Balance of XYL1 and XYL2 expression in different yeast chassis for improved xylose fermentation

Reducing xylitol formation is necessary in engineering xylose utilization in recombinant Saccharomyces cerevisiae for ethanol production through xylose reductase/xylitol dehydrogenase pathway. To balance the expression of XYL1 and mutant XYL2 encoding xylose reductase (XR) and NADP(+)-dependent xylitol dehydrogenase (XDH), respectively, we utilized a strategy combining chassis selection and direct fine-tuning of XYL1 and XYL2 expression in this study. A XYL1 gene under the control of various promoters of ADH1, truncated ADH1 and PGK1, and a mutated XYL2 with different copy numbers were constructed into different xylose-utilizing modules, which were then expressed in two yeast chassises W303a and L2612. The strategy enabled an improved L2612-derived recombinant strain with XYL1 controlled by promoter PGK1 and with two copies of XYL2. The strain exhibited a 21.3% lower xylitol yield and a 40.0% higher ethanol yield. The results demonstrate the feasibility of the combinatorial strategy for construction of an efficient xylose-fermenting S. cerevisiae.