Metabolic Engineering of the MEP Pathway in <i>Bacillus subtilis</i> for Increased Biosynthesis of Menaquinone-7

Yanwei, MA; McClure, Dale D.; Somerville, Mark V.; Proschogo, Nicholas; Dehghani, Fariba; Kavanagh, John M.; Coleman, Nicholas V.

doi:10.1021/acssynbio.9b00077

Cited by 44 publications

(47 citation statements)

References 70 publications

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“…In most bacterial species IPP form through the mevalonate-independent pathway called the MEP pathway (Frank and Groll, 2017). Overexpression of MEP and/or PP pathway genes increase MK production in B. subtilis, E. coli and E. meningoseptica (Kong and Lee, 2011;Ma et al, 2019;Yang et al, 2019). In line with these studies we achieved a doubling of the menaquinone content by overexpression of the mvk gene of the IPP-producing mevalonate pathway in L. lactis.…”

Section: Discussionsupporting

confidence: 69%

“…cremoris. Firstly, enhanced expression of menA result in around 2-fold higher MK production in E. coli, B. amyloliquefaciens, E. meningoseptica, and B.subtilis (Kong and Lee, 2011;Liu et al, 2017Liu et al, , 2018Xu et al, 2017;Ma et al, 2019;Yang et al, 2019) and we found that this also applies for L. lactis. More MenA activity will pull out products of both the menaquinone and MEV+PP pathways possibly leading to an increased flux through both pathways.…”

Section: Discussionmentioning

confidence: 62%

“…They include optimization of the fermentation process, increasing menaquinone secretion, improving bioreactor design, directed mutagenesis of strains or genetic engineering of strains (Ren et al, 2019). To enhance strain performance genomic changes has been made in order to maximize substrate pools, limit the production of by-products, overexpress key genes, express novel pathways, or combinations of the aforementioned strategies (Kong and Lee, 2011;Liu et al, 2017Liu et al, , 2018Xu et al, 2017;Ma et al, 2019;Yang et al, 2019). Most of these studies have been performed using B. subtilis, B. amyloliquefaciens, E. meningoseptica, or E. coli and seek to produce menaquinone on an industrial scale for use as a nutritional supplement or food additive.…”

Section: Discussionmentioning

confidence: 99%

“…In E. menigoseptica a 2.5-fold increase was achieved by combining overexpression of menA and a single MEP pathway gene (Liu et al, 2018). In B. subtilis a recent study reported 11-fold increase by overexpressing menA and three genes of the MEP pathway (Ma et al, 2019).…”

Section: Discussionmentioning

confidence: 99%

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Engineering Lactococcus lactis for Increased Vitamin K2 Production

Bøe

Holo

2020

Front. Bioeng. Biotechnol.

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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.

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Section: Discussionsupporting

confidence: 69%

Section: Discussionmentioning

confidence: 62%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Engineering Lactococcus lactis for Increased Vitamin K2 Production

Bøe

Holo

2020

Front. Bioeng. Biotechnol.

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“…This strategy is based on the overexpression of different combinations of rate-limiting enzymes involved in MK-7 biosynthetic pathways. Using these approaches in the B. subtilis 168 strain, yields of 50 mg/L [163] and 69.5 mg/L [164] of MK-7 were reported. This represents a considerable improvement on vitamin K2 production, and further optimization may open the perspective for new and affordable manufacturing processes allowing a considerable reduction of costs that will benefit the final consumer.…”

Section: Vitamin K2mentioning

confidence: 99%

Vitamin K as a Diet Supplement with Impact in Human Health: Current Evidence in Age-Related Diseases

et al. 2020

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Vitamin K health benefits have been recently widely shown to extend beyond blood homeostasis and implicated in chronic low-grade inflammatory diseases such as cardiovascular disease, osteoarthritis, dementia, cognitive impairment, mobility disability, and frailty. Novel and more efficient nutritional and therapeutic options are urgently needed to lower the burden and the associated health care costs of these age-related diseases. Naturally occurring vitamin K comprise the phylloquinone (vitamin K1), and a series of menaquinones broadly designated as vitamin K2 that differ in source, absorption rates, tissue distribution, bioavailability, and target activity. Although vitamin K1 and K2 sources are mainly dietary, consumer preference for diet supplements is growing, especially when derived from marine resources. The aim of this review is to update the reader regarding the specific contribution and effect of each K1 and K2 vitamers in human health, identify potential methods for its sustainable and cost-efficient production, and novel natural sources of vitamin K and formulations to improve absorption and bioavailability. This new information will contribute to foster the use of vitamin K as a health-promoting supplement, which meets the increasing consumer demand. Simultaneously, relevant information on the clinical context and direct health consequences of vitamin K deficiency focusing in aging and age-related diseases will be discussed.

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Temperature‐induced mutagenesis‐based adaptive evolution of Bacillus amyloliquefaciens for improving the production efficiency of menaquinone‐7 from starch

Liu

Ruan

et al. 2020

J of Chemical Tech & Biotech

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BACKGROUND Starch can be used as a carbon source for producing menaquinone‐7 (MK‐7) by Bacillus sp., but the final titer and productivity are unsatisfactory because starch cannot be effectively hydrolyzed to fermentable sugars. This article focuses on developing thermo‐tolerable MK‐7 high‐producing strains by temperature‐induced mutagenesis‐based adaptive evolution. RESULTS Here, we isolated a thermo‐tolerable MK‐7 high‐producing strain B. amyloliquefaciens MK50‐36 after multi‐rounds of acclimation at high temperature. Strain MK50‐36 showed a large growth with soluble starch as carbon source at 50 °C. Compared with the parental strain B. amyloliquefaciens H.β.D.R.‐5 cultivated at 37 °C, MK‐7 yield of strain MK50‐36 increased about twofold at 50 °C when soluble starch was used as the sole carbon resource (17.5 ± 1.5 mg L–1 versus 37.1 ± 2.1 mg L–1). In addition, strain MK50‐36 showed an improvement in starch utilization rate as compared with strain H.β.D.R.‐5 because α‐amylase activity of strain MK50‐36 was the highest at 50 °C. Moreover, MK‐7 yield of strain MK50‐36 was further increased by feeding fresh broth to control the amount of fermentation liquid in fed‐batch fermentation because fermentation broth will be easy to volatilize and thus affect the production of MK‐7 at high temperature, which reached up to 52.7 ± 4.6 mg L–1. CONCLUSION These results indicated that soluble starch can act as an effective carbon source for producing MK‐7, and strain MK50‐36 has a superior capacity to produce MK‐7 from soluble starch. © 2020 Society of Chemical Industry

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Metabolic Engineering of the MEP Pathway in Bacillus subtilis for Increased Biosynthesis of Menaquinone-7

Cited by 44 publications

References 70 publications

Engineering Lactococcus lactis for Increased Vitamin K2 Production

Engineering Lactococcus lactis for Increased Vitamin K2 Production

Vitamin K as a Diet Supplement with Impact in Human Health: Current Evidence in Age-Related Diseases

Temperature‐induced mutagenesis‐based adaptive evolution of Bacillus amyloliquefaciens for improving the production efficiency of menaquinone‐7 from starch

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