Xylose fermentation efficiency of industrial Saccharomyces cerevisiae yeast with separate or combined xylose reductase/xylitol dehydrogenase and xylose isomerase pathways

ABSTRACT In industrial fermentation processes, the yeast Saccharomyces cerevisiae is commonly used for ethanol production. However, it lacks the ability to ferment pentose sugars like d-xylose and l-arabinose. Heterologous expression of a xylose isomerase (XI) would enable yeast cells to metabolize xylose. However, many attempts to express a prokaryotic XI with high activity in S. cerevisiae have failed so far. We have screened nucleic acid databases for sequences encoding putative XIs and finally were able to clone and successfully express a highly active new kind of XI from the anaerobic bacterium Clostridium phytofermentans in S. cerevisiae. Heterologous expression of this enzyme confers on the yeast cells the ability to metabolize d-xylose and to use it as the sole carbon and energy source. The new enzyme has low sequence similarities to the XIs from Piromyces sp. strain E2 and Thermus thermophilus, which were the only two XIs previously functionally expressed in S. cerevisiae. The activity and kinetic parameters of the new enzyme are comparable to those of the Piromyces XI. Importantly, the new enzyme is far less inhibited by xylitol, which accrues as a side product during xylose fermentation. Furthermore, expression of the gene could be improved by adapting its codon usage to that of the highly expressed glycolytic genes of S. cerevisiae. Expression of the bacterial XI in an industrially employed yeast strain enabled it to grow on xylose and to ferment xylose to ethanol. Thus, our findings provide an excellent starting point for further improvement of xylose fermentation in industrial yeast strains.

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Decreased Xylitol Formation during Xylose Fermentation in Saccharomyces cerevisiae Due to Overexpression of Water-Forming NADH Oxidase

Applied and Environmental Microbiology ◽

10.1128/aem.06635-11 ◽

2011 ◽

Vol 78 (4) ◽

pp. 1081-1086 ◽

Cited By ~ 34

Author(s):

Guo-Chang Zhang ◽

Jing-Jing Liu ◽

Wen-Tao Ding

Keyword(s):

Saccharomyces Cerevisiae ◽

Xylose Fermentation ◽

Nadh Oxidase ◽

Xylose Reductase ◽

Control Strain ◽

Content Type ◽

Ethanol Yields ◽

Glycerol Yield ◽

Set Up ◽

And Control

ABSTRACTThe recombinant xylose-fermentingSaccharomyces cerevisiaestrain harboring xylose reductase (XR) and xylitol dehydrogenase (XDH) fromScheffersomyces stipitisrequires NADPH and NAD+, creates cofactor imbalance, and causes xylitol accumulation during growth ond-xylose. To solve this problem,noxE, encoding a water-forming NADH oxidase fromLactococcus lactisdriven by thePGK1promoter, was introduced into the xylose-utilizing yeast strain KAM-3X. A cofactor microcycle was set up between the utilization of NAD+by XDH and the formation of NAD+by water-forming NADH oxidase. Overexpression ofnoxEsignificantly decreased xylitol formation and increased final ethanol production during xylose fermentation. Under xylose fermentation conditions with an initiald-xylose concentration of 50 g/liter, the xylitol yields for of KAM-3X(pPGK1-noxE) and control strain KAM-3X were 0.058 g/g xylose and 0.191 g/g, respectively, which showed a 69.63% decrease owing tonoxEoverexpression; the ethanol yields were 0.294 g/g for KAM-3X(pPGK1-noxE) and 0.211 g/g for the control strain KAM-3X, which indicated a 39.33% increase due tonoxEoverexpression. At the same time, the glycerol yield also was reduced by 53.85% on account of the decrease in the NADH pool caused by overexpression ofnoxE.

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