Analysis and Reconstitution of the Menaquinone Biosynthesis Pathway in Lactiplantibacillus plantarum and Lentilactibacillus buchneri

In Lactococcus lactis and some other lactic acid bacteria, respiratory metabolism has been reported upon supplementation with only heme, leading to enhanced biomass formation, reduced acidification, resistance to oxygen, and improved long-term storage. Genes encoding a complete respiratory chain with all components were found in genomes of L. lactis and Leuconostoc mesenteroides, but menaquinone biosynthesis was found to be incomplete in Lactobacillaceae (except L. mesenteroides). Lactiplantibacillus plantarum has only two genes (menA, menG) encoding enzymes in the biosynthetic pathway (out of eight), and Lentilactobacillus buchneri has only four (menA, menB, menE, and menG). We constructed knock-out strains of L. lactis defective in menA, menB, menE, and menG (encoding the last steps in the pathway) and complemented these by expression of the extant genes from Lactipl. plantarum and Lent. buchneri to verify their functionality. Three of the Lactipl. plantarum biosynthesis genes, lpmenA1, lpmenG1, and lpmenG2, as well as lbmenB and lbmenG from Lent. buchneri, reconstituted menaquinone production and respiratory growth in the deficient L. lactis strains when supplemented with heme. We then reconstituted the incomplete menaquinone biosynthesis pathway in Lactipl. plantarum by expressing six genes from L. lactis homologous to the missing genes in a synthetic operon with two inducible promoters. Higher biomass formation was observed in Lactipl. plantarum carrying this operon, with an OD₆₀₀ increase from 3.0 to 5.0 upon induction.