The heterologous expression of a highly functional xylose isomerase pathway in Saccharomyces cerevisiae would have significant advantages for ethanol yield, since the pathway bypasses cofactor requirements found in the traditionally used oxidoreductase pathways. However, nearly all reported xylose isomerase-based pathways in S. cerevisiae suffer from poor ethanol productivity, low xylose consumption rates, and poor cell growth compared with an oxidoreductase pathway and, additionally, often require adaptive strain evolution. Here, we report on the directed evolution of the Piromyces sp. xylose isomerase (encoded by xylA) for use in yeast. After three rounds of mutagenesis and growth-based screening, we isolated a variant containing six mutations (E15D, E114G, E129D, T142S, A177T, and V433I) that exhibited a 77% increase in enzymatic activity. When expressed in a minimally engineered yeast host containing a gre3 knockout and tal1 and XKS1 overexpression, the strain expressing this mutant enzyme improved its aerobic growth rate by 61-fold and both ethanol production and xylose consumption rates by nearly 8-fold. Moreover, the mutant enzyme enabled ethanol production by these yeasts under oxygen-limited fermentation conditions, unlike the wild-type enzyme. Under microaerobic conditions, the ethanol production rates of the strain expressing the mutant xylose isomerase were considerably higher than previously reported values for yeast harboring a xylose isomerase pathway and were also comparable to those of the strains harboring an oxidoreductase pathway. Consequently, this study shows the potential to evolve a xylose isomerase pathway for more efficient xylose utilization.
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.
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.
Bioprospecting is an effective way to find novel enzymes from strains with desirable phenotypes. Such bioprospecting has enabled organisms such as Saccharomyces cerevisiae to utilize nonnative pentose sugars. Yet, the efficiency of this pentose catabolism (especially for the case of arabinose) remains suboptimal. Thus, further pathway optimization or identification of novel, optimal pathways is needed. Previously, we identified a novel set of xylan catabolic pathway enzymes from a superior pentose-utilizing strain of Ustilago bevomyces. These enzymes were used to successfully engineer a xylan-utilizing S. cerevisiae through a blended approach of bioprospecting and evolutionary engineering. Here, we expanded this approach to xylose and arabinose catabolic pathway engineering and demonstrated that bioprospected xylose and arabinose catabolic pathways from U. bevomyces offer alternative choices for enabling efficient pentose catabolism in S. cerevisiae. By introducing a novel set of xylose catabolic genes from U. bevomyces, growth rates were improved up to 85 % over a set of traditional Scheffersomyces stipitis pathway genes. In addition, we suggested an alternative arabinose catabolic pathway which, after directed evolution and pathway engineering, enabled S. cerevisiae to grow on arabinose as a sole carbon source in minimal medium with growth rates upwards of 0.05 h(-1). This pathway represents the most efficient growth of yeast on pure arabinose minimal medium. These pathways provide great starting points for further strain development and demonstrate the utility of bioprospecting from U. bevomyces.
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