Enhancing menaquinone-7 production in recombinant Bacillus amyloliquefaciens by metabolic pathway engineering

Xu, Jianzhong; Weiliu, Yan; Zhang, Weiguo

doi:10.1039/c7ra03388e

Cited by 34 publications

(26 citation statements)

References 41 publications

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“…Similar results were observed from the overexpression of 1,4-dihydroxy-2-naphthoate octaprenyltransferase(MenA) in B. subtilis 168 and E. coli increased the Vitamin K2 content, in which imply that the reaction of converting 1,4-Dihydroxy-2-naphthoate to 2-Demethylmenaquinone plays the most important role in increasing Vitamin K2 production [33][34][35]. Therefore, the enzyme MenA was listed as rate-limiting enzymes of the Menaquinone pathway.…”

Section: Discussionsupporting

confidence: 70%

Engineering Elizabethkingia meningoseptica sp. F2 for Vitamin K2 production guided by genome analysis.

Yang¹,

Zheng

Liu³

et al. 2020

Preprint

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Background The species in family Elizabethkingia meningoseptica are interesting strain for investigating Vitamin K2 metabolic analysis. However, their genomic sequence, metabolic pathway, potential abilities, and evolutionary status are still unknown. Results This study therefore aimed to perform a genome sequencing of Elizabethkingia meningoseptica sp. F2 and further accomplished comparative analysis with other Vitamin K2 strains reveals overall identifying its unique/shared metabolic genes across genomes. The 3,874,794–base pair sequence of Elizabethkingia meningoseptica sp. F2 is presented. Of 3,539 genes annotation was applied. Results of synteny block demonstrated Elizabethkingia meningoseptica sp. F2 shares high levels of synteny with Elizabethkingia meningoseptica ATCC 13253 and Elizabethkingia meningoseptica NBRC 12535. Identification of Vitamin K2 metabolic pathway in Elizabethkingia meningoseptica sp. F2 were also accomplished. In addition, Elizabethkingia meningoseptica sp. F2 was resistant to gentamicin, streptomycin, ampicillin and caramycin, consistent with the presence of multiple genes encoding diverse multidrug efflux pump protein in the genome. Furthermore, By co-overexpression experiments of MenA and MenG, we showed that Vitamin K2 content was enhanced by 37% compared with control strain. Conclusions The genome analysis of Elizabethkingia meningoseptica sp. F2 in conjunction with the comparative metabolic pathways analysis among the E.coli, Bacillus subtilis and Streptomyces provided a useful information on the Vitamin K2 biosynthetic pathway and other related pathways at systems level.

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Section: Discussionsupporting

confidence: 70%

Engineering Elizabethkingia meningoseptica sp. F2 for Vitamin K2 production guided by genome analysis.

Yang¹,

Zheng

Liu³

et al. 2020

Preprint

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“…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%

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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“…Neither of them has any catalytic activity unless they are combined . Overexpression of HepS in B. subtilis 168 could increase the MK‐7 production, while overexpression of HepS in B. amyloliquefaciens Y‐2 led to a greater increase in the MK‐7 production than that of other enzymes …”

Section: Resultsmentioning

confidence: 99%

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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Enhancing menaquinone-7 production in recombinant Bacillus amyloliquefaciens by metabolic pathway engineering

Abstract: Six key enzymes are vital for MK-7 production, but the same enzyme has different effect on MK-7 production in different cultivating methods. Thus, the high enzyme activity and high-traffic biosynthetic pathway are beneficial to synthesize MK-7.

Cited by 34 publications

References 41 publications

Engineering Elizabethkingia meningoseptica sp. F2 for Vitamin K2 production guided by genome analysis.

Engineering Elizabethkingia meningoseptica sp. F2 for Vitamin K2 production guided by genome analysis.

Engineering Lactococcus lactis for Increased Vitamin K2 Production

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

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