Xylulokinase overexpression in two strains of Saccharomyces cerevisiae also expressing xylose reductase and xylitol dehydrogenase and its effect on fermentation of xylose and lignocellulosic hydrolysate

Fermentation of the pentose sugar xylose to ethanol in lignocellulosic biom ass would make bioethanol production economically more competitive. Sacchar omyces cerevisiae, an efficient ethanol producer, can utilize xylose only w hen expressing the heterologous genes XYL1 (xylose reductase) and XYL2 (xyl itol dehydrogenase). Xylose reductase and xylitol dehydrogenase convert xyl ose to its isomer xylulose. The gene XKS1 encodes the xylulose-phosphorylat ing enzyme xylulokinase. In this study, we determined the effect of XKS1 ov erexpression on two different S. cerevisiae host strains, H158 and CEN.PK, also expressing XYL1 and XYL2. H158 has been previously used as a host stra in for the construction of recombinant xylose-utilizing S. cerevisiae strai ns. CEN.PK is a new strain specifically developed to serve as a host strain for the development of metabolic engineering strategies. Fermentation was carried out in defined and complex media containing a hexose and pentose su gar mixture or a birch wood lignocellulosic hydrolysate. XKS1 overexpressio n increased the ethanol yield by a factor of 2 and reduced the xylitol yiel d by 70 to 100% and the final acetate concentrations by 50 to 100%. However , XKS1 overexpression reduced the total xylose consumption by half for CEN. PK and to as little as one-fifth for H158. Yeast extract and peptone partly restored sugar consumption in hydrolysate medium. CEN.PK consumed more xyl ose but produced more xylitol than H158 and thus gave lower ethanol yields on consumed xylose. The results demonstrate that strain background and modu lation of XKS1 expression are important for generating an efficient xylose- fermenting recombinant strain of S. cerevisiae.