Improvement of menaquinone-7 production by Bacillus subtilis natto in a novel residue-free medium by increasing the redox potential

Wang, Han; Liu, Hui; Wang, Li; Zhao, Genhai; Tang, Hengfang; Sun, Xiaowen; Ni, Wenfeng; Yang, Qiang; Wang, Peng; Zheng, Zhiming

doi:10.1007/s00253-019-10044-5

Cited by 27 publications

(17 citation statements)

References 67 publications

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“…The increase in expression level of NADH dehydrogenases indicated the ratio of NADH/NAD + decreased. Our result was consistent with previous study, which found that the ratio of NADH/NAD + in B. subtilis was inversely proportional to the concentration of MK-7 [51]. Above study also found the ratio of NADH/NAD + can characterize the rate of intracellular electrons extracted, which con rmed that su cient electron supply and transfer are important for the e cient synthesis of MK-7 [51].…”

Section: Discussionsupporting

confidence: 93%

“…3A). Furthermore, Glu97 and Arg105 as well as pi-stacking from Tyr101 and Trp104 formed salt bridges to joined adjacent helices [51]. Therefore, aromatic residues Glu97, Tyr101, Trp104 and Arg105 in SinR, were replaced with Lys34, Leu38, Lys41 and Ser42 in SinI, may mimic the SinR tetramer interface with DNA binding.…”

Section: Discussionmentioning

confidence: 99%

“…Therefore, we speculate that there may be other processes that can donate large amounts of electrons. It has been reported that NADH is the most important electron donor which donates the electrons under action of NADH dehydrogenase and transfers electrons to the electron transport system (ETM) and pumps protons out of the cell [51]. The process of producing electrons is shown in Fig.…”

Section: Differential Gene Expression Related To Mk-7 Biosynthesismentioning

confidence: 99%

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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: Discussionsupporting

confidence: 93%

Section: Discussionmentioning

confidence: 99%

Section: Differential Gene Expression Related To Mk-7 Biosynthesismentioning

confidence: 99%

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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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“…It has been reported that NADH is the most important electron donor. It donates electrons under the action of NADH dehydrogenase and transfers electrons to the electron transport system (ETM) and pumps protons out of the cell [ 36 ]. The process of producing electrons is shown in Fig.…”

Section: Resultsmentioning

confidence: 99%

Site-directed mutagenesis of the quorum-sensing transcriptional regulator SinR affects the biosynthesis of menaquinone in Bacillus subtilis

Zhao

et al. 2021

Microb Cell Fact

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Background Menaquinone (MK-7) is a highly valuable vitamin K2 produced by Bacillus subtilis. Common static metabolic engineering approaches for promoting the production of MK-7 have been studied previously. However, these approaches caused an accumulation of toxic substances and reduced product yield. Hence, dynamic regulation by the quorum sensing (QS) system is a promising method for achieving a balance between product synthesis and cell growth. Results In this study, the QS transcriptional regulator SinR, which plays a significant role in biofilm formation and MK production simultaneously, was selected, and its site-directed mutants were constructed. Among these mutants, sinR knock out strain (KO-SinR) increased the biofilm biomass by 2.8-fold compared to the wild-type. SinRquad maximized the yield of MK-7 (102.56 ± 2.84 mg/L). To decipher the mechanism of how this mutant regulates MK-7 synthesis and to find additional potential regulators that enhance MK-7 synthesis, RNA-seq was used to analyze expression changes in the QS system, biofilm formation, and MK-7 synthesis pathway. The results showed that the expressions of tapA, tasA and epsE were up-regulated 9.79-, 0.95-, and 4.42-fold, respectively. Therefore, SinRquad formed more wrinkly and smoother biofilms than BS168. The upregulated expressions of glpF, glpk, and glpD in this biofilm morphology facilitated the flow of glycerol through the biofilm. In addition, NADH dehydrogenases especially sdhA, sdhB, sdhC and glpD, increased 1.01-, 3.93-, 1.87-, and 1.11-fold, respectively. The increased expression levels of NADH dehydrogenases indicated that more electrons were produced for the electron transport system. Electrical hyperpolarization stimulated the synthesis of the electron transport chain components, such as cytochrome c and MK, to ensure the efficiency of electron transfer. Wrinkly and smooth biofilms formed a network of interconnected channels with a low resistance to liquid flow, which was beneficial for the uptake of glycerol, and facilitated the metabolic flux of four modules of the MK-7 synthesis pathway. Conclusions In this study, we report for the first time that SinRquad has significant effects on MK-7 synthesis by forming wrinkly and smooth biofilms, upregulating the expression level of most NADH dehydrogenases, and providing higher membrane potential to stimulate the accumulation of the components in the electron transport system.

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“…Liu et al changed the state of the cell membrane in Escherichia coli , and the extracellular MK reached 10.71 ± 0.19 mg/L after 120 h of cultivation [49]. Wang et al improved the redox potential and the state of the cell membrane of B. subtilis natto, the MK-7 concentration increased to 43 mg/L in a 5-L bioreactor for 144 h [50]. Ma et al modified the MEP pathway in B. subtilis to enhance the biosynthesis of MK-7, resulting in 50 mg/L MK-7 after 120 h of cultivation [51].…”

Section: Discussionmentioning

confidence: 99%

Construction of a novel MK-4 biosynthetic pathway in Pichia pastoris through heterologous expression of HsUBIAD1

et al. 2019

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Background With a variety of physiological and pharmacological functions, menaquinone is an essential prenylated product that can be endogenously converted from phylloquinone (VK1) or menadione (VK3) via the expression of Homo sapiens UBIAD1 (HsUBIAD1). The methylotrophic yeast, Pichia pastoris, is an attractive expression system that has been successfully applied to the efficient expression of heterologous proteins. However, the menaquinone biosynthetic pathway has not been discovered in P. pastoris. Results Firstly, we constructed a novel synthetic pathway in P. pastoris for the production of menaquinone-4 (MK-4) via heterologous expression of HsUBIAD1. Then, the glyceraldehyde-3-phosphate dehydrogenase constitutive promoter (PGAP) appeared to be mostsuitable for the expression of HsUBIAD1 for various reasons. By optimizing the expression conditions of HsUBIAD1, its yield increased by 4.37 times after incubation at pH 7.0 and 24 °C for 36 h, when compared with that under the initial conditions. We found HsUBIAD1 expressed in recombinant GGU-23 has the ability to catalyze the biosynthesis of MK-4 when using VK1 and VK3 as the isopentenyl acceptor. In addition, we constructed a ribosomal DNA (rDNA)-mediated multi-copy expression vector for the fusion expression of SaGGPPS and PpIDI, and the recombinant GGU-GrIG afforded higher MK-4 production, so that it was selected as the high-yield strain. Finally, the yield of MK-4 was maximized at 0.24 mg/g DCW by improving the GGPP supply when VK3 was the isopentenyl acceptor. Conclusions In this study, we constructed a novel synthetic pathway in P. pastoris for the biosynthesis of the high value-added prenylated product MK-4 through heterologous expression of HsUBIAD1 and strengthened accumulation of GGPP. This approach could be further developed and accomplished for the biosynthesis of other prenylated products, which has great significance for theoretical research and industrial application.

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Improvement of menaquinone-7 production by Bacillus subtilis natto in a novel residue-free medium by increasing the redox potential

Cited by 27 publications

References 67 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

Site-directed mutagenesis of the quorum-sensing transcriptional regulator SinR affects the biosynthesis of menaquinone in Bacillus subtilis

Construction of a novel MK-4 biosynthetic pathway in Pichia pastoris through heterologous expression of HsUBIAD1

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