Systematic and evolutionary engineering of a xylose isomerase-based pathway in

Abstract

BackgroundEfficient xylose fermentation by yeast would improve the economical and sustainable nature of biofuels production from lignocellulosic biomass. However, the efficiency of xylose fermentation by the yeast Saccharomyces cerevisiae is suboptimal, especially in conversion yield, despite decades of research. Here, we present an improved performance of S. cerevisiae in xylose fermentation through systematic and evolutionary engineering approaches.ResultsThe engineering of S. cerevisiae harboring xylose isomerase-based pathway significantly improved the xylose fermentation performance without the need for intensive downstream pathway engineering. This strain contained two integrated copies of a mutant xylose isomerase, gre3 and pho13 deletion and XKS1 and S. stipitis tal1 overexpression. This strain was subjected to rapid adaptive evolution to yield the final, evolved strain (SXA-R2P-E) which could efficiently convert xylose to ethanol with a yield of 0.45 g ethanol/g xylose, the highest yield reported to date. The xylose consumption and ethanol production rates, 0.98 g xylose g cell−1 h−1 and 0.44 g ethanol g cell−1 h−1, respectively, were also among the highest reported. During this process, the positive effect of a pho13 deletion was identified for a xylose isomerase-containing strain and resulted in up to an 8.2-fold increase in aerobic growth rate on xylose. Moreover, these results demonstrated that low inoculum size and the cell transfer at exponential phase was found to be the most effective adaptation strategy during a batch culture adaptation process.ConclusionsThese results suggest that the xylose isomerase pathway should be the pathway of choice for efficient xylose fermentation in S. cerevisiae as it can outperform strains with the oxidoreductase pathway in terms of yield and ethanol production and xylose consumption rates. Consequently, the strain developed in this study could significantly improve the prospect of biofuels production from lignocellulosic biomass.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-014-0122-x) contains supplementary material, which is available to authorized users.

Unraveling the genetic basis of xylose consumption in engineered Saccharomyces cerevisiae strains

“…2a and b). The best evolved strain LVY34.4 presents a yield of 0.46 g ethanol/g xylose (0.51 g/g is the theoretical maximum ), similar or superior to most recently developed xylose-fermenting yeasts described in the literature222324.…”

“…In addition, adaptive evolution in xylose medium is typically conducted in batch or chemostat cultivation cycles to achieve the best results192021. Successful examples of strains developed through this approach have been reported222324, however, the specific mutations responsible for the acquired ability to metabolize xylose have not been well characterized.…”

Engineering a wild-type diploid Saccharomyces cerevisiae strain for second-generation bioethanol production

“…The chassis cell BSIF was diploid; therefore, to destroy two alleles of PHO13 , which would benefit xylose metabolism (Bamba et al 2016; Lee et al 2014; Shen et al 2012; Van Vleet et al 2008), two plasmids were assembled in the plasmid pUC19 as follows: pXIP1/2 (Additional file 1: Fig. S2a) containing two pairs of PHO13 -targeted recombinant arms, PHO13 - RA1 vs. PHO13 - RA2 and PHO13 - RA1 vs. PHO13 - RA3 .…”

“…Additionally, the inactivation of aldose reductase Gre3p, which is encoded by GRE3 , to decrease the formation of the byproduct xylitol (Bamba et al 2016; Fujitomi et al 2012; Hahn-Hägerdal et al 2007; Kuyper et al 2005a; Peng et al 2012), and the inactivation of alkaline phosphatase Pho13p, encoded by PHO13 , to reduce ATP waste (Bamba et al 2016; Lee et al 2014; Shen et al 2012; Van Vleet et al 2008), were also the considered strategies. Normally, xylose is absorbed non-specifically and insufficiently by hexose transporters and is competitively inhibited by glucose in S. cerevisiae (Subtil and Boles 2012).…”

Comparison of xylose fermentation by two high-performance engineered strains of Saccharomyces cerevisiae

“…In addition to the introduction and functional expression of two different metabolic pathways for xylose consumption (XR/XDH and XI), rational and evolutionary metabolic engineering strategies have been used to construct efficient xylose-fermenting S. cerevisiae strains [5], [7], [11], [12]. An outstanding question in the field is whether one pathway or the other would be preferred for large-scale fermentation [18].…”