高效表达内切葡聚糖酶酿酒酵母工程菌的构建

Construction of an engineered Saccharomyces cerevisiae expressing endoglucanase efficiently

摘要:

内切葡聚糖酶 (EG) 是纤维素酶的重要组分，在纤维素降解酶系中发辉重要作用。由于天然微生物来源的内切葡聚糖酶产量低，极大地制约了其生产和应用，所以对内切葡聚糖酶进行高效异源表达是解决这一问题的有效途径。为了获得高效内切葡聚糖酶酿酒酵母工程菌，本研究从纤维梭菌中克隆了内切葡聚糖酶 (EG) 基因，全长1 996 bp，编码440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法构建完整表达盒 (PαEGC)，通过整合rDNA的方法构建内切葡聚糖酶酿酒酵母的表达载体，在酿酒酵母中进行内切葡聚糖酶的随机多拷贝表达。利用微滴数字PCR鉴定内切葡聚糖酶拷贝数，并探索拷贝数与蛋白表达量之间的关系。通过rDNA整合法获得了拷贝数为1、3、4、7、9、11、15、16、19、21、22、23的内切葡聚糖酶酿酒酵母工程菌，结果表明当拷贝数为15时，酶活性最高，为351 U/mL。本研究成功构建了内切葡聚糖酶酿酒酵母工程菌，为其他工业酶异源高效表达提供参考和借鉴。

Abstract:

Endoglucanase (EG) is an important component of cellulases and play an important role in cellulose degradation. However, its application is limited due to the low yield of endoglucanase from natural microorganisms. Efficient heterologous expression of endoglucanase is an effective way to solve this problem. To obtain the engineered Saccharomyces cerevisiae for high-yield endoglucanase, endoglucanase gene was cloned from Clostridium cellulovorans, with a total length of 1 996 bp, encoding 440 amino acids, and the complete expression cassette (PαEGC) was constructed with the PGK promoter sequence from Saccharomyces cerevisiae, α-signal peptide sequence from pPIC9K plasmid and CYC1 terminator sequence from pSH65 plasmid by gene splicing by overlap extension PCR (SOE PCR), and the expression vector of endoglucanase in Saccharomyces cerevisiae was constructed by rDNA integration. The relationship between copy number and protein expression was explored. Random multicopy expression of endoglucanase was performed in Saccharomyces cerevisiae. The copy number of endoglucanase was identified by Droplet Digital PCR and explore the relationship between copy number and protein expression.The engineered Saccharomyces cerevisiae of endoglucanase with copy numbers of 1, 3, 4, 7, 9, 11, 15, 16, 19, 21, 22 and 23 were obtained by rDNA integration, respectively. The results showed that when the copy number was 15, the enzyme activity was the highest, namely 351 U/mL. The engineered strain of Saccharomyces cerevisiae for endoglucanase was successfully constructed, which can provide reference for the heterologous expression of other industrial enzymes.