Metabolic Engineering of Ammonium Assimilation in Xylose-Fermenting Saccharomyces cerevisiae Improves Ethanol Production

Abstract

Cofactor imbalance impedes xylose assimilation in Saccharomyces cerevisiae that has been metabolically engineered for xylose utilization. To improve cofactor use, we modified ammonia assimilation in recombinant S. cerevisiae by deleting GDH1, which encodes an NADPH-dependent glutamate dehydrogenase, and by overexpressing either GDH2, which encodes an NADH-dependent glutamate dehydrogenase, or GLT1 and GLN1, which encode the GS-GOGAT complex. Overexpression of GDH2 increased ethanol yield from 0.43 to 0.51 mol of carbon (Cmol) Cmol ؊1 , mainly by reducing xylitol excretion by 44%. Overexpression of the GS-GOGAT complex did not improve conversion of xylose to ethanol during batch cultivation, but it increased ethanol yield by 16% in carbon-limited continuous cultivation at a low dilution rate.In order to develop an efficient process for the production of bioethanol from lignocellulosic material, there have been many attempts to improve the conversion of xylose to ethanol by construction of recombinant Saccharomyces cerevisiae strains.Even though the open reading frames encoding the three first enzymes involved in xylose metabolism, i.e., xylose reductase (XR), xylitol dehydrogenase (XDH), and xylulose kinase (XK), have been identified in the genome of S. cerevisiae (10, 15), the genes are poorly expressed (or not expressed at all), and xylose cannot be metabolized without introduction of heterologous genes from naturally xylose-fermenting microorganisms and overexpression of endogenous XK (3,6,17). In the resulting strains, there is a low rate of xylose consumption and substantial xylitol secretion.Since xylose-metabolizing strains of S. cerevisiae harbor a NAD(P)H-dependent xylose reductase and a NAD ϩ -dependent xylitol dehydrogenase from Pichia stipitis, one of the main reasons for xylitol excretion can be explained by the redox imbalance occurring at the level of XR and XDH. Metabolic engineering of redox metabolism may therefore be a strategy for improving the conversion of xylose to ethanol. This has been illustrated by deletion of the zwf1 gene encoding glucose-6-phosphate dehydrogenase in a xylose-metabolizing strain of S. cerevisiae (7). The main source of NADPH originating from the oxidative part of the pentose phosphate pathway has thereby been reduced, and during growth on xylose, this has resulted in a significant improvement of ethanol yield, from 0.31 g g Ϫ1 (yield on consumed sugar) to 0.41 g g Ϫ1 with a concomitant reduction in the xylitol yield.In this study, we have taken a different approach to modulating the redox metabolism to favor xylose metabolism, namely through metabolic engineering of the ammonia assimilation. Ammonium assimilation in S. cerevisiae involves glutamate dehydrogenase and glutamine synthetase. Glutamate dehydrogenase catalyzes the synthesis of glutamate from ammonium and 2-ketoglutarate. The most important enzyme for ammonia assimilation is the NADPH-dependent glutamate dehydrogenase 1, encoded by GDH1. The other glutamate dehydrogenase that is present in S. cerevisiae is N...

Fermentation performance of engineered and evolved xylose‐fermenting Saccharomyces cerevisiae strains

“…Nevertheless, the principal stoichiometric redox problem was not solved (Bruinenberg et al, 1984;Hahn-Hägerdal et al, 2001), and thus xylitol formation will likely remain an inherent property of strains engineered with xylose reductase and xylitol dehydrogenase. Instead, conceptually new metabolic engineering strategies will be necessary such as the use of xylose isomerase (Kuyper et al, 2003;Walfridsson et al, 1996) or new biochemical routes to NADH reoxidation (Roca et al, 2003;Verho et al, 2003). Methods of mutagenesis and selection or more elaborate evolutionary engineering (Sauer, 2001) appear to be the most promising for initial introduction of a novel (Sonderegger and Sauer, 2003) or for improvement of a weak property (Steiner and Sauer, 2003).…”

Physiological Effects of GLT1 Modulation in Saccharomyces cerevisiae Strains Growing on Different Nitrogen Sources

“…Only very few manuscripts looking over the engineering of the central nitrogen metabolism of S. cerevisiae have been published [25,27,32,38]. In all these works, the redox intracellular status was unbalanced to decrease glycerol levels and to increase the ethanol production in aerobic or anaerobic batch, using ammonium sulfate as a sole nitrogen source.…”

Industrial systems biology

“…Expression of XR, XDH, and XK lead to highly inefficient xylose utilization due to a co-factor imbalance, where excess NADH must be regenerated via xylitol production resulting in reduced ethanol yield. Therefore, metabolic engineering of the ammonium assimilation through deletion of the NADPHdependent glutamate dehydrogenase (GDH1) and overexpression of the NADH-dependent glutamate dehydrogenase (GDH2) resulted in a 16% higher ethanol yield due to a 44% xylitol reduction (Grotkjaer et al, 2005;Roca et al, 2003). Using a reverse metabolic engineering approach, metabolic flux analysis (MFA) was used to characterize the intracellular fluxes for both strains based on experimental data of anaerobic continuous cultivations using a growth limited feed of 13 C-labeled glucose, confirming that XR activity shifted from being mostly NADPH to partly NADH dependent in the CPB.CR4 strain.…”