Modular Pathway Engineering of <i>Bacillus subtilis</i> To Promote <i>De Novo</i> Biosynthesis of Menaquinone-7

Yang, Shi‐Yao; Cao, Yingxiu; Sun, Liming; Li, Congfa; Lin, Xue; Cai, Zhigang; Zhang, Guoyin; Song, Hao

doi:10.1021/acssynbio.8b00258

Cited by 53 publications

(62 citation statements)

References 66 publications

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“…Five times increased MK production has been achieved by overexpression in E. coli and B. subtilis (Kong and Lee, 2011;Yang et al, 2019) similar to the fourfold increase reported here. Except from the clones overexpressing single mevalonate genes there was no change in growth yield in L. lactis overproducing vitamin K2.…”

Section: Discussionsupporting

confidence: 85%

“…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: 68%

“…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: 63%

“…A good strategic starting point for metabolic engineering of L. lactis could be to ensure increased chorismate levels and thereby elevate the supply of substrate for isochorismate synthase. However, efforts to increase chorismate levels by manipulating the shikimate pathway have proven unsuccessful in B. subtilis, probably due to increased feedback inhibition of the shikimate pathway caused by a concomitant rise in AAA levels (Tsukamoto et al, 2001;Yang et al, 2019). Moreover, by overexpressing enzymes involved in conversion of chorismate to folate Wegkamp et al (2007) obtained considerable increases in folate production in L. lactis.…”

Section: Discussionmentioning

confidence: 99%

“…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%

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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: 85%

Section: Discussionsupporting

confidence: 68%

Section: Discussionmentioning

confidence: 63%

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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Metabolic engineering of Bacillus subtilis for high‐titer production of menaquinone‐7

et al. 2019

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Menaquinone-7 (MK-7) is a member of vitamin K 2 used for prevention from osteoporosis and cardiovascular calcification. This study constructed Bacillus subtilis strains for hightiter production of MK-7 through metabolic engineering approaches. In B. subtilis, MK-7 biosynthesis was categorized into five modules: glycerol dissociation pathway, shikimate pathway, pyrimidine metabolic pathway, methylerythritol phosphate pathway, and MK-7 pathway. Overexpression of GlpK and GlpD (glycerol dissociation pathway) led to ã 10% increase in the MK-7 titer. Deletion of the genes mgsA and araM increased the MK-7 production by 15%. Furthermore, overexpression of AroG D146N (shikimate pathway), PyrG E156K (pyrimidine metabolic pathway), HepS (methylerythritol phosphate pathway), and VHb could also increase the MK-7 titer. Finally, we obtained a recombinant strain BSMK_11 with simultaneous overexpressing the genes glpK, glpD, aroG fbr , pyrG fbr , hepS, vgb, and knockouting the genes mgsA and araM, and the MK-7 titer reached 281.4 ± 5.0 mg/L (i.e., 12.0 mg/g DCW) in a 5 L fermenter.

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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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Modular Pathway Engineering of Bacillus subtilis To Promote De Novo Biosynthesis of Menaquinone-7

Cited by 53 publications

References 66 publications

Engineering Lactococcus lactis for Increased Vitamin K2 Production

Engineering Lactococcus lactis for Increased Vitamin K2 Production

Metabolic engineering of Bacillus subtilis for high‐titer production of menaquinone‐7

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

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