Production of xylitol and other five-carbon sugars and sugar alcohols from D-glucose with Saccharomyces cerevisiae

We constructed recombinant Saccharomyces cerevisiae strains deficient in either transketolase or phosphoglucose isomerase activities for production of xylitol, ribitol or ribose from D-glucose via the pentose phosphate pathway (PPP) intermediates D-xylulose 5-phosphate, D-ribulose 5-phosphate or D-ribose 5-phosphate. The transketolase-deficient strains accumulated PPP sugar-phosphates and excreted D-ribulose/D-ribose to the culture medium. Introduction of xylitol dehydrogenase encoding gene XYL2 from Pichia stipitis increased ribitol and xylitol production and concomitantly decreased the amount of D-ribulose/D-ribose. A sugar phosphate phosphatase Dog1p increased ribitol production 1.6-fold, but did not enhance xylitol excretion. Deletion of the xylulokinase encoding gene XKS1 increased the amount of xylitol produced to 50% of the total sugar alcohols (ribitol plus xylitol) produced. The highest yield of xylitol and ribitol obtained with the various transketolase deficient strains studied was 3.6% (w/w) of consumed D-glucose. S. cerevisiae strains deficient in phosphoglucose isomerase activity are unable to grow on D-glucose unless a NADPH-consuming reaction is introduced into the cell (1, 2). We introduced two NADPH-consuming reactions, a NADPH-utilizing glyceraldehyde 3-phosphate dehydrogenase of Bacillus subtilis (GapB) and a NAD-dependent glutamate dehydrogenase (GDH2) of S. cerevisiae into the pgi1-mutant strain in order to produce 5-carbon sugars and sugar alcohols from D-glucose via the PPP. Both of these enzymes enabled growth on D-glucose of the pgi1-mutant strains, as shown for the GDH2 overexpression previously (1). The Dog1p phosphatase was, however, needed for production of 5-carbon sugars and sugar alcohols. On higher D-glucose concentrations the expression of DOG1 together with either GapB or GDH2 reduced growth. The Dog1p may create a futile cycle of phosphorylation and dephosphorylation of glucose 6-phosphate resulting in ATP depletion.