Saccharomyces cerevisiae engineered for xylose metabolism requires gluconeogenesis and the oxidative branch of the pentose phosphate pathway for aerobic xylose assimilation

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 both NADH and NADPH, is hypothesized to reduce the cofactor imbalance, allowing xylose fermentation in this yeast. However, unadapted S. cerevisiae strains expressing this XR grow poorly on xylose, suggesting that 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 gluconeogenesis in S. cerevisiae. S. cerevisiae cells expressing the XR-XDH, but not a 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 Kluyveromyces lactis. When grown in 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 upregulating enzymes 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 enzymes correlated with increased tolerance to the NADPH-depleting compound diamide and the fermentation inhibitors furfural and hydroxymethyl furfural; S. cerevisiae was not able to increase enzyme activity for glucose-6-P production when grown in xylose medium and was more sensitive to these inhibitors in xylose medium compared to glucose. Published in

Cofactor engineering for advancing chemical biotechnology

“…Protein engineering of xylose reductase to a NADH-preferring enzyme gave high ethanol productivity [6]. A study with the NADH-NADPH utilizing Scheffersomyces stipitis xylose reductase suggested formation of glucose 6-phosphate via gluconeogenesis was limiting [4]. The expression of NADH oxidase to diminish cofactor imbalance lowered glycerol and xylitol yields while improving the ethanol yield [7].…”

“…Some examples illustrating the requirement for cofactor balance and availability include: the conversion of biomass feedstocks containing xylose to ethanol where the formation of xylitol is a problem [1][2][3][4][5][6][7]; as a driving force for more effective production of reduced compounds such as biofuels [8]; in using cytochrome P450s in specific oxidation reactions where the recycling of active enzyme is required [9][10][11]; and the production of chiral pharmaceutical intermediates where specific reductions require a certain cofactor [12,13]. Experimental studies along with more complete computational models have shown a global picture of the flow of reducing equivalents and its connection to cell physiology and allowed these insights to be considered for metabolic engineering purposes [14][15][16][17].…”

Organic acid production in Aspergillus niger and other filamentous fungi

“…For the purpose of ex vivo functional validation of the selected fungal transporters strain EBY.VW4000 was genetically modified to be able to metabolize xylose as a carbon source and laboratory-evolved for an enhanced growth on xylose. For this, strain EBY.VW4000, unable to grow on glucose, mannose, galactose or fructose as carbon source, was transformed with the plasmid pRH315, expressing the P. stipitis D-xylose reductase (XYL1) and xylitol dehydrogenase (XYL2) genes, and the S. cerevisiae xylulokinase (XKS1) gene (Hector et al, 2011). An isolated transformant of the EBY.VW.4000 strain expressing the xylose utilization pathway (EBY.XP) was subsequently transformed with a plasmid expressing the A.…”

“…Poor growth of xylose utilizing yeast transformant strains has been described before (Colabardini et al, 2014;Huang et al, 2015;Saloheimo et al, 2007), and this could be due to metabolic imbalances as a result of different cofactor specificities of the heterologous genes XYL1 and XYL2 present in the modified S. cerevisiae strain (Hector et al, 2011). To obtain a better growing xylose-utilizing host for routine identification of xylose transporters, the ability of the EBY.XP strain to metabolize xylose was improved through laboratory-evolution.…”

“…Verification of the identified glucose and xylose transporter candidates (Chapters 5 and 6) was facilitated by the existence of a Saccharomyces cerevisiae hexose transporter null mutant (Wieczorke et al, 1999), which, for experimental validation of the xylose transporters, was engineered to metabolise xylose (Hector et al, 2011). In this mutant strain, heterologous expression of the identified glucose and xylose transporter candidates opens a new door, and it is in the interest of the host organism to make use of this entry to obtain the carbon that is available.…”