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Menaquinone-7 (MK-7), a subtype of vitamin K 2 (VK 2 ), assumes crucial roles in coagulation function, calcium homeostasis, and respiratory chain transmission. The production of MK-7 via microbial fermentation boasts mild technological conditions and high biocompatibility. Nevertheless, the redox activity of MK-7 imposes constraints on its excessive accumulation in microorganisms. To address this predicament, an adaptive laboratory evolution (ALE) protocol was implemented in Bacillus subtilis BS011, utilizing vitamin K 3 (VK 3 ) as a structural analog of MK-7. The resulting strain, BS012, exhibited heightened tolerance to high VK 3 concentrations and demonstrated substantial enhancements in biofilm formation and total antioxidant capacity (T-AOC) when compared to BS011. Furthermore, MK-7 production in BS012 exceeded that of BS011 by 76% and 22% under static and shaking cultivation conditions, respectively. The molecular basis underlying the superior performance of BS012 was elucidated through genome and transcriptome analyses, encompassing observations of alterations in cell morphology, variations in central carbon and nitrogen metabolism, spore formation, and antioxidant systems. In summation, ALE technology can notably enhance the tolerance of B. subtilis to VK and increase MK-7 production, thus offering a theoretical framework for the microbial fermentation production of other VK 2 subtypes. Additionally, the evolved strain BS012 can be developed for integration into probiotic formulations within the food industry to maintain intestinal flora homeostasis, mitigate osteoporosis risk, and reduce the incidence of cardiovascular disease. KEY POINTS: • Bacillus subtilis was evolved for improved vitamin K tolerance and menaquinone-7 (MK-7) production • Evolved strains formed wrinkled biofilms and elongated almost twofold in length • Evolved strains induced sporulation to improve tolerance when carbon was limited., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

This research combined Whole-Genome sequencing, intraspecific comparative genomics and transposon mutagenesis to investigate the menaquinone-7 (MK-7) synthesis potential in Bacillus subtilis natto. First, Whole-Genome sequencing showed that Bacillus subtilis natto BN-P15-11-1 contains one single circular chromosome in size of 3,982,436 bp with a GC content of 43.85 %, harboring 4,053 predicted coding genes. Next, the comparative genomics analysis among strain BN-P15-11-1 with model Bacillus subtilis 168 and four typical Bacillus subtilis natto strains proves that the closer evolutionary relationship Bacillus subtilis natto BN-P15-11-1 and Bacillus subtilis 168 both exhibit strong biosynthetic potential. To further dig for MK-7 biosynthesis latent capacity of BN-P15-11-1, we constructed a mutant library using transposons and a high throughput screening method using microplates. We obtained a YqgQ deficient high MK-7 yield strain F4 with a yield 3.02 times that of the parent strain. Experiments also showed that the high yield mutants had defects in different transcription and translation regulatory factor genes, indicating that regulatory factor defects may affect the biosynthesis and accumulation of MK-7 by altering the overall metabolic level. The findings of this study will provide more novel insights on the precise identification and rational utilization of the Bacillus subtilis subspecies for biosynthesis latent capacity., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: Menaquinone-7 (MK-7), which is associated with complex and tightly regulated pathways and redox imbalances, is produced at low titres in Bacillus subtilis. Synthetic biology provides a rational engineering principle for the transcriptional optimisation of key enzymes and the artificial creation of cofactor regeneration systems without regulatory interference. This holds great promise for alleviating pathway bottlenecks and improving the efficiency of carbon and energy utilisation., Results: We used a bottom-up synthetic biology approach for the synthetic redesign of central carbon and to improve the adaptability between material and energy metabolism in MK-7 synthesis pathways. First, the rate-limiting enzymes, 1-deoxyxylulose-5-phosphate synthase (DXS), isopentenyl-diphosphate delta-isomerase (Fni), 1-deoxyxylulose-5-phosphate reductase (DXR), isochorismate synthase (MenF), and 3-deoxy-7-phosphoheptulonate synthase (AroA) in the MK-7 pathway were sequentially overexpressed. Promoter engineering and fusion tags were used to overexpress the key enzyme MenA, and the titre of MK-7 was 39.01 mg/L. Finally, after stoichiometric calculation and optimisation of the cofactor regeneration pathway, we constructed two NADPH regeneration systems, enhanced the endogenous cofactor regeneration pathway, and introduced a heterologous NADH kinase (Pos5P) to increase the availability of NADPH for MK-7 biosynthesis. The strain expressing pos5P was more efficient in converting NADH to NADPH and had excellent MK-7 synthesis ability. Following three Design-Build-Test-Learn cycles, the titre of MK-7 after flask fermentation reached 53.07 mg/L, which was 4.52 times that of B. subtilis 168. Additionally, the artificially constructed cofactor regeneration system reduced the amount of NADH-dependent by-product lactate in the fermentation broth by 9.15%. This resulted in decreased energy loss and improved carbon conversion., Conclusions: In summary, a "high-efficiency, low-carbon, cofactor-recycling" MK-7 synthetic strain was constructed, and the strategy used in this study can be generally applied for constructing high-efficiency synthesis platforms for other terpenoids, laying the foundation for the large-scale production of high-value MK-7 as well as terpenoids., (© 2022. The Author(s).)

