Wang Yukun, Zhang Sitong, Chen Guang
Key Laboratory of Straw Biology and Utilization, Ministry of Education, Jilin Agricultural University, Changchun 130118, Jilin, China.
College of Life Sciences, Jilin Agricultural University, Changchun 130118, Jilin, China.
Sheng Wu Gong Cheng Xue Bao. 2020 Oct 25;36(10):2193-2205. doi: 10.13345/j.cjb.200110.
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.
内切葡聚糖酶（EG）是纤维素酶的重要组成部分，在纤维素降解中发挥着重要作用。然而，由于天然微生物产生的内切葡聚糖酶产量较低，其应用受到限制。内切葡聚糖酶的高效异源表达是解决这一问题的有效途径。为了获得高产内切葡聚糖酶的工程酿酒酵母，从食纤维梭菌中克隆了内切葡聚糖酶基因，全长1996 bp，编码440个氨基酸，并通过重叠延伸PCR（SOE PCR）将来自酿酒酵母的PGK启动子序列、来自pPIC9K质粒的α信号肽序列和来自pSH65质粒的CYC1终止子序列构建成完整的表达盒（PαEGC），通过rDNA整合构建了内切葡聚糖酶在酿酒酵母中的表达载体。探索了拷贝数与蛋白表达之间的关系。在酿酒酵母中进行了内切葡聚糖酶的随机多拷贝表达。通过液滴数字PCR鉴定内切葡聚糖酶的拷贝数，并探索拷贝数与蛋白表达之间的关系。通过rDNA整合分别获得了拷贝数为1、3、4、7、9、11、15、16、19、21、22和23的内切葡聚糖酶工程酿酒酵母。结果表明，当拷贝数为15时，酶活性最高，即351 U/mL。成功构建了内切葡聚糖酶工程酿酒酵母菌株，可为其他工业酶的异源表达提供参考。
