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.
|Conference||3rd European Federation of Biotechnology Conference |
|Period||13/06/07 → 16/06/07|