Abstract
BackgroundLactococcus lactis is one of the most extensively characterized lactic acid bacteria, from physiological traits to industrial exploitation. Since last decade, L. lactis has been developed into cell factories for the production of bioactive compounds such as enzymes, vaccine antigens and natural products. However, its precise and efficient genome editing tools is still required to make L. lactis more suitable candidate for engineered functionality.ResultsA high active recombinase, RecT of Enterococcus faecalis ATCC14506, was selected from six candidates and mediated homologous recombination between single-stranded DNA (ssDNA) and the L. lactis chromosomal rpoB locus with an efficiency of 100% after rifampin selection. To screen mutants without an externally selectable phenotype, the CRISPR/Cas9 system was used for counterselection, yielding an upp mutant with an efficiency of 46%. By optimization of the copy number of plasmid carrying the CRISPR/Cas9 system and the length of spacer sequence, the off-target efficiency of the recA, galK, hemN and noxD genes were eliminated. The ability of this optimized tool to perform sequential point mutation was demonstrated using the upp and galK gene loci as targets with improved efficiencies > 75%. Moreover, seamless genomic DNA deletions (50/100 bp) or insertion (a loxP site, 34 bp) was efficiently accomplished within 72 h.ConclusionsThe work provided a rapid, versatile and precise tool for L. lactis genomic engineering by combination of ssDNA recombineering with improved CRISPR/Cas9 counterselection. This tool will simplify the production of isogenic strains for assessment of gene function or construction of biosynthetic host.
Highlights
Lactococcus lactis is one of the most extensively characterized lactic acid bacteria, from physiological traits to industrial exploitation
Selection of recombinase suitable for single-stranded DNA (ssDNA) recombineering in L. lactis The activities of six characterized or predicated prophage-derived recombinases were tested by evaluating their function to mediate ssDNA recombineering of the rpoB H486N mutation conferring cells with rifampin resistance [19]
In L. lactis NZ9000, the nisin of 10 ng/ mL was used to induce the expression of the six candidates: Redβ, recombinase of E. coli λ-Red system; RecT, derived from E. faecalis ATCC14506; LCABL_13050 and Lp_0641, recombinases from Lb. casei BL23 and Lb. plantarum WCFS1, respectively; phiJB_00020, a predicated ssDNA binding protein derived from Lactobacillus prophage; pi12, a putative recombinase identified on the basis of protein homology from L. lactis IL1403 [4, 5, 25,26,27]
Summary
Lactococcus lactis is one of the most extensively characterized lactic acid bacteria, from physiological traits to industrial exploitation. Lactic acid bacteria (LAB) are a group of non-sporulating Gram-positive bacterium that catabolize water soluble carbohydrates mainly into lactic acid, including Lactococcus, Lactobacillus, Streptococcus, Enterococcus, Pediococcus, Leuconostoc and Oenococcus. They are highly diverse in genetic composition and physiological metabolism [1]. Several modified approaches have been developed to increase the screening efficiency by counterselectable markers, such as the genes upp, oroP, and pheS [10,11,12] These protocols are still imperative performed in two steps (vector integration and co-integrate resolution) and labor-intensive, timeconsuming (usually 3 weeks) to generate a prospective mutant
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