双启动子过表达蔗糖转化酶Suc2酿酒酵母菌株的构建

Construction of double promoter drived invertase Suc2 overexpression Saccharomyces cerevisiae strain

为了提高蔗糖转化酶Suc2在酿酒酵母中的表达水平,本研究利用串联双启动子联合启动Suc2基因表达,构建高效菊糖基乙醇的生产菌株。首先构建含有串联磷酸甘油酸激酶组成型强启动子PGK1p的Suc2重组表达载体pYC230-DP-mSuc2,电击转化至酿酒酵母BY4741中,成功构建重组菌BY-DP。经实时荧光定量PCR、高效气相色谱和摇瓶培养验证,证明了在含15%菊糖的培养基中发酵48 h后,重组菌BY-DP具有更高的Suc2基因转录水平、酶活以及乙醇产量,与含单个PGK1p启动子的对照重组菌BY-mS相比,分别提高了30.0%、18.6%和25.3%。实验通过构建串联双PGK1p启动子,有效强化了Suc2的表达,提高了重组菌发酵菊糖生产乙醇能力。

To improve the expression level of sucrose converting enzyme Suc2 in Saccharomyces cerevisiae,tandem double promoters were used to jointly initiate the expression of Suc2 gene and an efficient strain for producing inulin based ethanol was constructed.A recombinant expression vector pYC230-DP-m Suc2 containing a strong promoter PGK1p composed of tandem phosphoglycerate kinase was constructed,which was transformed into Saccharomyces cerevisiae BY4741 by electric shock,to construct the recombinant strain BY-DP.Through real-time fluorescence quantitative PCR,high-performance gas chromatography and shake flask culture validation,it was demonstrated that the recombinant strain BY-DP had higher gene transcription levels of Suc2,enzyme activity and ethanol production after fermentation for 48 h in a medium containing 15%inulin,increased by 30.0%,18.6%,and 25.3%,respectively,compared with the control recombinant strain BY-mS containing a single PGK1p promoter.The expression of Suc2 was effectively enhanced,and the ability of recombinant bacteria to ferment inulin and produce ethanol was improved by constructing a tandem double PGK1p promoter.