Optimal yeast strains for the production of fuels and chemicals from lignocellulosic biomass

The microbial production of fuels and chemicals has recently received much attention as an alternative to the limited fossil fuels. To become an economically viable option, the bioprocess must use non-food plants, called cellulosic biomass. Such renewable biomass, however, contains both hexose and pentose sugars, mainly glucose and xylose, which are not efficiently fermented by microbes. Despite numerous efforts for improving xylose fermentation during the last two decades, a method for engineering efficient xylose-fermenting strains has not been finalized yet. In this thesis study, by elucidating fundamental bottlenecks impeding the xylose metabolism, I identified and implemented powerful genetic perturbation targets for developing an efficient xylose-fermenting Saccharomyces cerevisiae strain. First, I used a genome library screening method to investigate the limiting steps in xylose metabolism by a S. cerevisiae strain expressing a heterologous xylose-assimilating pathway consisting of xylose reductase (XR), xylitol dehydrogenase (XDH), and xylulose kinase (XK), coded by XYL1, XYL2, and XYL3, respectively. Finding XYL2 as an overexpression target, I optimized the expression levels of XYL1/XYL2/XYL3 to maximize the ethanol yield with a minimum xylitol yield. Later, I confirmed that the optimized expression level of the xylose-assimilating pathway resulted efficient xylose fermentation regardless of the unbalanced cofactor requirements between XR and XDH: NADPH-specific XR (GRE3) was successfully substituted for NAD(P)H-specific XR (XYL1) in a S. cerevisiae strain expressing NAD+-specific XDH. Through evolutionary engineering of the engineered strain with the optimized xylose-assmilating pathway, deletion of PHO13 was found to be a key element in improving the growth rate on xylose. Lastly, after disruption of a major acetaldehyde dehydrogenase (ALD6) to prevent acetate accumulation, the final strain was able to ferment xylose efficiently under various conditions including dilute-acid hydrolyzates of cellulosic biomass. Using this engineered S. cerevisiae strain capable of fermenting xylose efficiently, we will be able to produce various fuels and chemicals from cellulosic biomass hydrolyzates