酿酒酵母菌中过量表达BAT2和缺失PDC6提高异丁醇产率

Overexpression of BAT2 and deletion of PDC6 to increase isobutanol in Saccharomyces cerevisiae

利用酿酒酵母发酵生产生物质能源异丁醇越来越受到学术界和工业界的重视。本研究通过DNA重组技术,提出一种提高酿酒酵母合成异丁醇的产率的方法。通过对酿酒酵母异丁醇合成代谢途径分析,过量表达了编码分支氨基酸转氨酶的BAT2基因和缺失编码内源性丙酮酸脱羧酶的PDC6基因,同时构建过量表达编码乙酰乳酸合酶ILV2基因和编码二羟基戊酸脱水酶的ILV3基因的表达质粒。将所构建突变株HAZL-7pILV2pILV3(W303-1A PGK1p-BAT2pdc6::R YEplac195-ILV3p-PGK1p-ILV3 YEplac181-PGK1p-ILV2)进行厌氧发酵。厌氧发酵60h,发酵液中异丁醇的产率从0.035g/L到0.4g/L,提高了11.4倍,乙醇的产率减少,乙酸的产率升高,甘油的产率基本稳定。酿酒酵母菌中过表达BAT2基因和缺失PDC6基因,同时过量表达ILV2和ILV3的表达质粒载体的基因修饰,对异丁醇的代谢流有明显的影响,异丁醇的产率提高11.4倍。这些研究为异丁醇产量的进一步提高奠定了理论基础。

Biomass isobutanol produced by Saccharomyces cerevisiae has been paid more and more attention.In this research,a method of increasing isobutanol yieldwas put forward by recombinant DNA technology.By the analysis of isobutanol synthesis pathway in Saccharomyces cerevisiae ,BAT2 （encoding branched chain amino acids transaminase）,ILV2 （encoding acetolactate synthase）and ILV3 （encoding dihydroxy-acid dehydratase）were overpressed in this study.Meanwhile,endogenous pyruvate de-carboxylase encoded by PDC6 was deleted.Anaerobic fermentation of the mutant HAZL-7 pILV2 pILV3 （W303-1A PGK1p-BAT2 pdc6 ：：R YEplac195-ILV3p-PGK1p-ILV3 YEplac181-PGK1p-ILV2 ）were performed.After fermentation 60 hours, isobutanol production was from 0.035 g/L to 0.4 g/L,11.4-fold increase,ethanol production was decrease,acetic acid produc-tion was increase,and glycerol production was stable in the fermentation broth.In Saccharomyces cerevisiae ,the modification of overexpressed BAT2 ,deleted PDC6 and constructed expression plasmid of ILV2 and ILV3 have a significant effect on metabolic flux of isobutanol.Isobutanol production was increased by 11.4 times.These results provide a theoretical basis for the further improvement of the yield of isobutanol.