The GRE3 encoded aldo-keto reductase and its influence on xylose fermentation in recombinant Saccharomyces cerevisiae strains

Sammanfattning: The aim of the work presented in this thesis was to improve ethanol formation from xylose in recombinant Saccharomyces cerevisiae strains. This was achieved by decreasing the xylitol by-product formation. Gre3p is the major xylose-reducing enzyme in S. cerevisiae. Ypr1p, Gcy1p and the protein encoded by YJR096w also have the ability to reduce xylose in cell extracts. Furthermore, Gre3p, Ypr1p, Gcy1p and the protein encoded by YJR096w have the ability to reduce arabinose, with Ypr1p being most active. The GRE3 gene, which encodes an aldo-keto reductase, was deleted and generated recombinant S. cerevisiae with no detectable xylose reductase activity and significantly decreased xylitol formation. In recombinant S. cerevisiae strains producing xylose isomerase (XI) from the thermophilic bacterium Thermus thermophilus, the ethanol yield was 0.21 g/g consumed xylose when the GRE3 gene was deleted compared with 0.12 g/g consumed xylose when the GRE3 gene was intact. This may be due to less inhibition of XI by xylitol and to that the carbon flux being redirected towards ethanol. Three different cold-adapted T. thermophilus XI mutants were produced in S. cerevisiae. A higher ethanol yield (0.43 g/g compared with 0.14 g/g consumed xylose) was obtained with the recombinant S. cerevisiae strain producing XI M-1021, than with the reference strain producing the wild-type XI. Regarding xylose consumption in recombinant S. cerevisiae producing XI, it seems important that the KM for xylose remains low for XI. The GRE3 gene was deleted from the xylose-utilising recombinant S. cerevisiae strain TMB3001(expressing XYL1 and XYL2 from Pichia stipitis and overexpressing endogenous XKS1). In continuous culture using mineral medium with glucose and xylose, the resulting strain, TMB3120, formed half the amount of xylitol and 60% more ethanol than TMB3001. Recombinant S. cerevisiae expressing GRE3 at two different levels in combination with XYL2 and XSK1 generated xylose-fermenting strains. This shows that Gre3p is capable of xylose reduction in vivo. Xylose consumption and ethanol formation increased in the recombinant strain where Gre3p was overproduced, compared with the recombinant strain producing native Gre3p, showing that XR activity was limiting in these strains. The oxidative pentose phosphate pathway was disrupted in TMB3001, generating new recombinant S. cerevisiae strains displaying lower xylitol yield but also lower xylose consumption. Xylose consumption was restored by additional expression of the XYL1 gene (encoding xylose reductase (XR)), but the glycerol yield increased significantly. This might be because XR from P. stipitis was able to reducedihydroxy acetone phosphate (DHAP). XR showed a KM for DHAP of 78 mM compared with 91.7 mM for xylose, and the activity with DHAP was half that obtained with xylose.