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A Single-Plasmid Genome Editing System for Metabolic Engineering of Lactobacillus casei.

ABSTRACT: Genome engineering of Lactobacillus casei, an important industrial microorganism for dairy fermented product, currently relies on inefficient and time-consuming double crossover events. In this study, we developed an easy-to-use genome engineering strategy for metabolic engineering of L. casei for acetoin production. Plasmid pMSP456-Cre, that contains prophage recombinase operon LCABL_13040-50-60 driven by the nisin-controlled inducible expression (NICE) system and the site-specific recombinase gene cre under the control of the promoter of the lactose operon from L. casei, was constructed. Using this plasmid, integration of a hicD3 gene linear donor cassette (up-lox66-cat-lox71-down) was catalyzed by the LCABL_13040-50-60 recombinase and the cat gene was excised by the Cre/lox system with an efficiency of 60%. To demonstrate this system for sequential and iterative knocking out genes in L. casei, another three genes (pflB, ldh and pdhC) related to acetoin production were deleted with the efficiencies of 60, 40, and 60%, respectively. The yielding quadruple mutant could produce a ?18-fold higher amount of acetoin than the wild-type and converted 59.8% of glucose to acetoin in aerobic. Therefore, these results proved this simple genome engineering strategy have potential in metabolic engineering of L. casei for production of high value-added metabolites.

SUBMITTER: Xin Y

PROVIDER: S-EPMC6289983 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Project description:Lactobacillus casei has drawn increasing attention as a health-promoting probiotic, while effective genetic manipulation tools are often not available, e.g., the single-gene knockout in L. casei still depends on the classic homologous recombination-dependent double-crossover strategy, which is quite labor-intensive and time-consuming. In the present study, a rapid and precise genome editing plasmid, pLCNICK, was established for L. casei genome engineering based on CRISPR-Cas9D10A In addition to the P23-Cas9D10A and Pldh-sgRNA (single guide RNA) expression cassettes, pLCNICK includes the homologous arms of the target gene as repair templates. The ability and efficiency of chromosomal engineering using pLCNICK were evaluated by in-frame deletions of four independent genes and chromosomal insertion of an enhanced green fluorescent protein (eGFP) expression cassette at the LC2W_1628 locus. The efficiencies associated with in-frame deletions and chromosomal insertion is 25 to 62%. pLCNICK has been proved to be an effective, rapid, and precise tool for genome editing in L. casei, and its potential application in other lactic acid bacteria (LAB) is also discussed in this study.IMPORTANCE The lack of efficient genetic tools has limited the investigation and biotechnological application of many LAB. The CRISPR-Cas9D10A nickase-based genome editing in Lactobacillus casei, an important food industrial microorganism, was demonstrated in this study. This genetic tool allows efficient single-gene deletion and insertion to be accomplished by one-step transformation, and the cycle time is reduced to 9 days. It facilitates a rapid and precise chromosomal manipulation in L. casei and overcomes some limitations of previous methods. This editing system can serve as a basic technological platform and offers the possibility to start a comprehensive investigation on L. casei As a broad-host-range plasmid, pLCNICK has the potential to be adapted to other Lactobacillus species for genome editing.

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A Single-Plasmid Genome Editing System for Metabolic Engineering of Lactobacillus casei.

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