Construction of a CRISPR/nCas9-assisted genome editing system for exopolysaccharide biosynthesis in Streptococcus thermophilus. (August 2022)

• Record Type: Journal Article
• Authors: Kong, Linghui; Song, Xin; Xia, Yongjun; Ai, Lianzhong; Xiong, Zhiqiang
• Is Part Of: Food research international. Volume 158(2022)
• Publication Date: 2022-08
• Subjects: Lactic acid bacteria -- Streptococcus thermophilus -- CRISPR/nCas9 -- Gene editing -- Exopolysaccharide biosynthesis -- Eps gene cluster; Food -- Analysis -- Periodicals; Food industry and trade -- Periodicals; Food industry and trade -- Canada -- Periodicals; Food Technology -- Periodicals; Food -- Periodicals; Food-Processing Industry -- Periodicals; Aliments -- Industrie et commerce -- Périodiques; Aliments -- Industrie et commerce -- Canada -- Périodiques; Aliments -- Recherche -- Périodiques; Food industry and trade; Canada; Periodicals; Electronic journals; 664.005
• DOI: 10.1016/j.foodres.2022.111550
• Languages: English
• ISSNs: 0963-9969

Abstract

Graphical abstract: Highlights: A CRISPR/nCas9 genome editing method was developed for single and multiplex gene editing in Streptococcus thermophilus. The epsA, epsB and epsE were key genes in exopolysaccharide biosynthesis based on CRISPR/nCas9 analysis. The epsC, epsE and epsG could alter molecular weight and monosaccharide composition of exopolysaccharide, respectively. Abstract: Streptococcus thermophilus is an economically prominent starter for common dairy products due to its potential health and nutritional benefits. However, lack of precise genetic manipulation approaches has greatly hampered the industrial application of S. thermophilus .. Herein, we developed an efficient genome editing toolbox (pKLH353) based on CRISPR/nCas9 (Cas9 nickase) in S. thermophilus to seamlessly edit single or multiple genes. A native constitutive promoter library was used to optimize the nCas9 and sgRNA expression with gene deletion efficiencies of 14–60%. The epsA, epsB and epsE were identified as key genes affecting exopolysaccharide (EPS) biosynthesis in S. thermophilus S-3 using the CRISPR/nCas9 toolbox. Moreover, compared to the wild-type, knockout of epsC, epsE or epsG led to a decrease of EPS titer with reducing in its molecular weight (>2.5-fold) and intrinsic viscosity (>19.8-fold). The ratio of monosaccharide composition of the mutants has also changed, suggesting that these eps genes are involved in the chain length synthesis and repeat unit assembly. Taken together, this CRISPR/nCas9 system can serve as a basic toolkit for precise genetic engineering of S. thermophilus and facilitate strain engineering to produce bio-based products.