Abstract
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.
Highlights
Lactic acid bacteria (LAB) are non-respiring, fermentative anaerobic oxygen-tolerant bacteria and are typically cultivated underanaerobic conditions
Including L. lactis and L. mesenteroides, a respiration-like behavior was observed upon addition of heme to the medium [1], and actual respiration was shown in L. lactis [2]
To verify the functionality of the existing menaquinone biosynthesis genes from Lactipl. plantarum WCFS1 and Lent. buchneri DSM 20057, we constructed respective knock-out strains in L. lactis using the Cre-loxP system with selection marker removal by Cre recombinase [17]
Summary
Lactic acid bacteria (LAB) are non-respiring, fermentative anaerobic oxygen-tolerant bacteria and are typically cultivated under (micro)anaerobic conditions. Since LAB do not produce heme [10] due to an incomplete heme biosynthesis pathway [11], it must be supplemented for all species Supplementation of both heme and menaquinone leads to respiratory behavior in several additional species, indicating an incomplete respiratory chain lacking quinones [1,11]. Buchneri DSM 20057 contains menE, menB, menA, and menG, with the genes encoding the first four steps (menFDHC) in the pathway being absent. Both genomes contain the genes for the cytochrome oxidase (data from Joint Genome Institute, www.jgi.doe.gov, 9 May. 2016, pathway analysis using the pathway maps of the Kyoto Encyclopedia of Genes and Genomes, www.genome.jp, 9 May 2016). We determined the functionality of the remaining menaquinone pathway genes in Lactipl. plantarum WCFS1 and Lent. buchneri DSM 20057 using classical methods of gene inactivation and genetic complementation for the investigation of a biosynthetic pathway and analyzed their growth behavior under aerobic conditions with heme supplementation
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