高效组成型分泌表达木素过氧化物酶酿酒酵母工程菌的构建

Construction of an engineered Saccharomyces cerevisiae for lignin peroxidase production

摘要:

[目的]为了获得高产木素过氧化物酶酿酒酵母工程菌。[方法]本研究从黄孢原毛平革菌中克隆了木素过氧化物酶（lignin peroxidases，LiP）基因，全长2193 bp，编码371个氨基酸，并与来源于酿酒酵母的PGK启动子序列、来源于pPIC9K质粒的α-信号肽序列以及来源于pSH65质粒的CYC1终止子序列通过重叠延伸PCR构建完整表达盒（PαLiC），利用rDNA整合法构建木素过氧化物酶酿酒酵母表达载体，实现木素过氧化物酶在酿酒酵母中的多拷贝表达。利用数字微滴PCR技术对拷贝数进行鉴定，探究拷贝数与蛋白表达量之间的关系。[结果]通过rDNA整合法得到拷贝数为1、2、4、5、6、7、8、9、10、11、12和13的木素过氧化物酶酿酒酵母工程菌，通过对其酶活测定，表明当拷贝数为7时，酶活力最高，为367 U/L。[结论]本研究在酿酒酵母中表达了木素过氧化物酶，研究了其基因拷贝数与酶活性的关系，对木质素降解技术的发展具有重要意义。

关键词:酿酒酵母;木素过氧化物酶;rDNA整合法;微滴数字PCR技术

Abstract:

[Objective] To obtain engineered Saccharomyces cerevisiae with high-yield of lignin peroxidase. [Methods] We cloned a constitutive promoter PGK, an exogenous protein secretion signal peptide α-factor, lignin peroxidases (LiP) genes and a terminator CYC1. We constructed complete expression box (PαLC) by overlap extension PCR method. Then, we established expression vector of lignin peroxidase S. cerevisiae via rDNA integration method, to achieve multi-copy expression of lignin peroxidase in S. cerevisiae. Then, we identified the copy number via droplet digital PCR technology, to explore the relationship between the copy number and protein expression amount. [Results] We got the engineered strain of S. cerevisiae to produce lignin peroxidase with the copy number of 1, 2, 4, 5, 6, 7, 8, 9, 10, 11, 12 and 13 via rDNA integration method, through enzymatic activity determination, it shows that the enzymatic activity is as highest as 367 U/L when the copy number is 7. [Conclusion] In this study, lignin peroxidase was expressed in S. cerevisiae, and the relationship between gene copy number and enzyme activity was studied, which is of great significance to the development of lignin degradation technology.

Key words:Saccharomyces cerevisiae;lignin peroxidase;rDNA integration method;droplet digital PCR technology