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
付彤 1刘瑞曦 1王碧莹 1倪新 1杨帆1
作者信息
- 1. 大连工业大学 生物工程学院,辽宁 大连 116034
- 折叠
摘要
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.
Abstract
To improve the expression level of sucrose converting enzyme Suc2 in Saccharomyces cerevisiae,tandem double promoters were used to jointly initiate the expression of Suc 2 gene and an efficient strain for producing inulin based ethanol was constructed.A recombinant expression vector pYC230-DP-mSuc2 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.
关键词
酿酒酵母/蔗糖转化酶/双启动子/菊糖/乙醇Key words
Saccharomyces cerevisiae/invertase/double promoter/inulin/ethanol引用本文复制引用
基金项目
辽宁省教育厅科学技术研究项目(J2020041)
辽宁省自然科学基金项目(2020-MS-276)
出版年
2024
国家科技期刊平台
NSTL
万方数据