利用 Cpf1 与 Ku/LigD 对谷氨酸棒杆菌进行基因组编辑。

Genome editing of Corynebacterium glutamicum mediated with Cpf1 plus Ku/LigD.

机构信息

Key Laboratory of Veterinary Chemical Drugs and Pharmaceutics, Ministry of Agriculture, Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai, 200241, China.

出版信息

Biotechnol Lett. 2021 Dec;43(12):2273-2281. doi: 10.1007/s10529-021-03195-x. Epub 2021 Oct 20.

DOI:10.1007/s10529-021-03195-x

PMID:34669078

Abstract

OBJECTIVES

Corynebacterium glutamicum (C. glutamicum) has been harnessed for multi-million-ton scale production of glutamate and lysine. To further increase its amino acid production for fermentation industry, there is an acute need to develop next-generation genome manipulation tool for its metabolic engineering. All reported methods for genome editing triggered with CRISPR-Cas are based on the homologous recombination. While, it requires the generation of DNA repair template, which is a bottle-neck for its extensive application.

RESULTS

In this study, we developed a method for gene knockout in C. glutamicum via CRISPR-Cpf1-coupled non-homologous end-joining (CC-NHEJ). Specifically, CRISPR-Cpf1 introduced double-strand breaks in the genome of C. glutamicum, which was further repaired by ectopically expressed two NHEJ key proteins (Mycobacterium tuberculosis Ku and ligase D). We provide the proof of concept, for CC-NHEJ, by the successful knockout of the crtYf/e gene in C. glutamicum with the efficiency of 22.00 ± 5.56%, or something like that.

CONCLUSION

The present study reported a novel genome manipulation method for C. glutamicum.

摘要

目的

谷氨酸棒杆菌（Corynebacterium glutamicum）已被用于生产数百万吨规模的谷氨酸和赖氨酸。为了进一步提高其在发酵工业中的氨基酸产量，迫切需要开发下一代基因组操作工具用于其代谢工程。所有基于 CRISPR-Cas 的报道的基因组编辑方法都是基于同源重组的。然而，这需要生成 DNA 修复模板，这是其广泛应用的瓶颈。

结果

在本研究中，我们通过 CRISPR-Cpf1 偶联的非同源末端连接（CC-NHEJ）开发了一种在谷氨酸棒杆菌中进行基因敲除的方法。具体来说，CRISPR-Cpf1 在谷氨酸棒杆菌的基因组中引入双链断裂，然后通过异位表达两个 NHEJ 关键蛋白（结核分枝杆菌 Ku 和连接酶 D）进行修复。我们通过成功敲除谷氨酸棒杆菌中的 crtYf/e 基因证明了 CC-NHEJ 的概念，效率为 22.00±5.56%，或类似水平。