Metabolic engineering of recombinant S. cerevisiae

Based on their positive "fuel and environmental" properties both ethanol and refined in the form of methyl-t-butyl ester represent the most promising alternatives to currently used transportation fuels from fossil origin. The increasing interest on alternative fuels is mainly based on the enactment of the fuel directive (2003/30/EU) which forms the statutory basis for the addition of fuel substitutes to fossil fuels and a portion of 5.75% is intended to be achieved by 2010. The application of biomass from plants in form of lignocellulosic materials for the production of ethanol is a promising alternative to traditionally used industrial crops. The complete utilization of the sugar fraction in these materials formed predominantly by glucose and by xylose is required to guarantee economic attractive production of ethanol. Saccharomyces cerevisiae, which is traditionally used in the ethanol production process, has to be metabolically modified to enable ethanol production from xylose, as the yeast is not able to utilize xylose. Combined expression of the enzymes xylose reductase, xylitol dehydrogenase and xylulokinase qualified the yeast to convert xylose to ethanol under exclusion of oxygen but with moderate ethanol yield and strong accumulation of undesired xylitol. These inefficiencies are due to opposite coenzyme specificities of xylose reductase (prefers NADPH) and of xylitol dehydrogenase (NAD-specific) which avoid recycling of coenzymes during xylose assimilation. Balancing coenzyme utilization in the cell is necessary for efficient ethanol production. Coenzyme specificities have been changed for xylose reductase and xylitol dehydrogenase by preliminary works in our working group which offers the unique opportunity to focus directly at the optimization of the first steps of xylose assimilation. For this purpose six yeast strains have been designed and constructed such that they prefer either NADH/NAD or NADPH/NADP for coenzyme recycling. Moreover NADPH/NADP specific strains differ in their relative coenzyme preference which enables adaptation to the optimal coenzyme balance in the cell. The phenotype exhibiting improved ethanol production will be characterized in comparison to the reference strain by using metabolic network analysis based on metabolite balancing and 13C-isotopomer analysis in the course of the scholarship at the ETH Zürich at the Institute of Molecular Systems Biology under the direction of Prof. Dr. Uwe Sauer. Determination of metabolic net flux rates in the central carbon metabolism together with the identification of metabolic network topologies is essential for a comprehensive understanding of the resulted phenotypes which in turn represents the molecular basis for further development of a yeast strain capable for industrial use.