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Title:   Author  item
                                            Hector, Ronald

                                                         - *Ron*item
                                            Mertens, Jeffreyitem
                                            Bowman, Michaelitem
                                            Nichols, Nancyitem
                                            Cotta, Michaelitem
                                            Hughes, Stephen
  Submitted to: Yeast
                                    Publication Type: Peer Reviewed Journal
                                    Publication Acceptance Date: 6/14/2011
                                    Publication  Date: 7/1/2011
                                    Citation: Hector, R.E., Mertens, J.A., Bowman, M.J., Nichols, N.N., Cotta, M.A., Hughes, S.R. 2011. Saccharomyces cerevisiae engineered for xylose metabolism requires gluconeogenesis and the oxidative branch of the pentose phosphate pathway for aerobic xylose assimilation. Yeast. 28:645-660.Interpretive Summary: Intense interest in achieving energy security and reducing carbon emissions has led to a global research effort towards developing renewable sources of liquid transportation fuels. Most of the bioethanol produced in the United States is from fermentation of corn starch. Expanding biofuel production further in the United States will largely depend upon developing lignocellulosic materials as feedstocks. Industrial-scale fermentation of lignocellulosic materials will require commercially-robust strains that are genetically stable and possess high ethanol yields and productivities. Additionally, they must be capable of fermenting all biomass-derived sugars, including the five-carbon sugar, xylose. The objective of this study was to further investigate metabolic limitations on xylose utilization. Yeast strains deficient in genes hypothesized to be involved in xylose metabolism were deleted and growth in xylose medium was determined. We show that engineered xylose-fermenting yeast require the production of glucose from xylose to grow using xylose as a carbon source. Additionally, natural xylose-utilizing yeasts were able to induce proteins required to produce glucose from xylose, while engineered yeast did not. Failure to induce these proteins when growing in xylose medium also resulted in increased sensitivity of the engineered yeast to fermentation inhibitors. These results identify a new area for further strain development for improving xylose-fermenting.
                                Technical Abstract: Saccharomyces strains engineered to ferment xylose using Scheffersomyces stipitis xylose reductase (XR) and xylitol dehydrogenase (XDH) genes appear to be limited by metabolic imbalances due to differing cofactor specificities of XR and XDH. The S. stipitis XR, which uses nicotinamide adenine dinucleotide (NADH) almost as well as nicotinamide adenine dinucleotide phosphate (NADPH), is hypothesized to reduce this cofactor imbalance, allowing xylose fermentation. However, S. cerevisiae cells expressing this XR grow poorly on xylose, suggesting metabolism is still imbalanced, even under aerobic conditions. In this study, we investigated the possible reasons for this imbalance by deleting genes required for NADPH production and involved in gluconeogenesis in S. cerevisiae. S. cerevisiae cells expressing the XR/XDH, but not the xylose isomerase, pathway required the oxidative branch of the pentose phosphate pathway (PPP) and gluconeogenic production of glucose-6-P for xylose assimilation. The requirement for generating glucose-6-P from xylose was also shown for the natural xylose-assimilating yeast Kluyveromyces lactis. When grown on xylose medium, both K. lactis and S. stipitis showed increases in enzyme activity required for producing glucose-6-P. Thus, natural xylose-assimilating yeast respond to xylose, in part, by up regulating genes required for recycling xylose back to glucose-6-P for the production of NADPH via the oxidative branch of the PPP. Finally, we show that induction of these genes correlated with increased tolerance to the NADPH-depleting compound diamide and the fermentation inhibitor furfural; S. cerevisiae was not able to increase genes for glucose-6-P when grown in xylose medium and was more sensitive to these inhibitors in xylose medium compared to glucose.


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