Development of CRISPR Interference (CRISPRi) Platform for Metabolic Engineering of Leuconostoc citreum and Its Application for Engineering Riboflavin Biosynthesis
| Autor: | Jaewoo Son, Ji Won Cha, Seung Hoon Jang, Ki Jun Jeong |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2020 |
| Předmět: |
0106 biological sciences
0301 basic medicine Operon Streptococcus pyogenes Riboflavin medicine.disease_cause 01 natural sciences Catalysis Article Inorganic Chemistry Metabolic engineering lcsh:Chemistry Leuconostoc citreum 03 medical and health sciences Plasmid 010608 biotechnology medicine CRISPR Humans Lactic Acid Physical and Theoretical Chemistry Molecular Biology Gene lcsh:QH301-705.5 Spectroscopy CRISPR interference Chemistry Cas9 Probiotics Organic Chemistry CRISPRi General Medicine Computer Science Applications Cell biology lactic acid bacteria 030104 developmental biology lcsh:Biology (General) lcsh:QD1-999 Metabolic Engineering synthetic biology CRISPR-Cas Systems Leuconostoc Plasmids RNA Guide Kinetoplastida |
| Zdroj: | International Journal of Molecular Sciences Volume 21 Issue 16 International Journal of Molecular Sciences, Vol 21, Iss 5614, p 5614 (2020) |
| ISSN: | 1422-0067 |
| Popis: | Leuconostoccitreum, 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 L. citreum. For reliable expression, the expression system of synthetic single guide RNA (sgRNA) and the deactivated Cas9 of Streptococcus pyogenes (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&ndash 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 ribF and folE genes was downregulated (3.3-fold and 5.6-fold decreases, respectively), thereby improving riboflavin production. In addition, the co-expression of the rib 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. |
| Databáze: | OpenAIRE |
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