Details

Autor(en) / Beteiligte

• Chen, Rui
• Shi, Feng
• Xiang, Youhe
• Lai, Wenmei
• Ji, Guohui

Titel

Establishment of CRISPR-Cpf1-assisted gene editing tool and engineering of 4-hydroxyisoleucine biosynthesis in Corynebacterium glutamicum

Ist Teil von

• World journal of microbiology & biotechnology, 2023-10, Vol.39 (10), p.266-266, Article 266

Ort / Verlag

Dordrecht: Springer Netherlands

Erscheinungsjahr

2023

Link zum Volltext

Quelle

Alma/SFX Local Collection

Beschreibungen/Notizen

• Corynebacterium glutamicum , an important industrial producer, is a model microorganism. However, the limited gene editing methods and their defects limit the efficient genome editing of C. glutamicum . To improve the screening efficiency of second-cross-over strains of traditional SacB editing system, a universal pCS plasmid which harbors CRISPR-Cpf1 system targeting kan gene of SacB system was designed and established to kill the false positive single-cross-over strains remained abundantly after the second-cross-over events. The lethality of pCS plasmid to C. glutamicum carrying kan gene on its genome was as high as 98.6%. In the example of P odhA ::P ilvBNC replacement, pCS plasmid improved the screening efficiency of second-cross-over bacteria from 5% to over 95%. Then this pCS-assisted gene editing system was applied to improve the supply of precursors and reduce the generation of by-products in the production of 4-hydroxyisoleucine (4-HIL). The 4-HIL titer of one edited strain SC01- TD5 I M reached 137.0 ± 33.9 mM, while the weakening of lysE by promoter engineering reduced Lys content by 19.0–47.7% and 4-HIL titer by 16.4–64.5%. These editing demonstrates again the efficiency of this novel CRISPR-Cpf1-assisted gene editing tool, suggesting it as a useful tool for improving the genome editing and metabolic engineering in C. glutamicum .