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Saccharomyces Cerevisiae Engineered for Xylose Metabolism Yeast Yeast 2011; 28: 645–660. Published online 1 August 2011 in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/yea.1893 Research Article Saccharomyces cerevisiae engineered for xylose metabolism requires gluconeogenesis and the oxidative branch of the pentose phosphate pathway for aerobic xylose assimilation§ Ronald E. Hector1*, Jeffrey A. Mertens1, Michael J. Bowman1, Nancy N. Nichols1, Michael A. Cotta1 and Stephen R. Hughes2 1Bioenergy Research Unit, National Center for Agricultural Utilization Research, Peoria, IL, USA 2Renewable Product Technology Research Unit, National Center for Agricultural Utilization Research, Peoria, IL, USA *Correspondence to: Abstract Ronald E. Hector, Bioenergy Research Unit, National Center Saccharomyces strains engineered to ferment xylose using Scheffersomyces stipitis for Agricultural Utilization xylose reductase (XR) and xylitol dehydrogenase (XDH) genes appear to be limited Research, Agricultural Research by metabolic imbalances, due to differing cofactor specificities of XR and XDH. The Service, US Department of S. stipitis XR, which uses both NADH and NADPH, is hypothesized to reduce the Agriculture†,1815North cofactor imbalance, allowing xylose fermentation in this yeast. However, unadapted University Street, Peoria, IL S. cerevisiae strains expressing this XR grow poorly on xylose, suggesting that 61604, USA. metabolism is still imbalanced, even under aerobic conditions. In this study, we E-mail: investigated the possible reasons for this imbalance by deleting genes required for [email protected] NADPH production and gluconeogenesis in S. cerevisiae. S. cerevisiae cells expressing †Mention of trade names or the XR–XDH, but not a xylose isomerase, pathway required the oxidative branch of commercial products in this the pentose phosphate pathway (PPP) and gluconeogenic production of glucose-6-P article is solely for the purpose of for xylose assimilation. The requirement for generating glucose-6-P from xylose was providing scientific information also shown for Kluyveromyces lactis. When grown in xylose medium, both K. lactis and does not imply and S. stipitis showed increases in enzyme activity required for producing glucose-6- recommendation or endorsement P. Thus, natural xylose-assimilating yeast respond to xylose, in part, by upregulating by the US Department of enzymes required for recycling xylose back to glucose-6-P for the production of Agriculture. USDA is an equal NADPH via the oxidative branch of the PPP. Finally, we show that induction of opportunity provider and employer. these enzymes correlated with increased tolerance to the NADPH-depleting compound diamide and the fermentation inhibitors furfural and hydroxymethyl furfural; §This article is a US Government S. cerevisiae was not able to increase enzyme activity for glucose-6-P production when work and is in the public domain grown in xylose medium and was more sensitive to these inhibitors in xylose medium in the USA. compared to glucose. Published in 2011 by John Wiley & Sons, Ltd. Keywords: xylose; fermentation; Saccharomyces; gluconeogenesis; NADPH; Received: 16 March 2011 imbalance Accepted: 14 June 2011 Introduction five-carbon sugar, is the second most abundant sugar in biomass hydrolysates; only glucose is Development of economical processes for con- present at a higher concentration. Although C6 and verting lignocellulosic feedstocks to renewable C5 sugars could be potentially split into separate products will require efficient utilization of all carbon streams, many of the proposed processes available sugars (Sassner et al., 2008). Xylose, a for production of biofuels and other renewable Published in 2011 by John Wiley & Sons, Ltd. 646 R. E. Hector et al. products require the combined use of xylose and phosphate pathway (PPP) intermediate xylulose- glucose. Saccharomyces cerevisiae would be the 5-phosphate (Figure 1). Most xylose reductases preferred organism for biofuels production, due to identified show a strict dependence on NADPH as the availability of a robust genetic transformation the cofactor, while xylitol dehydrogenase is specific system along with a long history of use in for NAD+. This imbalance of cofactors can lead to industrial fermentation processes. Unfortunately, depletion of NADPH and excess NADH (Van Vleet xylose metabolism presents a unique challenge for and Jeffries, 2009). Aerobically, excess NADH S. cerevisiae. can be re-oxidized by the mitochondria. Under Most natural xylose-assimilating yeasts, such as Scheffersomyces stipitis (formerly known as Pichia anaerobic conditions, excess NADH accumulates stipitis; Kurtzman and Suzuki, 2010) metabolize and xylose utilization slows, or in some cases xylose by a reduction/oxidation pathway. Xylose