Functional Expression of a Bacterial Xylose Isomerase in Saccharomyces cerevisiae

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.

Engineering of yeast hexose transporters to transport d -xylose without inhibition by d -glucose

“…Conversion of xylose to xylulose is achieved either by a reductive/oxidative pathway using xylose reductase and a xylitol dehydrogenase (2) or by direct isomerization using a xylose isomerase (XI) (3)(4)(5). In both cases, xylulose is then phosphorylated by an endogenous or heterologous xylulokinase (XKS) and channeled through the nonoxidative branch of the pentose phosphate pathway (PPP) into glycolysis.…”

Can Microbially Derived Advanced Biofuels Ever Compete with Conventional Bioethanol? A Critical Review

“…There have been several works to solve this redox imbalance including the heterologous expression of transhydrogenase enzymes (Kuyper et al 2005). Later efforts in expressing xylose isomerase in S. cerevisiae partially resolved this problem, as the enzyme can directly convert xylose to xylulose, which can directly enter the pentose phosphate pathway (Brat et al 2009;Matsushika et al 2009). This approach was adapted in genetically engineered S. cerevisiae for the production of farnesane from a mixture of glucose and xylose (Amyris 2013).…”

125th Anniversary Review: Fuel Alcohol: Current Production and Future Challenges

“…Further information on this genetic engineering approach is available from references 18,21,47,59,120,134 . The yields of ethanol from xylose by GM strains of S. cerevisiae have been reported at 0.43 g/g, with maximum ethanol concentrations achieved at 46.5 g/L 101 .…”