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

Wu, Jiang; Li, Wei; Zhao, Su; Senhe, Qian; Zhou, Wang; Zhou, Mengjie; Hu, Wenchao; Wang, Jian; Hu, Liu-xiu; Liu, Yan; Xue, Zhenglian

doi:10.1186/s12934-021-01603-5

Cited by 18 publications

(5 citation statements)

References 65 publications

(72 reference statements)

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“…It was found that the synthesis of 1 mol DMK-7 requires 19 mol glycerol and 10 mol NADPH (accompanied by the production of 11 mol NADH). From the overall reaction equation, the higher the production of MK-7, the higher the level of intracellular accumulation of NADH; this conclusion was supported by the findings of a previous study [ 2 ]. However, when the intracellular reducing power is unbalanced, MK-7 synthesis is inhibited, resulting in a decrease in MK-7 levels.…”

Section: Resultssupporting

confidence: 84%

Bottom-up synthetic biology approach for improving the efficiency of menaquinone-7 synthesis in Bacillus subtilis

et al. 2022

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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.

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Section: Resultssupporting

confidence: 84%

Bottom-up synthetic biology approach for improving the efficiency of menaquinone-7 synthesis in Bacillus subtilis

et al. 2022

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“…Triple-fusion PCR was applied for the connection of the upstream DNA sequence, lox71-lox66 cassettes, promoter P D4 sequence, target gene sequence, and downstream sequence. 36 GenScript Biotech (Nanjing, China) was employed to detect the sequence of the PCR fusion fragments. After the sequence validation by GenScript Biotech (Nanjing, China), we integrated the fusion segments into the genome.…”

Section: ■ Materials and Methodsmentioning

confidence: 99%

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

Zhao

et al. 2022

ACS Synth. Biol.

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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.

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“…In our previous studies, we also found that the formation of wrinkles could promote the connection of channel networks in the membrane and reduce the resistance of liquid ow, so that the pressure in the pipeline was lower than the atmospheric pressure outside. Such a pressure gradient could drive and promote the ow of nutrients through the bio lm (Wu et al 2021) and nally affect the synthesis of the secondary metabolite MK-7. As a nonionic surfactant, PEG-200 does not ionize and generate ions in aqueous solution, possessing the characteristics of wetting and emulsifying.…”

Section: Screening and Optimization Of Surfactantsmentioning

confidence: 99%

Improvement of Vitamin K2 Production by Bacillus subtilis mutant with leakage fermentation

Zhou

et al. 2022

Preprint

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Menaquinone-7 (MK-7), a highly valuable member of the vitamin K2 series, is an essential nutrient for humans. It plays an important role in the treatment of coagulation, osteoporosis, promotion of liver function recovery and prevention of cardiovascular diseases. In this study, in order to further improve the metabolic synthesis of MK-7 by the mutant strain, the effect of metabolic synthesis of MK-7 by mutant strain Bacillus subitilis 168 KO-SinR (BS168 KO-SinR) was analyzed by adding surfactants. The 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, as indicated by scanning electron microscopy and flow cytometry. 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%. qRT-PCR showed that the addition of surfactant significantly increased the expression level of MK-7 synthesis-related genes, and the results of electron microscope showed that the addition of surfactant changed the permeability of cell membrane. The research results of this paper provide a certain reference value for the industrial development of MK-7 prepared by fermentation.

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

Cited by 18 publications

References 65 publications

Bottom-up synthetic biology approach for improving the efficiency of menaquinone-7 synthesis in Bacillus subtilis

Bottom-up synthetic biology approach for improving the efficiency of menaquinone-7 synthesis in Bacillus subtilis

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

Improvement of Vitamin K2 Production by Bacillus subtilis mutant with leakage fermentation

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