异源转氢酶NNT对酿酒酵母木糖代谢的影响

表达有毕赤酵母木糖还原酶(XR)和木糖醇脱氢酶(XDH)的重组酿酒酵母,能代谢木糖.但是XR和XDH分别偏好辅酶NADPH和NAD⁺,造成辅酶的不平衡和副产物的积累,所以重组酿酒酵母利用木糖产生乙醇的效率很低.转氢酶可以催化辅酶NADPH和NADH之间的相互转化,因此本实验将黑曲霉的转氢酶基因NNT转入到重组酿酒酵母中,通过实验确定了NNT基因的表达蛋白在酵母细胞内定位于线粒体中,NNT基因分别用pPGK1、pCCW12和pHXT7启动子进行表达,在微好氧的木糖发酵条件下,NNT基因的导入使酿酒酵母甘油产量下降,乙醇产率提高,在由pCCW12和pHXT7表达NNT基因的重组酿酒酵母中,木糖醇产率分别下降86.3%和49.3%,乙醇产率提高16.7%和12.7%,说明转氢酶NNT的存在改善了木糖代谢的辅酶不平衡,提高了乙醇的转化率.

Effects on Xylose Fermentation in Recombinant Saccharomyces cerevisiae with the Heterologous Transhydrogenase NNT

The recombinant Saccharomyces cerevisiae which encoded the genes xylose reductase (XR) and xylitol dehydrogenase(XDH) from Pichia stipitis, can utilize xylose. However, XR and XDH prefer NADPH and NAD⁺ respectively, create cofactor imbalance. As a result, xylose fermentation to ethanol with recombinant Saccharomyces cerevisiae was ineffective. NADPH and NADH can be converted to one another by transhydrogenase. So in this study, the gene NNT from Aspergillus niger was introduced into recombinant S.cerevisiae. The result of experiment demonstrated the expression protein of gene NNT was positioned at chondriosome. Gene NNT was expressed by the promoters pPGK1, pCCW12 and pHXT7 respectively. Under micro aerobic condition of xylose fermentation, the formation of glycerol was decreased and yield of ethanol was increased in the yeast carrying NNT. In recombinant yeasts with promoter pPGK1 and pCCW12, the yield of xylitol decreased by 86.3% and 49.3%, and the yields of ethanol increased by 16.7% and 12.7% respectively, which illustrated the transhydrogenase improved cofactor imbalance of xylose fermentation and increased ethanol conversion.