(490y) Engineering the Cofactor Preference of Xylose Reductase and Xylitol Dehydrogenase to Enhance Xylose Fermentation by Recombinant Saccharomyces Cerevisiae

Green house gases, supply instability and high oil prices have led to an increased emphasis on renewable source of energy. Bioethanol is a CO₂-neutral alternative to fossil fuels. Currently it is mainly produced from glucose which leads to the controversial debate about food and feed versus energy. Lignocellulose based feedstocks from agricultural wastes are cost efficient carbon sources that would help to eliminate the ethical concerns over utilization of biomass in biofuel production. Xylose is the main constituent of hemicellulosic hydrolyzates and has to be utilized in order to make fermentation processes of lignocellulosic material economically viable. Despite its high productivity and ethanol tolerance, Saccharomyces cerevisiae is unable to utilize xylose.

Insertion of a heterologous pathway consisting of xylose reductase (XR) and xylitol dehydrogenase (XDH), as found in other yeasts, enables isomerisation of xylose into xylulose. In turn a xylulose kinase (XK) converts xylulose to xylulose 5-phosphate which then enters the pentose phosphate pathway. A fundamental problem of this oxidoreductive pathway is the different cofactor specificity of XR and XDH. XR is a NADPH preferring enzyme, while XDH depends on NAD⁺. The pathway therefore accumulates NADH at the expense of NADPH. Overall this leads to the formation of xylitol and glycerol as by-products and a reduced ethanol yield. In order to overcome these obstacles, we constructed a set of mutants of XR from Candida tenuis [1] and XDH from Galactocandida mastotermitis [2] that showed altered coenzyme preference. Combinations of XR and XDH that should provide better coenzyme recycling than the wild-type enzymes do were integrated into the genome of S. cer. An additional copy of the endogenous XK was also integrated with the aim of increasing the flux towards the pentose phosphate pathway.

Anaerobic conversion in a bioreactor with a strain expressing the wild type XDH and a doubly mutated XR (K247R-N276D) showed a 42% enhanced ethanol yield in comparison to the reference strain expressing wild type XR. The production of xylitol and glycerol was reduced by 52% and 57%, respectively. The xylose uptake rate was not affected [3]. It seems that alteration of XR coenzyme specificity caused a global metabolic response improving the distribution of fermentation products without introducing an extra kinetic bottleneck. A quintuple mutant (D202LVES206 → A202ΔΔPR206) of XDH shows a 4 times higher catalytic efficiency with NADP⁺ than with NAD⁺. The mutant displayed with NADP⁺ about 70% of the specific activity of the wild type enzyme with NAD⁺. The coenzyme preference of this XDH mutant is close to the preference of NADPH compared with NADH in the K247R-N276D (0.8) and the N276D (4) mutant of XR. Xylose conversions using the strains comprising these combinations showed an up to 50 % decreased glycerol yield in comparison to the reference strain. This was however not accompanied by enhancement of the overall ethanol yield. Combining concentrations of internal metabolites with kinetic parameters of the XDH enzymes suggests that under in vivo conditions the XDH mutant is a significantly slower enzyme than the wild type. The observed product distribution might therefore be due to an unfavourable ratio between the in vivo activities of the XR and XDH mutants. Batch fermentations of mixed glucose and xylose substrates showed the expected diauxic conversion with glucose being metabolized before xylose. Though, towards glucose depletion xylose uptake seemed to increase to rates even exceeding xylose uptake in fermentations where xylose is the sole carbon source. Fed batch fermentations under high xylose concentrations (up to 50 g/L) and limiting glucose concentrations (<0.3 g/L) strongly supported the hypothesis of an enhanced xylose uptake rate in the presence of low amounts of glucose.

[1] Petschacher, B.; Leitgeb, S.; Kavanagh, K. L.; Wilson, D. K.; Nidetzky, B., Biochem. J. 2005, 385, 75-83.

[2] Krahulec, S.; Klimacek, M.; Nidetzky, B., Biotechnol. J. 2009, in press.

[3] Petschacher, B.; Nidetzky, B., Microb. Cell Fact. 2008, 7, 9.