Engineering Saccharomyces cerevisiae for mixed-sugar fermentation

Abstract: Efficient fermentation of all the monomeric sugars derived from lignocellulose is crucial to increase the economy of bioethanol production, since they can account for a considerable fraction of the fermentable sugars in the raw material. This thesis describes the engineering of Saccharomyces cerevisiae strains for improved xylose, galactose and/or arabinose utilization. S. cerevisiae is the preferred microorganism for commercial ethanolic fermentation, but the wild type cannot utilize xylose or arabinose. In addition, the fermentation rate and the transcriptional response of glycolytic genes is higher on a glucose substrate than on galactose. The conversion of xylose to xylulose has been attempted by engineering S. cerevisiae strains with the xylose reductase-xylitol dehydrogenase (XR-XDH) pathway and the xylose isomerase (XI) pathway. The results presented in this thesis compared isogenic recombinant xylose-utilizing strains engineered via these pathways. The specific ethanol productivity from the fermentation of xylose was twice as high in the XR-XDH-engineered strain, while the XI-carrying strain provided a higher ethanol yield. Also the XR-XDH encoding genes could be integrated in the chromosomal DNA, whereas the construction of a functional XI-carrying S. cerevisiae strain required high-level expression of XI under the control of a strong promoter in a multicopy plasmid. Finally different promoters and XYL1 genes were evaluated under both aerobic and anaerobic conditions in isogenic strains engineered with the XR-XDH pathway. The fermentation of galactose was also investigated, either alone or in combination with other sugars. PGM2 overexpression reduced the total fermentation time by 34% while the anaerobic growth rate increased by 42%. Overexpression of PGM2 by integration of an additional copy was sufficient to give these positive effects, and allow for the construction of stable industrial strains. In addition, PGM2 overexpression improved xylose utilization. In mixed-sugar fermentation in galactose-containing medium, PGM2 overexpression, or the presence of glucose, reduced the time required to deplete all the sugar. Improved xylose and arabinose co-utilization was achieved with an industrial S. cerevisiae strain engineered via the fungal XR-XDH pathway and the bacterial arabinose pathway by evolutionary engineering. This resulted in increased levels of the heterologous xylose pathway enzymes, increased consumption rate of both xylose and arabinose, and increased transport capacity of xylose and arabinose.