Bacillus subtilis natto is a GRAS bacterium. Nattokinase, with fibrinolytic and antithrombotic activities, is one of the major products of this organism. It is being gradually recognized that B. subtilis natto can also be used as a biosynthetic strain for vitamin K2, which has phenomenal benefits, such as effects in the prevention of cardiovascular diseases and osteoporosis along with antitumor effects. Knocking out of the aprN gene by homologous recombination could improve the redox potential and slightly increase the concentration of MK-7. By detecting the change in redox potential during the growth of B. subtilis natto, a good oxygen supply and state of the cell membrane were found to be beneficial to vitamin K2 synthesis. A two-step RSM was used to optimize the operation parameters and substrate concentration in the new residue-free fermentation culture. The optimal conditions for the residue-free medium and control were determined. The optimum concentrations of soybean flour, corn flour, and peptone were 78.9, 72.4, and 24.8 g/L, respectively. The optimum rotational speed and volume of the culture medium using a shaking flask were 117 rpm and 10%, respectively. The state and composition of the cell membranes were more stable when engineered bacteria were cultured in this residue-free fermentation medium. Finally, the concentration of MK-7 increased by 37% to 18.9 mg/L, and the fermentation time was shortened by 24 h.

In this study, seven microbial materials (entomogenous fungi Bb3088 mycelia, entomogenous fungi Bb3088 spores, entomogenous fungi Ma2677 mycelia, entomogenous fungi Ma2677 spores, Bacillus subtilis 8204, Staphylococcus aureus 6725, and Saccharomyces cerevisiae 1025) were used to measure electromagnetic (EM) signal extinction. They were subjected to light absorption and reflection measurements in the range of 4000-400 cm(-1) (2.5-25 µm) using Fourier transform infrared spectroscopy. The specular reflection spectrum method was used to calculate the real (n) and imaginary (k) parts of the complex refractive index. The complex refractive index with real part n and imaginary part k in the infrared band satisfies the following conditions n ≥ 1 and k ≥ 0. The mass extinction coefficient was calculated based on Mie theory. Entomogenous fungi Ma2677 spores and entomogenous fungi Bb3088 spores were selected as EM signal extinction materials in the smoke box test. The transmittances of entomogenous fungi Bb3088 spores and entomogenous fungi Ma2677 spores were 11.63% and 5.42%, and the mass extinction coefficients were 1.8337 m(2)/g and 1.227 m(2)/g. These results showed that entomogenous fungi Bb3088 spores and entomogenous fungi Ma2677 spores have higher extinction characteristics than other microbial materials., (© The Author(s) 2016.)

Botrytis cinerea is one of the most serious post-harvest pathogens of fruits and vegetables. Volatiles generated by Bacillus subtilis JA significantly inhibited both spore germination and elongation of germ tubes in Botrytis cinerea using a two-compartment agar-plate assay. The volatiles caused protoplasm retraction from the hyphal tips to the spores.

