The expression of aPichia stipitis xylose reductase mutant with higherKM for NADPH increases ethanol production from xylose in recombinantSaccharomyces cerevisiae

Abstract

Xylose fermentation by Saccharomyces cerevisiae requires the introduction of a xylose pathway, either similar to that found in the natural xylose-utilizing yeasts Pichia stipitis and Candida shehatae or similar to the bacterial pathway. The use of NAD(P)H-dependent XR and NAD(+)-dependent XDH from P. stipitis creates a cofactor imbalance resulting in xylitol formation. The effect of replacing the native P. stipitis XR with a mutated XR with increased K(M) for NADPH was investigated for xylose fermentation to ethanol by recombinant S. cerevisiae strains. Enhanced ethanol yields accompanied by decreased xylitol yields were obtained in strains carrying the mutated XR. Flux analysis showed that strains harboring the mutated XR utilized a larger fraction of NADH for xylose reduction. The overproduction of the mutated XR resulted in an ethanol yield of 0.40 g per gram of sugar and a xylose consumption rate of 0.16 g per gram of biomass per hour in chemostat culture (0.06/h) with 10 g/L glucose and 10 g/L xylose as carbon source.

Combinatorial Design of a Highly Efficient Xylose-Utilizing Pathway in Saccharomyces cerevisiae for the Production of Cellulosic Biofuels

“…The error bars represent standard deviations (n ϭ 3). Statistical significance: *, n ϭ 3, P Ͻ 0.05; **, n ϭ 3, P Ͻ 0.005. and activities of XR and XDH (22,28,29), and glycerol is a common by-product in anaerobic glucose fermentation and is related to the regeneration of NAD ϩ (30). Although the limited oxygen supply and growth in complex media can alleviate the cofactor imbalance reducing xylitol production, the significant reduction of the by-products and higher ethanol yield among the selected recombinants demonstrates the effects of pathway balancing.…”

Limitations in Xylose-FermentingSaccharomyces cerevisiae, Made Evident through Comprehensive Metabolite Profiling and Thermodynamic Analysis

“…A buffered (100 mM citrate, pH 5.5) mineral medium containing 20 g/liter glucose was used. The medium was supplemented with vitamins and trace elements (23), ergosterol (8 mg/liter), Tween 80 (336 mg/liter), and polypropylene glycol 2000 (1 ml/liter of a 10% [vol/vol] aqueous emulsion). Xylose was added to a final concentration of 20 g/liter when glucose was completely consumed.…”

Progress in Metabolic Engineering of Saccharomyces cerevisiae

“…1). To rescue cells from redox factor imbalance, several attempts have been made to identify xylose reductase mutants that prefer NADH over NADPH (142,386).…”