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

Abstract

Numerous lactic acid bacteria (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 the 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 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.