is stops (Bruinenberg et al., 1984). NADPH must be first converted to xylitol using a xylose reductase regenerated through metabolic routes. When glu- (XR). Xylitol dehydrogenase (XDH) converts xyl- cose is available, most of the NADPH required itol to xylulose, which can then be phosphorylated for anabolic reductive reactions and tolerance to by xylulokinase (XK) to generate the pentose inhibitors is generated through the oxidative branch A Oxidative PPP Glucose 2 XYLOSE ATP NADPH (XR) XYL1 NADPH + CO NADP+ NADPH NADP+ NADP+ 6-phospho- 6-phospho- Ribulose-5-P xylA Glucose-6-P gluconate Xylitol ZWF1 glucono SOL3 GND1 (XI) (G6PD) lactone SOL4 GND2 PGI1 RKI1 RPE1 NAD+ (XDH) XYL2 ATP NADH Fructose-6-P ATP Fructose-6-P non-oxidative Xylulose Pentose FBP1 PFK1 XKS1 PFK2 Glyceraldehyde-3-P Phosphate (XK) Pathway Fructose-1,6-BP FBA1 aromatic Histidine and Dihydroxy Glyceraldehyde-3-P acetone-P TPI1 amino nucleic acids acids glycerol ETHANOL B NADP+ NADPH NADP+ NADPH Acetaldehyde Acetate Isocitrate α-ketoglutarate ALD6 IDP2 Figure 1. (A) Representation of glucose and xylose metabolism via glycolysis, pentose phosphate pathway and gluconeogenesis. Abbreviations/gene names and associated pathways are as follows. Glycolysis: PGI1, phosphoglucose isomerase; PFK1 and PFK2, phosphofructokinase; FBA1, fructose 1,6-bisphosphate aldolase; TPI1, triose phosphate isomerase. Oxidative PPP: ZWF1, glucose-6-phosphate dehydrogenase (G6PD); SOL3 and SOL4, 6-phosphogluconolactonase; GND1 and GND2, 6-phosphogluconate dehydrogenase. Non-oxidative PPP: RKI1, ribose-5-phosphate ketol-isomerase; RPE1, D-ribulose-5-phosphate 3-epimerase; TKL1, transketolase; TAL1,transaldolase.Initial xylose metabolism: xylA,xylose isomerase (XI); XYL1, xylose reductase (XR); XYL2, xylitol dehydrogenase (XDH); XKS1,xylulokinase(XK).Gluconeogenesis: PGI1, phosphoglucose isomerase; FBP1, fructose 1,6-bisphosphate phosphotase; FBA1, fructose 1,6-bisphosphate aldolase. (B) Minor sources of NADPH in S. cerevisiae. ALD6, cytosolic NADP+-dependent aldehyde dehydrogenase; IDP2, cytosolic NADP+-dependent isocitrate dehydrogenase Published in 2011 by John Wiley & Sons, Ltd. Yeast 2011; 28: 645–660. DOI: 10.1002/yea S. cerevisiae requires the PG11 and ZWF1 genes for xylose assimilation 647 of the PPP (Lagunas and Gancedo, 1973; Nogae for genes of the oxidative branch of the PPP during and Johnston, 1990). anaerobic growth on xylose, suggesting that regula- S. stipitis appears to be unique among xylose- tion of these pathways may be important for xylose assimilating yeasts as under oxygen-limited con- metabolism (Runquist et al., 2009). This idea was ditions, it can ferment xylose to produce ethanol. further enhanced when it was found that deletion The ability of S. stipitis to ferment xylose has been of an activating transcription factor for PPP genes, attributed to the ability of its XR to use NADH Stb5p,inS. cerevisiae led to impaired NADPH almost as well as NADPH, thus avoiding an imbal- production (Cadiere` et al., 2010). ance of cofactors (Bruinenberg et al., 1984). The To gain further insight into the requirement S. stipitis XR–XDH pathway has been expressed of these pathways for xylose metabolism in in S. cerevisiae and confers the ability to metabo- S. cerevisiae, we deleted genes for recycling lize xylose (Figure 1; reviewed in Hahn-Hagerdal¨ fructose-6-phosphate (fructose-6-P) back to et al., 2007). Natural xylose-assimilating yeasts do glucose-6-phosphate (glucose-6-P) and through the not exhibit (under aerobic conditions) a cofac- oxidative branch of the PPP. We also deleted tor imbalance and grow well on xylose medium. genes for alternative sources of NADPH and anal- S. stipitis can grow in synthetic xylose medium ysed each strain’s ability to assimilate xylose. with an aerobic growth rate of µ = 0.45 h−1 and We show that S. cerevisiae engineered for xylose quickly reach optical densities >12 OD660. While metabolism via an XR–XDH pathway, but not a some further adapted and modified Saccharomyces xylose isomerase (XI) pathway, requires the pro- yeasts with the XR–XDH pathway show signif- duction of glucose-6-P from fructose-6-P for the icant increases in growth on xylose, unadapted generation of NADPH by the oxidative branch S. cerevisiae strains expressing this pathway grow of the PPP. Requirement for the oxidative branch poorly on xylose, indicating that even under aer- of the PPP for xylose metabolism is not unique obic conditions, metabolism is not properly bal- to S. cerevisiae. Both of the xylose-assimilating anced. For example, the unadapted S. cerevisiae yeasts Scheffersomyces stipitis and Kluyve- laboratory strain CEN.PK2-1C, expressing the romyces lactis induce glucose-6-P dehydrogenase S. stipitis XR–XDH pathway, has a much slower and phosphoglucose isomerase activity
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