Design of a Saccharomyces cerevisiae strain capable of simultaneously utilizing cellobiose and xylose

Saccharomyces cerevisiae has been widely utilized as a platform microorganism for bioethanol production from lignocelluloses. However, glucose repression limits efficient ethanol production because glucose in lignocellulosic hydrolysates inhibits xylose and other sugars’ utilization. As a result, it is attractive to construct a glucose derepressed S. cerevisiae strain for efficient utilization of lignocellulosic sugars. In this thesis, we proposed and constructed an artificial cellobiose assimilating pathway consisting of a cellobiose transporter and a β-glucosidase in S. cerevisiae. A total of six different cellobiose assimilating pathways were constructed and compared in a laboratory S. cerevisiae strain capable of xylose utilization and the one with best fermentation performance was selected. The resultant yeast strain showed significantly improved cellobiose and xylose consumption ability and ethanol productivity in both shake-flask and bioreactor fermentation. The xylose consumption rate was enhanced by 42% to 0.68 g L-1 h-1 in the engineered laboratory strain, and a maximum ethanol productivity of 0.49 g L-1 h-1was obtained, with no obvious glucose repression phenomenon observed. The maximum ethanol yield achieved was 0.39 g per g sugar. In addition, the best cellobiose assimilating pathway was also transferred to an industrial yeast strain and the resultant industrial strain showed greatly improved fermentation performance. The ethanol productivity was 0.64 g L-1 h-1, the ethanol yield was 0.42 g per g sugar, and the cellobiose consumption rate was more than 1.77 g L-1 h-1, which enables fast and efficient ethanol production from lignocelluloses. Thus this approach has been demonstrated to be a promising method to overcome glucose repression and at the same time enhance ethanol productivity. iii It was found that a small amount of glucose was accumulated during either cellobiose fermentation or cellobiose and xylose co-fermentation, which inevitably decreased the ethanol yield and productivity. To address this limitation, the role of mutarotase, also called aldose 1-epimerase, which is capable of converting glucose between two anomers was investigated. Three endogenous mutarotase genesYHR210c, YNR071c and GAL10 were identified in S. cerevisiae s288c wild type strain. The natural cellobiose assimilating strain Neurospora crassa also has a mutarotase gene named NCU09705. Overexpression of both S. cerevisiae and N. crassa aldose 1-epimerases showed improved sugar consumption and ethanol production in cellobiose assimilating S. cerevisiae strains and aldose 1-epimerase disrupted S. cerevisiae strains derived from the s288c strain showed significant drawbacks in cellobiose utilization