Enhanced vitamin K2 production by engineered Bacillus subtilis during leakage fermentation.

Menaquinone-7 (MK-7), a valuable member of the vitamin K2 series, is an essential nutrient for humans. It is used for treating coagulation disorders, and osteoporosis, promoting liver function recovery, and preventing cardiovascular diseases. In this study, to further improve the metabolic synthesis of MK-7 by the mutant strain, the effect of surfactants on the metabolic synthesis of MK-7 by the mutant strain Bacillus subtilis 168 KO-SinR (BS168 KO-SinR) was analyzed. The scanning electron microscopy and flow cytometry results showed that the addition of surfactants changed the permeability of the cell membrane of the mutant strain and the structural components of the biofilm. When 0.7% Tween-80 was added into the medium, the extracellular and intracellular synthesis of MK-7 reached 28.8 mg/L and 59.2 mg/L, respectively, increasing the total synthesis of MK-7 by 80.3%. Quantitative real-time PCR showed that the addition of surfactant significantly increased the expression level of MK-7 synthesis-related genes, and the electron microscopy results showed that the addition of surfactant changed the permeability of the cell membrane. The research results of this paper can serve as a reference for the industrial development of MK-7 prepared by fermentation.

Poly-γ-Glutamic Acid Production by Engineering a DegU Quorum-Sensing Circuit in Bacillus subtilis.

As a natural biological macromolecule, γ-polyglutamic acid (γ-PGA) plays a significant role in medicine, food, and cosmetic industries owing to its unique properties of biocompatibility, biodegradability, water solubility, and viscosity. Although many strategies have been adopted to increase the yield of γ-PGA in Bacillus subtilis, the effectiveness of these common approaches is not high because the strong viscosity affects cell growth. However, dynamic regulation based on quorum sensing (QS) has been extensively applied as a fundamental tool for fine-tuning gene expression in reaction to changes in cell density without adding expensive inducers. A modular PhrQ-RapQ-DegU QS system is developed based on promoter PD4, which is upregulated by phosphorylated DegU (DegU-P). In this study, first, we analyzed the DegU-based gene expression regulation system in B. subtilis 168. We constructed a promoter library of different abilities, selected suitable promoters from the library, and performed mutation screening on the selected promoters and degU region. Furthermore, we constructed a PhrQ-RapQ-DegU QS system to dynamically control the synthesis of γ-PGA in BS168. Cell growth and efficient synthesis of the target product can be dynamically balanced by the QS system. Our dynamic adjustment approach increased the yield of γ-PGA to 6.53-fold of that by static regulation in a 3 L bioreactor, which verified the effectiveness of this strategy. In summary, the PhrQ-RapQ-DegU QS system has been successfully integrated with biocatalytic functions to achieve dynamic metabolic pathway control in BS168, which can be stretched to a large number of microorganisms to fine-tune gene expression and enhance the production of metabolites.

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

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.

Identification of six important amino acid residues of MenA from Bacillus subtilis natto for enzyme activity and formation of menaquinone.

The enzyme 1, 4-dihydroxy-2-naphthoic acid (DHNA) prenyltransferase (MenA) is a critical player in determining the efficiency of the menaquinone (MK) synthesis pathway and is an attractive target for the development of novel chemotherapeutics against pathogenic Gram-positive bacteria. However, there has been no report on structural properties or active region of MenA. To solve this challenge, we predicted the three-dimensiona structure and critical amino acid sites of MenA by bioinformatics analysis. Six amino acid sites were chosen by alligning the amino acid sequence of MenA from Bacillus subtilis natto with 4-hydroxybenzoate octaprenyl transferase (UbiA) from Escherichia coli, Aeropyrum pernix and Archaeoglobus fulgidus. Among them, four Asp sites located in two Asp-rich motifs (D78XXXXXD84 and D208XXXD212) were found to be indispensable amino acid residues in maintaining MenA activity. Site-directed mutagenesis of two other sites (Q67th, N74th) positively affected the catalytic activity of MenA and the MK titer. Q67R resulted in more than a 5-fold increase in specific 2-demethylmenaquinone (DMK) content (YP1/x) compared to wild-type, and the hydrophobic interaction between Cys63 and Arg67 could be the main reason according to the three-dimensional structure analysis. Moreover, a dramatic increase in specific MK content (YP2/x) was realized by co-expressing menG in EcMenA (Q67R). The results obtained could be useful not only in developing novel chemotherapeutics to combat potentially pathogenic Gram-positive bacteria, but also in regulating and optimizating E. coli mutant cultures for the efficient production of MK metabolites.

Bioconversion of farnesol and 1,4-dihydroxy-2-naphthoate to menaquinone by an immobilized whole-cell biocatalyst using engineered Elizabethkingia meningoseptica.

Menaquinone (MK) has important applications in the pharmaceutical and food industries. To increase the production rate (QP) of MK-4, we developed a straightforward biotransformation method for MK-4 synthesis directly from its precursors 1,4-dihydroxy-2-naphthoate (DHNA) and farnesol using whole cells of genetically engineered Elizabethkingia meningoseptica. Results showed that MK-4 can be produced directly from farnesol and DHNA using both free and immobilized FM-D198 cells. MK-4 yield peaked at 29.85 ± 0.36 mg/L in the organic phase and 24.08 ± 0.33 mg/g DCW after 12 h of bioconversion using free cells in a two-phase conversion system. MK-4 yield reached 26.34 ± 1.35 mg/L and 17.44 ± 1.05 mg/g DCW after 8 h using immobilized cells. Although this yield was lower than that using free cells, immobilized cells can be re-used for MK-4 production via repeated-batch culture. After ten batch cultures, efficient MK-4 production was maintained at a yield of more than 20 mg/L. After optimizing the catalysis system, the MK-4 yield reached 26.91 ± 1.27 mg/L using the immobilized cells and had molar conversion rates of 58.56 and 76.90% for DHNA and farnesol, respectively.

The change of the state of cell membrane can enhance the synthesis of menaquinone in Escherichia coli.

Menaquinone (MK) was an attractive membrane-bound intracellular chemical. To enhance its production, we tried to find the relationship between its synthesis and the state of cell membrane in producing strain. Due to non-ionic surfactant-polyoxyethylene oleyl ether (POE) and plant oil-cedar wood oil (CWO) can typically increase extracellular secretion and intracellular synthesis of MK respectively, the effect of these two substances on cell morphology, physical properties of cell membrane was investigated. Finally, two engineering strains were constructed to verify whether the state of cell membrane can enhance MK synthesis. The result showed that the edge of cells was broken when POE added in the medium. Other physical properties such as total fatty acid content decreased by 40.7% and the ratio of saturated fatty acids to unsaturated fatty acids decreased from 1.58 ± 0.05 to 1.31 ± 0.04. Meanwhile, cell membrane leakage was enhanced from 7.14 to 64.31%. Different from POE group, cell membrane was intact in CWO group. Moreover, the ratio of saturated fatty acids to unsaturated fatty acids increased from 1.58 ± 0.05 to 1.78 ± 0.04 and the average lipid length decreased from 16.05 ± 0.08 to 15.99 ± 0.10. Two constructed strains, especially Escherichia coli DH5α FatB, exhibited strong MK secretion ability and the extracellular MK reached 10.71 ± 0.19 mg/L. An understanding of these functionary mechanisms could not only provide a new idea for the synthesis of MK, but also provide a reference to increase the yield of intracellular membrane-bound metabolites.