Metabolic engineering of Saccharomyces cerevisiae for conversion of D-glucose to xylitol and other five-carbon sugars and sugar alcohols

Abstract

Recombinant Saccharomyces cerevisiae strains that produce the sugar alcohols xylitol and ribitol and the pentose sugar D-ribose from D-glucose in a single fermentation step are described. A transketolase-deficient S. cerevisiae strain accumulated D-xylulose 5-phosphate intracellularly and released ribitol and pentose sugars (D-ribose, D-ribulose, and D-xylulose) into the growth medium. Expression of the xylitol dehydrogenase-encoding gene XYL2 of Pichia stipitis in the transketolase-deficient strain resulted in an 8.5-fold enhancement of the total amount of the excreted sugar alcohols ribitol and xylitol. The additional introduction of the 2-deoxy-glucose 6-phosphate phosphatase-encoding gene DOG1 into the transketolase-deficient strain expressing the XYL2 gene resulted in a further 1.6-fold increase in ribitol production. Finally, deletion of the endogenous xylulokinase-encoding gene XKS1 was necessary to increase the amount of xylitol to 50% of the 5-carbon sugar alcohols excreted.

FIG. 1.

(a) The pentose phosphate pathway of S. cerevisiae showing how xylitol could be formed from d-glucose. TKL, transketolase isoenzymes 1 and 2. (b) The suggested routes to xylitol, ribitol, and d-ribose in the metabolically engineered S. cerevisiae strains created in this study. RPE1, d-ribulose-phosphate 3-epimerase; RKI1, d-ribose-5-phosphate keto-isomerase; Ptase, sugar phosphate phosphatase (e.g., Dog1p); XDH, xylitol dehydrogenase; AldR, nonspecific aldose reductase (e.g., Gre3p). All sugars presented are in d-configuration.

FIG. 2.

Volumetric production (mg liter⁻¹) of extracellular pentose sugars and sugar alcohols by the transketolase-deficient strain H1055 (a) and the transketolase-deficient strain H1506 containing the XYL2 gene integrated to the genome (b), grown on YSC medium with 20 g liter⁻¹ d-glucose as a carbon source in an aerobic flask cultured with shaking. Production levels of sugar alcohols ribitol (white) and xylitol (gray) and of d-ribulose plus d-ribose (black) at 55, 78, and 100 h of culture are shown. The 5-carbon sugars were analyzed by HPLC (see Materials and Methods).

FIG. 3.

Volumetric consumption of d-glucose (in g liter⁻¹; circles) and production of biomass (dry weight [DW]; squares), ethanol (in g liter⁻¹; diamonds), ribitol (in g liter⁻¹; triangles), and xylitol (in g liter⁻¹; crosses and stars) by the transketolase-deficient strain H1506 expressing the XYL2 gene integrated into the genome, in aerobic flasks with shaking on YSC medium with 20 g liter⁻¹ d-glucose as a carbon source. Open and closed symbols indicate two identical, independent experiments.