Susanne, 2000;Ye et al, 2010) and FBA1 (Sang et al, 2016), and a newly selected TEF1, resulting in strains E9H1, E9F1, and E9T1, respectively.…”

“…We previously developed a set of S. cerevisiae promoters to regulate the xylose metabolic pathway including HXT7, HXT4, TPI1, FBA1, CCW12, ACO1, HSP26, HSP70, PGK1, and ADH1, resulting in some rational promoter elements like HXT7 and FBA1 (Sang et al, 2016). We replaced the ADH1 promoter with two previously proposed inducible promoters, HXT7 (Friedrich and Susanne, 2000;Ye et al, 2010) and FBA1 (Sang et al, 2016), and a newly selected TEF1, resulting in strains E9H1, E9F1, and E9T1, respectively. These strains (Figures 2A-C) together with E9 ( Figure 2D) were evaluated in the YPX medium to assess effects of XYL1 expression driven by different promoters on the xylose fermentation, and the results at 120 h are summarized in Table 1.…”