Bacillus subtilis JA produces a broad spectrum of bioactive lipopeptides with great potential applications in agriculture and industry. Crude lipopeptides were extracted with 100% methanol from the precipitate which was obtained by adding 6 mol/L HCl to the cell-free culture broth. The crude extract was separated on a Diamonsil C18 column (5 microm, 250 mm x 4.6 mm) in reversed-phase high performance liquid chromatography (HPLC) system to separate the lipopeptide homologues. Five peaks were eluted from HPLC. Electrospray ionization mass spectrometry (ESI-MS) was used to analyze each HPLC fraction. The results showed three series of ion peaks. According to the values of m/z, the three series of ions were classified into surfactin, iturin and fengycin homologues, which were well-known biosurfactants produced by B. subtilis strains. The major ions were structurally characterized using tandem mass spectrometry. This study suggested a reasonable method for the isolation and identification of lipopeptides produced by B. subtilis strains.

Lipopeptides produced by Bacillus subtilis JA antagonized a broad spectrum of plant fungal pathogens. The purification and identification of the lipopeptide antibiotics plays an important role for further research. Crude lipopeptides were extracted with methanol from the precipitate, which was obtained by adding 6mol/L HCl to the cell-free culture broth and then stored at 4 degrees C overnight. The crude extract was run on reversed-phase HPLC system with a Diamonsil C18 column (250 mm x 4.6 mm, Dikma) to separate the lipopeptides. Two antifungal compounds, which had strong inhibitory activity against various plant fungal pathogens, such as Fusarium graminearum, were purified. The molecular weights of two compounds were determined by electrospray ionization mass spectrometry (ESI/MS). Two compounds, with molecular weights of 1042.4 Da and 1056.5 Da, were homologues differed by a structure of -CH2. ESI collision induced dissociation mass spectrometry analysis was used to sequence the structure of purified compounds. Typical b- and y- type fragments showed that compound 1 (with a molecular weight of 1042.4 Da) had a primary structure of Pro-Asn-Tyr-PAA-Asn-Tyr-Asn-Gln (PAA represented beta-amino acid), which was consistent with lipopeptide iturin A. Compound 2 was a homologue of iturin A.

Bacillus subtilis is one of the few strains that can secrete synthetic menaquinone-7 (MK-7) to the outside of the cell, and its purpose and mechanism have not been clearly studied. As an amphiphilic protein naturally synthesized by Bacillus subtilis, the BslA protein may be involved in the inversion of extracellular vitamin K2 solubility. The protein structure in UniProt was used to search for the possible binding sites of MK-7, and the analysis of the higher ranking results of the genetic algorithm showed that the ASP166 residue was likely to be the binding site. They could form a stable hydrogen bond connection through ASP166, and approximately 7 proteins formed the conformation of a fixed naphthoquinone ring. We isolated and obtained the BslA protein by Ni-NTA affinity chromatography. Then, MK-7 was modified by BslA in vitro. A series of experiments, such as SEM, XPS, and WCA, showed that MK-7 and BslA proteins can realize self-assembly and transform from fat-soluble to water-soluble complexes. When the bslA protein in Bacillus subtilis natto was overexpressed, its MK-7 synthesis ability was further improved, especially the extracellular MK-7 content, which increased by 16%. This finding suggests that the BslA protein in Bacillus subtilis is likely to be involved in the extracellular secretion of MK-7 as a receptor. [ABSTRACT FROM AUTHOR]

• It is the first report on the purification of menaquinone-7 from Bacillus subtilis natto fermentation. • A large of menaquinone-7 was extracted from wet-cell by two-stage extraction with ethanol. • A simple, economic, and efficient preparation of manquinone-7 from Bacillus subtilis natto fermentation. • The method possesses great potential for large-scale preparation of vitamin K 2. Meanquinone-7 (MK-7) can be produced by the fermentation of Bacillus subtilis (natto), which is known for its nutraceutical and pharmacological properties. This study focused on the separation and purification of meanquinone-7 from wet cells produced by Bacillus subtilis (natto) fermentation. Using ethanol to extract menaquinone-7 twice directly from the crude cells after fermentation produced a menaquinone-7 yield of 1.47mg/g. The crude extract was purified using macroporous resin and subsequent crystallization. After treatment with HPD722 resin, the purification of menaquinone-7 were 7.17-fold increased with recovery yields of 97.2%. Menaquinone-7 with purities of more than 96% were produced with recovery yield of 99.3% by recrystallized with 95% ethanol. The developed procedure can produce high-purity of menaquinone-7 as well as high recoveries. This is an economical, efficient and simple process to large-scale preparation of high-purity menaquinone-7. [ABSTRACT FROM AUTHOR]