为探究酵母菌以木糖为底物生产D-阿拉伯糖醇的可行性,该研究以热带假丝酵母(Candida tropicalis)CU-208为出发菌株,通过代谢工程构建利用木糖生产D-阿拉伯糖醇的菌株。在验证了热带假丝酵母内源D-阿拉伯糖醇脱氢酶(D-arabitol dehydrogenase,ARD)功能的基础上,进行如下代谢改造:先敲除木酮糖激酶(xyluki- nase,XKS)基因,以阻断木糖进入磷酸戊糖途径,同时过表达上游木糖醇脱氢酶(xylitol dehydrogenase,XDH)基因,构建菌株FYM01;再敲除ARD基因ard,减少菌株对D-阿拉伯糖醇的消耗,构建菌株FYM02;然后,过表达来源于圆红冬孢酵母菌的外源D-阿拉伯糖醇脱氢酶(D-arabitol dehydrogenase,ADH)基因,从木酮糖合成D-阿拉伯糖醇,构建菌株FYM04;最后,过表达内源葡萄糖-6-磷酸脱氢酶(glucose-6-phosphate dehydrogenase,ZWF)基因,以增强辅酶NADPH供应,构建菌株FYM05。结果发现,基因ard 参与代谢D-阿拉伯糖醇;代谢改造获得的菌株FYM05,以葡萄糖为辅助碳源,利用木糖生产D-阿拉伯糖醇,菌株FYM05摇瓶发酵的D-阿拉伯糖醇产量为11.35 g/L,是出发菌株的11.7倍。该研究通过代谢工程手段成功构建了1株能够利用木糖生产D-阿拉伯糖醇的重组热带假丝酵母,为热带假丝酵母工业化生产D-阿拉伯糖醇奠定基础。
In order to explore the feasibility of yeast in producing D-arabitol using xylose as a substrate, based on Candida tropicalis CU-208, an engineered strain for D-arabitol production was constructed by means of metabolic engineering in this study. Based on the verification of the endogenous D-arabitol dehydrogenase(ARD)function of C. tropicalis, the following metabolic modifications were conducted. Firstly, xks gene was knocked out to block xylose to enter the pentose-phosphate pathway, and xdh gene was overexpressed to construct strain FYM01. Then, ard gene was knocked out to block D-arabitol consumption to construct strain FYM02. Next, adh gene was overexpressed to construct strain FYM04. Finally, zwf gene was overexpressed to enhance the NADPH coenzyme supply, and strain FYM05 was construct. The results showed that ard gene was involved in the catabolism of D-arabitol. Besides, the strain FYM05 obtained via metabolic transformation could not only utilize glucose as an auxiliary carbon source to provide growth energy and reducing power, but also use xylose to produce D-arabitol. The yield of D-arabitol in shake flask by strain FYM05 reached 11.35 g/L, which is 11.7 times higher than that of the parental strain. In brief, in this study, a recombinant C. tropicalis that is capable of producing D-arabitol from xylose was successfully constructed through metabolic engineering, which laid the foundation for the industrial production of D-arabitol by C. tropicalis.
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