CRISPR/dCas9-based metabolic pathway engineering for the systematic optimization of exopolysaccharide biosynthesis in Streptococcus thermophilus

Streptococcus thermophilus is used extensively in the dairy industry and has shown great promise as a chas-sis cell for the biosynthesis of high-value metabolites. However, metabolic engineering in S. thermophilus lacks effective genetic modification tools to modu-late gene expression to relieve metabolic burden and maximize the production of desired compounds. Here, we developed a clustered regularly interspaced short palindromic repeats interference (CRISPRi) system for efficient gene transcriptional modulation in S. ther-mophilus. Our CRISPRi system typically achieved 66 to 98% knockdown of single or multiple gene expres-sion. We used CRISPRi for the biosynthesis of a new exopolysaccharide (EPS) as a paradigm model. Repres-sion of galK at module of uridine diphosphate glucose sugar metabolism and overexpression of epsA and epsE at EPS synthesis module resulted in an approximately 2-fold increase in EPS titer (277 mg/L) when com-pared with a control strain. This study demonstrated the effectiveness of CRISPRi as a powerful metabolic engineering tool and synthetic biology strategy for S. thermophilus.