Triple deletion of clpC, porB, and mepA enhances production of small ubiquitin-like modifier-N-terminal pro-brain natriuretic peptide in Corynebacterium glutamicum
Triple deletion of clpC, porB, and mepA enhances production of small ubiquitin-like modifier-N-terminal pro-brain natriuretic peptide in Corynebacterium glutamicum
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clpC、porB 和 mepA 的三重缺失增强了谷氨酸棒杆菌中小泛素样修饰剂 N 端脑利钠肽前体的产生
DOI：
10.1007/s10295-018-2091-8
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发表时间：
2019-01-01
影响因子：
3.4
通讯作者：
Bai, Zhonghu
中科院分区：
文献类型：
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作者：
Peng, Feng;Liu, Xiuxia;Bai, Zhonghu
在我们之前的工作中，构建了用于谷氨酸棒杆菌基因组编辑的双质粒CRISPR/Cas9系统。为了提高转化效率并简化质粒固化步骤，构建了用于高效基因组编辑的一体化CRISPR/Cas9系统。此外，为了研究重组蛋白生产过程中的蛋白水解，并获得重组蛋白增强表达的宿主，利用该系统对C. 15647，分别编码Clp蛋白酶亚基ClpC、阴离子选择性通道蛋白B和金属肽酶A。在对不同质粒和宿主进行评估后，成功地在三突变clpCporBmepA中表达了用于诊断轻度心力衰竭的重要蛋白-小泛素样修饰物-N-末端脑钠肽前体（SUMO-NT-proBNP），其蛋白表达水平是野生型的3倍。总之，我们在C语言中创建了一个简化的CRISPR基因组编辑工具。glutamicum等，提供了一种产生用于增强表达重组蛋白的宿主的方法，并在C.谷氨酸。该工具和方法将极大地促进这一重要平台生物的基因工程和代谢优化。
In our previous work, a two-plasmid CRISPR/Cas9 system was constructed for genome editing in Corynebacterium glutamicum. To increase the transformation efficiency and simplify the plasmid curing steps, an all-in-one CRISPR/Cas9 system was constructed for efficient genome editing. In addition, to research proteolysis during the production of recombinant proteins and generate a host for enhanced expression of recombinant proteins, the system was used to delete three genes, clpC, porB, and mepA in C. glutamicum CGMCC1.15647, which encoded the Clp protease subunit ClpC, anion selective channel protein B, and metallopeptidase A, respectively. After the evaluation of different plasmids and hosts, small ubiquitin-like modifier-N-terminal pro-brain natriuretic peptide (SUMO-NT-proBNP), an important protein used for the diagnosis of mild heart failure was successfully expressed in the triple mutant clpCporBmepA, which exhibit threefold higher levels of protein expression compared with the wild-type. In conclusion, we created a simplified CRISPR tool for genome editing in C. glutamicum, provided a method to generate a host for enhanced expression of recombinant proteins and successfully expressed SUMO-NT-proBNP in C. glutamicum. This tool and method will greatly facilitate genetic engineering and metabolic optimization of this important platform organism.