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

Jaewoo Son,Ki Jun Jeong,Seung Hoon Jang,Ji Won Cha

doi:10.3390/ijms21165614

Abstract

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–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.

Highlights

Leuconostoc spp. is a hetero-fermentative type of lactic acid bacteria that plays a significant role in the fermentation of products such as kimchi, milk, vegetables and meat [1]
It is generally known that a high concentration of single guide RNA (sgRNA) in cells is required for the effective operation of CRISPR interference (CRISPRi) [26]
For the reliable expression of SpdCas9, the expression cassette was constructed in a bicistronic design (BCD) platform, which we had previously developed to induce the reliable expression of recombinant proteins in L. citreum [20]

Summary

Introduction

Leuconostoc spp. is a hetero-fermentative type of lactic acid bacteria that plays a significant role in the fermentation of products such as kimchi, milk, vegetables and meat [1]. The engineering of bacterial hosts, including Leuconostoc spp., can enhance their performance by giving them desired properties, which can be achieved by the extensive manipulation of the host genome, such as the elimination or downregulation of unnecessary genes and/or the overexpression of essential genes. Indirect genome modification tools using catalytically deactivated Cas (dCas9) can make it possible to effectively regulate the expression of target genes without permanently removing the genes from chromosomes This system, called CRISPR interference (CRISPRi), can regulate the expression of a target gene by blocking RNA polymerase through the interaction of dCas with synthetic guide RNA, which has a complementary sequence to the target gene (Figure 1A) [17,18]. Most of these tools are available only for Lactococcus spp. and Lactobacillus spp.; genome-editing tools that can be optimized and used for Leuconostoc spp. are very limited

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Conclusion