Overexpression of NADH-dependent fumarate reductase improves d-xylose fermentation in recombinant Saccharomyces cerevisiae

Abstract

Deviation from optimal levels and ratios of redox cofactors NAD(H) and NADP(H) is common when microbes are metabolically engineered. The resulting redox imbalance often reduces the rate of substrate utilization as well as biomass and product formation. An example is the metabolism of D-xylose by recombinant Saccharomyces cerevisiae strains expressing xylose reductase and xylitol dehydrogenase encoding genes from Scheffersomyces stipitis. This pathway requires both NADPH and NAD(+). The effect of overexpressing the glycosomal NADH-dependent fumarate reductase (FRD) of Trypanosoma brucei in D-xylose-utilizing S. cerevisiae alone and together with an endogenous, cytosol directed NADH-kinase (POS5Δ17) was studied as one possible solution to overcome this imbalance. Expression of FRD and FRD + POS5Δ17 resulted in 60 and 23 % increase in ethanol yield, respectively, on D-xylose under anaerobic conditions. At the same time, xylitol yield decreased in the FRD strain suggesting an improvement in redox balance. We show that fumarate reductase of T. brucei can provide an important source of NAD(+) in yeast under anaerobic conditions, and can be useful for metabolic engineering strategies where the redox cofactors need to be balanced. The effects of FRD and NADH-kinase on aerobic and anaerobic D-xylose and D-glucose metabolism are discussed.

Xylose-induced dynamic effects on metabolism and gene expression in engineered Saccharomyces cerevisiae in anaerobic glucose-xylose cultures

“…MAL2-8 c . SUC2), ura3::XYL1 XYL2, xks1::XKS1], and VTT C-10880 (Salusjärvi et al 2013). In both strains, the XYL1 and XYL2 genes of S. stipitis were chromosomally integrated into the URA3 locus under PGK1 and ADH1 promoters.…”

“…The correct integration of URA3 was verified by PCR from genomic DNA of transformants. In both VTT C-10880 and VTT C-10883, XKS1 of S. cerevisiae was integrated into the XKS1 locus, as described previously for VTT C-10880 (Salusjärvi et al 2013).…”

“…The S. cerevisiae strains used in the study were VTT C-10883 originating from VTT C-10880 (Salusjärvi et al 2013), i.e.…”

Physiological effects of over-expressing compartment-specific components of the protein folding machinery in xylose-fermenting Saccharomyces cerevisiae

“…There are, however, FR enzymes that use NADH as co-factor instead of FADH 2 . The NADH-dependent FR enzyme from Trypanosoma brucei has recently been expressed in a xylose-utilizing S. cerevisiae strain which resulted in increased ethanol yield and decreased xylitol yield when fermenting xylose [47]. Based on these results we conclude that the FR enzymes from S. cerevisiae do not influence the regeneration of cytosolic NAD.…”

Accumulation of metabolic side products might favor the production of ethanol in Pho13 knockout strains

“…2) and an increase in the NADH/NAD + ratio, which inhibits glyceraldehyde-3-P dehydrogenase, thereby hampering glycolysis and ethanol formation. This interpretation is supported by the finding that xylose conversion to ethanol may be improved by ( 1 ) reducing the cellular capacity to synthesize NADPH via a suppression or reduction of the activity of enzymes of the pentose phosphate pathway such as glucose-6-P dehydrogenase, 6-P-gluconate dehydrogenase or phosphoglucose isomerase 7; ( 2 ) reversing the dependency of xylitol dehydrogenase from NAD + to NADP + 8; ( 3 ) using a mutated xylose reductase that preferentially uses NADH instead of NADPH 9; ( 4 ) expressing an NADH-dependent fumarate reductase, which eliminates the excess of NADH production 10.…”