Identification and functional analysis of potential prophage-derived recombinases for genome editing in Lactobacillus casei

Abstract

Numerous LAB bacteriophage genomes have been sequenced, while the functional genes are yet to be exploited. In this study, a λ Red-like recombinase operon LCABL_13040–50-60 was identified from a prophage PLE3 in Lactobacillus casei BL23 genome, and its recombination function was confirmed by replacement of a 167 bp galK fragment with chloramphenicol-resistant gene (cat) in the L. casei BL23 genome. Further functional analysis showed that LCABL_13040 and LCABL_13060 were analogs to the host nuclease inhibitor (Redγ) and 5'-3' exonuclease (Redα/RecE), respectively. After optimization of recombineering conditions, including induction, homology length, recovery time and double-strand DNA (dsDNA) substrates quantity, the recombineering efficiency reached ∼2.2 × 10⁻⁷. Subsequently, combining cre-lox technology, the optimal LCABL_13040–50-60 proteins could catalyze markerless deletion of a 167 bp galK fragment and insertion of the gfp gene as well as precision point mutation of rpoB gene in the L. casei BL23 genome, suggesting the LCABL_13040–50-60 operon encoded for three recombineering proteins. Moreover, with the assistance of Redγ, the LCABL_13040–50-60 proteins also showed recombinase activity in six other L. casei strains, L. paracasei OY and L. plantarum WCSF1. All the results demonstrated that the prophage-associated recombinases LCABL_13040–50-60 have great potential to be used for genome editing in LAB.