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.
Highlights
Vitamin K is a family of essential, fat-soluble vitamins required for blood coagulation, and involved in deposition and removal of calcium in various tissues (Flore et al, 2013; Schwalfenberg, 2017)
The second gene chosen for overexpression, menA, encodes the DHNA polyprenyltransferase catalyzing the joining of the prenyl diphosphate chain and DHNA resulting in demethylated menaquinone
Biosynthesis of menaquinones require over 20 enzymatic reactions and it is reasonable to expect that an elevated level of a single enzyme is insufficient to dramatically increase the amount of pathway product
Summary
Vitamin K is a family of essential, fat-soluble vitamins required for blood coagulation, and involved in deposition and removal of calcium in various tissues (Flore et al, 2013; Schwalfenberg, 2017). The family comprises two naturally active vitamers: vitamin K1 (phylloquinone) produced by plants, and vitamin K2 (menaquinone, MK-n where n represents the number of isoprene units). In humans and other mammals vitamin K is essential for its role as a cofactor for the enzyme γ-glutamyl carboxylase (Furie et al, 1999). This enzyme carboxylates glutamine residues on certain proteins into γ-carboxyglutamic acid (Gla) residues. Gla-proteins have increased affinity for calcium and are involved in protein-protein interactions (through Ca2+), cell membrane interactions and processes that promote correct deposition of calcium in bone and prevents deposition in soft tissues like arteries, cartilage, and heart valves (Wen et al, 2018)
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