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.

Keywords: Lactococcus lactis; MK-3; MK-8; MK-9; menaquinone; mevalonate kinase; prenyl diphosphate synthase; vitamin K2.

Figure 1

The lactococcal biosynthetic pathways for menaquinone(s). Intermediates that are substrates for competing essential pathways are shown as branched points (chorismate and FPP). IPP, isopentenyl pyrophosphate; FPP, farnesyl pyrophosphate; UPP, undecaprenyl pyrophosphate.

Figure 2

(A) Analytical HPLC chromatograms of quinones from recombinant L. lactis MG1363 cultures expressing additional lactococcal isochorismate synthase (encoded by menF) or DHNA polyprenyltransferase (encoded by menA) from P32 promoter of pMG36e. L. lactis containing empty vector (pMG36e) is shown in the upper panel for comparison. Peaks K1, MK-7 to MK-9 were identified based on retention times (RTs) compared to RTs of standards. (B) Plot of isoprenyl unit lengths vs. log₁₀ (net RT in min) for standards MK-4 and MK-7. The linear function of the graph between 4 and 7 isoprenyl units was used to calculate the isoprenyl unit number of MK-X. The dashed line represents extrapolation of the graph. MK-X (RT = 2.75) is designated using a triangle and corresponds to 3 isoprenyl units (MK-3). (C) Quantification of MK levels from (a). Average and standard error of the means are shown from at least 3 independent experiments. The * and *** represent a p-value below 0.05 and 0.0005 respectively. The p-values were obtained using a two-tailed T-test where the strains overexpressing menF or menA were compared to the control strain carrying empty pMG36e.

Figure 3

(A) PreA is a functional polyprenyl PP synthase in L. lactis ssp. cremoris. Strain NZ9000 containing empty pNZ8037 or expressing either gerCA+ispB or preA after induction of P_nisA using increasing concentrations of nisin. (B) Combined overexpression of all mevalonate and polyprenyl pathway genes in one transcript [genes were cloned in cis after the constitutive P32 promoter and inserted into pHH145 (pMEV-PP)]. Strain L. lactis ssp. cremoris MG1363 transformed with pMEV-PP or pCTR. (C) Overexpression of mevalonate and polyprenyl pathway genes from the P32 promoter of pMG36e. Strain L. lactis ssp. cremoris MG1363 transformed with pHmcM, pThiL-MvaA, pMvk, pMvaD-Pmk, pFni or pIspA. (D) Specific concentrations of vitamin K2 (nmol MK/g DW) when overexpressing mevalonate or polyprenyl pathway genes from the P32 promoter of pMG36e. (E) Combined overexpression of preA and menA and its effect on menaquinone production. Strain L.lactis NZ9000 containing empty pNZ8037 or transformed with pPreA-MenA after induction of P_nisA with increasing concentrations of nisin. (F) Combined overexpression of preA, menA, and mvk and its effect on menaquinone production. Strain L. lactis NZ9000 containing empty pNZ8037 and empty pMG36e or transformed with pMvk and pPreA-MenA. Induction of P_nisA with 2 ng/ml nisin. All strains were cultivated in GM17 and statically incubated at 30°C over night. Quantification of MK-3, MK7-9 and MK-3+MK7-9 levels from average of at least 3 independent experiments. Error bars represent standard error of the means.

Figure 4

Vitamin K2 content in milk fermented by strains overexpressing key genes of the biosynthetic menaquinone pathway. Fermentation was carried out for 20 h at 30°C in heat-sterilized skimmed milk supplemented with 0.5% glucose and 1% tryptone. Nisin (2 ng/ml) was added to NZ9000 strains 1 h after inoculation. Quantification of MK-3, MK7-9 and MK-3+MK7-9 levels from average of at least 3 independent experiments. Error bars represent standard error of the means.