Research On The Expression Strategy Of Xylose Metabolism Relative Genes In Saccharomyces Cerevisiae And Its Application

Xylose, as a fermentable sugar, is the second most abundant of the lignocellulose hydrolysis product. The ethanol production from xylose is of great significance to alleviate oil crisis. Saccharomyces cerevisiae could not use xylose to produce ethanol directly. To construct xylose metabolic pathway in S. cerevisiae is a way for ethanol production from lignocellulose, and how to construct an efficient metabolic pathway is a key problem. In this study, xylose metabolism genes were differentially expressed in S. cerevisiae using the osmo-responsive promoters from Candida glycerinogenes, to improve the activity of xyltiol dehydrogenase and reduce the accumulation of xylitol(by-product), promoting the xylose synthesis and ethanol consumption.The xylose reductase(XR) gene XYL1 and xylitol dehydrogenase(XDH) gene XYL2 were obtained from C. glycerinogene, and xyluose kinase gene XKS1 obtained from S. cerevisiae. To construct the xylose metabolism recombinant strain, the multi-copy plasmid pYX212 was used to overexpress XYL2, while the single-copy plasmid p414 was used to express XYL1 and XKS1. The activity ratio of XR: XDH: XK in the recombinant strain was 1: 5: 4. The recombinant strain was able to consume 8.59 g·L-1 xylose at 72 h, while ethanol and xylitol were 2.58 g·L-1 and 4.30 g·L-1, respectively, indicating that the low XDH activity was still the main reason for xylitol accumulation. Meanwhile, XR was proved to prefer NADPH 5 times more than NADH, and XDH was exclusively depended on NAD+.To improve the activity of xylitol dehydrogenase, recombinant strains were constructed to express XYL2 using osmo-responsive promoters PCgSTL3, PCgZWF and PCgGPD from C. glycerinogenes by the multi-copy plasmid pYX212. It showed that the recombinant strain whose XYL2 gene was expressed under osmo-responsive promoter PCgGPD exhibited the most sensitivity to osmotic pressure. And the XYL2 transcriptional level and XDH activity were increased by 3.5 and 1.7 times, respectively, when it was cultured with 100 g·L-1 xylose in YEPX. Furthermore, XYL2 transcriptional level and XDH activity were increased by 4.8 and 2.8 times, respectively, when 0.4 mol·L-1 NaCl was added in the culture. The result provided a new method for the high expression of XYL2.The fermentation performance of recombinant strain with XYL2 gene was expressed under osmo-responsive promoter PCgGPD was analyzed. When the recombinant was cultured in YEPX with 0.4 mol·L-1 NaCl, it was able to consume 20 g·L-1 xylose, while the ethanol was increased from 5.65 g·L-1 to 7.95 g·L-1 and the xylitol yield was decreased by 52.0%, and the conversion rate of xylose to ethanol was 0.40 g·g-1. When the recombinant was cultured in YEPDX with 0.4 mol·L-1 NaCl, the xylose consumption was increased by 62.3%, while the ethanol yield was increased from 0.31 g·g-1 to 0.34 g·g-1. The results indicated that the XDH activity can be significantly improved by the overexpression of XYL2 gene using an osmo-responsive promoter PCgGPD. This study provided a reference for improving the capacity to efficiently convert xylose to ethanol by S. cerevisiae.