Developing a RecT-assisted endogenous CRISPR/SzCas9 system for precise genome editing in Streptococcus zooepidemicus.

Autor: Zhang X; State Key Laboratory of Microbial Technology, Shandong University, Qingdao 266237, People's Republic of China., Wang F; State Key Laboratory of Microbial Technology, Shandong University, Qingdao 266237, People's Republic of China., Guo X; Bloomage Biotechnology Corporation Limited, Jinan 250101, People's Republic of China., Qiao L; Bloomage Biotechnology Corporation Limited, Jinan 250101, People's Republic of China., Luo X; Bloomage Biotechnology Corporation Limited, Jinan 250101, People's Republic of China., Cui L; Bloomage Biotechnology Corporation Limited, Jinan 250101, People's Republic of China., Liang Q; State Key Laboratory of Microbial Technology, Shandong University, Qingdao 266237, People's Republic of China. Electronic address: liangquanfeng@sdu.edu.cn., Liu M; State Key Laboratory of Microbial Technology, Shandong University, Qingdao 266237, People's Republic of China. Electronic address: liummeng@sdu.edu.cn., Qi Q; State Key Laboratory of Microbial Technology, Shandong University, Qingdao 266237, People's Republic of China.
Jazyk: angličtina
Zdroj: International journal of biological macromolecules [Int J Biol Macromol] 2024 Dec 12; Vol. 291, pp. 138758. Date of Electronic Publication: 2024 Dec 12.
DOI: 10.1016/j.ijbiomac.2024.138758
Abstrakt: Streptococcus zooepidemicus is the most commonly utilized strain for industrial-scale hyaluronic acid (HA) production due to its capacity to produce high-quality HA. However, the lack of efficient genetic manipulation approaches has significantly hindered the industrial application of this species. In this study, we developed a RecT-assisted endogenous CRISPR/SzCas9 system to enable markerless gene deletion, gene substitution, stop codon insertion, and iterative editing in the industrially significant strain S. zooepidemicus for hyaluronic acid production. A variety of editing outcomes were achieved, including a 3186-bp deletion of the hylb gene, replacement of VHb, and insertion of stop codons into the sagA gene. Ultimately, we successfully developed an engineered strain, designated SD-3, which exhibited reduced toxicity and non-hemolytic properties. The hyaluronic acid production of SD-3 increased by 39.7%, reaching 10.8 g/L in a 5-L fermenter. This achievement represents the highest titer of high molecular weight hyaluronic acid to date, characterized by a uniform and high molecular weight of up to 2.8 × 10 6  Da.
Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
(Copyright © 2024 Elsevier B.V. All rights reserved.)
Databáze: MEDLINE
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