Zhang Wei, Zhao Zihao, Yang Yankun, Liu Xiuxia, Bai Zhonghu
National Engineering Laboratory for Cereal Fermentation Technology, Jiangnan University, Wuxi 214122, China; The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China.
National Engineering Laboratory for Cereal Fermentation Technology, Jiangnan University, Wuxi 214122, China; The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China.
Plasmid. 2017 Nov;94:1-6. doi: 10.1016/j.plasmid.2017.09.001. Epub 2017 Oct 3.
Corynebacterium glutamicum is an attractive host for the production of heterologous proteins despite its traditional use in fermentative production of amino acids. To enhance the expression levels of target genes, the development of useful promoters is required in the construction of expression systems. Here, we developed a new promoter, the aph promoter from aminoglycoside-3'-phosphotransferase gene, and used it to construct monocistronic and bicistronic expression systems that host different ribosome binding site (RBS) sequences. First, the expression level of the reporter protein, enhanced green fluorescent protein (EGFP), varied with changes in the RBS sequences in the constructed vectors. The results showed that the fluorescence intensities of the bicistronic group were higher than those of the monocistronic group and that RM3E showed the highest fluorescence intensity, which was 42-fold higher than the lowest (RA2E') among these groups. Next, taking advantage of the optimized aph promoter, we successfully employed this aph promoter for α-amylase and VH (camelid antibody fragment) expression. The secretion of α-amylase improved 1.5-fold after promoter mutation. This promoter will be useful for heterologous protein production in C. glutamicum cells.
尽管谷氨酸棒杆菌传统上用于氨基酸的发酵生产，但它仍是生产异源蛋白的理想宿主。为提高目标基因的表达水平，在构建表达系统时需要开发有用的启动子。在此，我们开发了一种新的启动子，即来自氨基糖苷-3'-磷酸转移酶基因的aph启动子，并用它构建了宿主不同核糖体结合位点（RBS）序列的单顺反子和双顺反子表达系统。首先，报告蛋白增强型绿色荧光蛋白（EGFP）的表达水平随构建载体中RBS序列的变化而变化。结果表明，双顺反子组的荧光强度高于单顺反子组，且RM3E的荧光强度最高，比这些组中最低的（RA2E'）高42倍。接下来，利用优化后的aph启动子，我们成功地将该aph启动子用于α-淀粉酶和VH（骆驼科抗体片段）的表达。启动子突变后，α-淀粉酶的分泌提高了1.5倍。该启动子将有助于谷氨酸棒杆菌细胞中异源蛋白的生产。
