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

Streptococcus thermophilus is used extensively in the dairy industry and has shown great promise as a chassis cell for the biosynthesis of high-value metabolites. However, metabolic engineering in S. thermophilus lacks effective genetic modification tools to modulate 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. thermophilus. Our CRISPRi system typically achieved 66 to 98% knockdown of single or multiple gene expression. We used CRISPRi for the biosynthesis of a new exopolysaccharide (EPS) as a paradigm model. Repression 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 compared with a control strain. This study demonstrated the effectiveness of CRISPRi as a powerful metabolic engineering tool and synthetic biology strategy for S. thermophilus.