Influence of site-directed mutagenesis of UbiA, overexpression of dxr, menA and ubiE, and supplementation with precursors on menaquinone production in Elizabethkingia meningoseptica

Liu, Yan; Yang, Ziming; Xue, Zhenglian; Senhe, Qian; Zhou, Wang; Hu, Liu-xiu; Wang, Jin; Zhu, Hao; Ding, Xiu-min; Yu, Fei

doi:10.1016/j.procbio.2018.01.022

Cited by 12 publications

(9 citation statements)

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“…Finally, menA and menG, which could combine the isoprene side chain and naphthoquinone ring and then catalyzed the methylation to form MK-7, were both up-regulated by 2.03-and 0.72-fold for SinR quad strain. It is consistent with our previous studies that overexpression of the menG/ubiE in Elizabethkingia meningoseptica also enhanced the MK content by 1.41-fold [25]. These results provided a new idea for further understanding the effect of SinR quad on the biosynthesis of MK-7 at the transcriptional level.…”

Section: Differential Gene Expression Related To Mk-7 Biosynthesissupporting

confidence: 92%

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Comparative transcriptional analysis site-directed mutagenesis of transcriptional regulator SinR boosts productivity of menaquinone synthesis in Bacillus subtilis

Wei

Zhao

et al. 2021

Preprint

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Background: MK-7 is a highly valuable vitamin K2 produced by Bacillus subtilis. Biofilm forming caused by quorum sensing (QS) system was beneficial for MK-7 synthesis. However, the specific regulator in QS system which play the most significant role in biofilm formation and MK production have not been revealed at genetic level, and this limits the possibility of further increasing MK-7 production.Results: In this study, the influence of different mutants in QS system on biofilm formation and MK-7 production was investigated. The transcriptional regulator SinR was chosen finally because the complete knocking out of sinR (KO-SinR) maximized the biofilm biomass and increased the yield of MK-7. However, KO-SinR led to a large number of spore and wrinkle forming, which slow down even stop the MK-7 biosynthesis. Five SinR mutants (E97K, Y101L, W104K, R105S, SinRquad) were constructed by site-directed mutagenesis of four aromatic residues (Glu97, Tyr101, Trp104 and Arg105). Among these, SinRquad formed more wrinkly but smooth biofilm, and obtained the maximum MK-7 value (102.56±2.84 mg/L), which was ten times of the parent strain. Comparative transcriptional analysis showed that SinRquad promoted the synthesis of extracellular polymeric substances and inhibited the swimming motility of late-flagellar. In addition, SinRquad upregulated the expression level of ctaC-G operator and qcrA-C operator which encode the cytochromes. Upregulation of components in the oxidative respiratory chain of B. subtilis was due to the upregulation of NADH dehydrogenases. Most NADH dehydrogenases especially sdhA-C and glpD, increased 1.01-, 3.93-, 1.87-, 1.11- fold, respectively. The increase in expression level of NADH dehydrogenases indicated the ratio of NADH/NAD+ decreased and more electrons produced for the electron transport system (ETM). Wrinkly and smooth biofilm formed a network of interconnected channels with low resistance to liquid flow was beneficial to obtain a more stable state of electron transport chain, which facilitate the metabolic flux of four modules of MK-7 synthesis pathways.Conclusions: In this study, we report for the first time that SinRquad has significant effects on the MK-7 synthesis by forming wrinkly and smooth biofilm, upregulating the expression level of most NADH dehydrogenases, providing more stable state of the electron transport.

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Section: Differential Gene Expression Related To Mk-7 Biosynthesissupporting

confidence: 92%

“…Three replicates were examined and data were analyzed by Student's t-test. The MK-7 detection method was the same with our previous research [25].…”

Section: Determination Of Bio Lm Biomass and Mk-7 Yieldmentioning

confidence: 99%

Comparative transcriptional analysis site-directed mutagenesis of transcriptional regulator SinR boosts productivity of menaquinone synthesis in Bacillus subtilis

Wei

Zhao

et al. 2021

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%

“…An additive effect on MK production when increasing the precursor pool for the prenyl diphosphate synthase combined with increased MenA activity has also been reported by others. 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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Combinatorial metabolic engineering of Bacillus subtilis for menaquinone‐7 biosynthesis

Sun,

Bi,

et al. 2024

Biotech & Bioengineering

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Menaquinone‐7 (MK‐7), a form of vitamin K2, supports bone health and prevents arterial calcification. Microbial fermentation for MK‐7 production has attracted widespread attention because of its low cost and short production cycles. However, insufficient substrate supply, unbalanced precursor synthesis, and low catalytic efficiency of key enzymes severely limited the efficiency of MK‐7 synthesis. In this study, utilizing Bacillus subtilis BSAT01 (with an initial MK‐7 titer of 231.0 mg/L) obtained in our previous study, the glycerol metabolism pathway was first enhanced to increase the 3‐deoxy‐arabino‐heptulonate 7‐phosphate (DHAP) supply, which led to an increase in MK‐7 titer to 259.7 mg/L. Subsequently, a combination of knockout strategies predicted by the genome‐scale metabolic model etiBsu1209 was employed to optimize the central carbon metabolism pathway, and the resulting strain showed an increase in MK‐7 production from 259.7 to 318.3 mg/L. Finally, model predictions revealed the methylerythritol phosphate pathway as the major restriction pathway, and the pathway flux was increased by heterologous introduction (Introduction of Dxs derived from Escherichia coli) and fusion expression (End‐to‐end fusion of two enzymes by a linker peptide), resulting in a strain with a titer of 451.0 mg/L in a shake flask and 474.0 mg/L in a 50‐L bioreactor. This study achieved efficient MK‐7 synthesis in B. subtilis, laying the foundation for large‐scale MK‐7 bioproduction.

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Influence of site-directed mutagenesis of UbiA, overexpression of dxr, menA and ubiE, and supplementation with precursors on menaquinone production in Elizabethkingia meningoseptica

Cited by 12 publications

References 36 publications

Comparative transcriptional analysis site-directed mutagenesis of transcriptional regulator SinR boosts productivity of menaquinone synthesis in Bacillus subtilis

Comparative transcriptional analysis site-directed mutagenesis of transcriptional regulator SinR boosts productivity of menaquinone synthesis in Bacillus subtilis

Engineering Lactococcus lactis for Increased Vitamin K2 Production

Combinatorial metabolic engineering of Bacillus subtilis for menaquinone‐7 biosynthesis

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