Dataset Information

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

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.

SUBMITTER: Son J

PROVIDER: S-EPMC7460652 | biostudies-literature | 2020 Aug

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

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

Son Jaewoo J Jang Seung Hoon SH Cha Ji Won JW Jeong Ki Jun KJ

International journal of molecular sciences 20200805 16

Leuconostoc citreum, 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 sin ...[more]

PMID: 32764465

Similar Datasets

Project description:Fructophilic lactic acid bacteria (FLAB), composed of Fructobacillus spp., Lactobacillus kunkeei, and Lactobacillus apinorum, are unique in that they prefer d-fructose over d-glucose as a carbon source. Strain F192-5, isolated from the peel of a satsuma mandarin and identified as Leuconostoc citreum, grows well on d-fructose but poorly on d-glucose and produces mainly lactate and acetate, with trace amounts of ethanol, from the metabolism of d-glucose. These characteristics are identical to those of obligate FLAB. However, strain F192-5 ferments a greater variety of carbohydrates than known FLAB. Comparative analyses of the genomes of strain F192-5 and reference strains of L. citreum revealed no signs of specific gene reductions, especially genes involved in carbohydrate transport and metabolism, in the genome of F192-5. The bifunctional alcohol/acetaldehyde dehydrogenase gene (adhE) is conserved in strain F192-5 but is not transcribed. This is most likely due to a deletion in the promoter region upstream of the adhE gene. Strain F192-5 did, however, ferment d-glucose when transformed with a plasmid containing the allochthonous adhE gene. L. citreum F192-5 is an example of a pseudo-FLAB strain with a deficiency in d-glucose metabolism. This unique phenotypic characteristic appears to be strain specific within the species L. citreum This might be one of the strategies lactic acid bacteria use to adapt to diverse environmental conditions.IMPORTANCE Obligate fructophilic lactic acid bacteria (FLAB) lack the metabolic pathways used in the metabolism of most carbohydrates and differ from other lactic acid bacteria in that they prefer to ferment d-fructose instead of d-glucose. These characteristics are well conserved at the genus or species level. Leuconostoc citreum F192-5 shows similar growth characteristics. However, the strain is metabolically and genomically different from obligate FLAB. This is an example of a strain that evolved a pseudo-FLAB phenotype to adapt to a fructose-rich environment.

Dataset Information

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

Publications

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

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets