葡萄糖-6-磷酸脱氢酶基因的Candida tropicalis过量表达及其对木糖醇合成代谢的影响

Cloning and Overexpression of Glucose-6-phosphate dehydrogenase in Candida tropicalis and Its Influence on Xylitol Biosynthesis

研究葡萄糖-6-磷酸脱氢酶基因g6pd过量表达对Candida tropicalis木糖醇生物合成代谢的影响。克隆Candida tropicalis CT16的g6pd基因,并将其与表达载体pYES-pgk重组连接,构建重组载体pYES-pgk-g6pd,LiAc/ssDNA/PEG方法转化导入C.tropicalis CT16,筛选阳性转化子,实现g6pd基因的过量表达。结果表明:发酵62 h,阳性转化子C.tropicalis SYG5的葡萄糖-6-磷酸脱氢酶活力提高了300%,发酵液中木糖醇质量浓度达到79.90 g/L,较野生型对照菌株的木糖醇产量提高了12.41%,木糖醇产率提高了44.94%。因此,葡萄糖-6-磷酸脱氢酶在C.tropicalis木糖醇的合成代谢途径中发挥重要作用,增强g6pd基因的表达,可以明显提高菌体NADPH供应量和还原力,有利于木糖醇的生物合成。

The influence of overexpression of glucose 6-phosphate dehydrogenase （G6PDH） gene,g6pd,on xylitol biosynthesis in Candida tropicalis was investigated.The gene g6pd was cloned from Candida tropicalis CT16 and inserted into a yeast expression vector pYES-pgk,generating a recombinant expression vector pYES-pgk-g6pd,which was then introduced into C.tropicalis CT16 by the LiAc/ssDNA/PEG transformation method,resulting in over-expression of the g6pd gene.The fermentation results showed that the G6PDH activity was increased by 300％,and a maximum xylitol yield of 79.90 g/L was achieved in the recombinant strain SYG5 harboring pYES-pgk-g6pd after 62 h of fermentation.Compared to the wild type strain C.tropicalis CT16,the yield and productive rate of xylitol in strain SYG5 were increased by 11.30％ and 44.91％,respectively.These results indicate that increasing the expression level of the gene g6pd significantly enhances xylitol production and that the G6PDH plays a key role in the biosynthesis pathway of xylitol in C.tropicalis.