Engineering Lactococcus lactis for Increased Vitamin K2 Production

Abstract

Cheese produced with Lactococcus lactis is the main source of vitamin K2 in the Western diet. Subclinical vitamin K2 deficiency is common, calling for foods with enhanced vitamin K2 content. In this study we describe analyses of vitamin K2 (menaquinone) production in the lactic acid bacterium L. lactis ssp. cremoris strain MG1363. By cloning and expression from strong promoters we have identified genes and bottlenecks in the biosynthetic pathways leading to the long-chained menaquinones, MK-8 and MK-9. Key genes of the biosynthetic menaquinone pathway were overexpressed, singly or combined, to examine how vitamin K2 production can be enhanced. We observed that the production of the long menaquinone polyprenyl side chain, rather than production of the napthoate ring (1,4-dihydroxy-2-naphtoic acid), limits total menaquinone synthesis. Overexpression of genes causing increased ring formation (menF and menA) led to overproduction of short chained MK-3, while overexpression of other key genes (mvk and llmg_0196) resulted in enhanced full-length MK-9 production. Of two putatively annotated prenyl diphosphate synthases we pinpoint llmg_0196 (preA) to be important for menaquinone production in L. lactis. The genes mvk, preA, menF, and menA were found to be important contributors to menaquinone levels as single overexpression of these genes double and more than triple the total menaquinone content in culture. Combined overexpression of mvk, preA, and menA increased menaquinone levels to a higher level than obtained individually. When the overproducing strains were applied for milk fermentations vitamin K2 content was effectively increased 3-fold compared to the wild type. The results provide a foundation for development of strains to ferment foods with increased functional value i.e., higher vitamin K2 content.

Comparative transcriptional analysis site-directed mutagenesis of transcriptional regulator SinR boosts productivity of menaquinone synthesis in Bacillus subtilis

“…More glyceraldehyde 3-phosphate entering MEP pathway is bene cial to the formation of polyprenyl side chain, and entering SA pathway is conducive to the synthesis of naphthoquinone ring. According to the previous research, the biosynthesis of the polyprenyl side chain, rather than biosynthesis of the naphthoquinone ring (1,4-dihydroxy-2-naphtoic acid), limited total MK production in Lactococcus lactis [53]. However, in our study, we found most of the genes in the biosynthesis of naphthoquinone ring were up-regulated and more glyceraldehyde (15,54).…”

“…SinI is an antagonist of SinR [52] by forming SinI-SinR dead-end complex and sequestering free SinR, thereby ensuring bio lm formation. Strong sequence similarity between SinI and the oligomerization domain of SinR suggested that SinI displaces a SinR monomer from its homotetramer complex [53]. Despite sequence and structural similarities, the four key residues (Glu97, Tyr101, Trp104 and Arg105) which involved in SinR tetramer formation are signi cantly different in SinI (Fig.…”

Site-directed mutagenesis of the quorum-sensing transcriptional regulator SinR affects the biosynthesis of menaquinone in Bacillus subtilis

“…More glyceraldehyde 3-phosphate entering the MEP pathway is beneficial for the formation of the polyprenyl side chain, and entering the SA pathway is conducive to the synthesis of the naphthoquinone ring. According to previous research, the biosynthesis of the polyprenyl side chain, rather than biosynthesis of the naphthoquinone ring (1,4-dihydroxy-2-naphtoic acid), limited total MK production in Lactococcus lactis [ 64 ]. However, in our study, we found most of the genes in the biosynthesis of the naphthoquinone ring were upregulated and more glyceraldehyde 3-phosphate entered the SA pathway.…”

New aspects of microbial vitamin K2 production by expanding the product spectrum

“…In the Western diet, cheese and curd produced with Lactococcus lactis are the most important sources of vitamin K2. Bøe and Holo [ 42 ] co-overexpressed mvk , preA , and menA to increase the vitamin K2 yield (mainly MK-3, MK-7, and MK-9) (Table 1 ). When milk was fermented using the modified K2-overproducing strains, the vitamin K2 yield was effectively increased threefold compared to the wild type, which provides a foundation for the development of strains to ferment food with increased functional value [ 42 ].…”

“…When milk was fermented using the modified K2-overproducing strains, the vitamin K2 yield was effectively increased threefold compared to the wild type, which provides a foundation for the development of strains to ferment food with increased functional value [ 42 ].…”