Development of CRISPR Interference (CRISPRi) Platform for Metabolic Engineering of Leuconostoc citreum and Its Application for Engineering Riboflavin Biosynthesis

• Published in: International journal of molecular sciences 2020-08, Vol.21 (16), p.5614
• Main Authors: Son, Jaewoo, Jang, Seung Hoon, Cha, Ji Won, Jeong, Ki Jun
• Format: Article
• Language: English
• Subjects: Bacteria; Binding sites; Biosynthesis; Cell growth; Chromosomes; Cloning; CRISPR; CRISPR-Cas Systems - genetics; Cytotoxicity; Gene expression; Gene manipulation; Genetic engineering; Genomes; Growth rate; Humans; Lactic acid; Lactic Acid - metabolism; Lactic acid bacteria; Leuconostoc - genetics; Leuconostoc - metabolism; Metabolic Engineering; Plasmids; Plasmids - genetics; Probiotics; Probiotics - metabolism; Proteins; Riboflavin; Riboflavin - biosynthesis; Riboflavin - genetics; RNA polymerase; RNA, Guide, CRISPR-Cas Systems - genetics; Streptococcus infections; Streptococcus pyogenes - genetics; Vitamin B
• ISSN: 1422-0067; 1661-6596; 1422-0067
• DOI: 10.3390/ijms21165614

, a hetero-fermentative type of lactic acid bacteria, is a crucial probiotic candidate because of its ability to promote human health. However, inefficient gene manipulation tools limit its utilization in bioindustries. We report, for the first time, the development of a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) interference (CRISPRi) system for engineering . For reliable expression, the expression system of synthetic single guide RNA (sgRNA) and the deactivated Cas9 of (SpdCas9) were constructed in a bicistronic design (BCD) platform using a high-copy-number plasmid. The expression of SpdCas9 and sgRNA was optimized by examining the combination of two synthetic promoters and Shine-Dalgarno sequences; the strong expression of sgRNA and the weak expression of SpdCas9 exhibited the most significant downregulation (20-fold decrease) of the target gene (sfGFP), without cell growth retardation caused by SpdCas9 overexpression. The feasibility of the optimized CRISPRi system was demonstrated by modulating the biosynthesis of riboflavin. Using the CRISPRi system, the expression of and genes was downregulated (3.3-fold and 5.6-fold decreases, respectively), thereby improving riboflavin production. In addition, the co-expression of the operon was introduced and the production of riboflavin was further increased up to 1.7 mg/L, which was 1.53 times higher than that of the wild-type strain.