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

Abstract

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.

Menaquinone Production by Engineering a DegU Quorum Sensing Circuit in Bacillus subtilis

“…As an important type of dynamic regulation, quorum sensing overcomes the limitations of biosensors and metabolite response promoters (Lv et al 2019) and achieve a global regulation of bacteria without the additional inducers in the process. At present, there are many operations to improve MK-7 synthesis by knockout or overexpression of the key genes in regulatory pathways, such as Ding (Ding et al 2022), who successively overexpressed the rate-limiting enzymes Dxs, Fni, Dxr, MenF and MenA, and improved NADH conversion e ciency, the nal output was 53.07 mg/L; Wang (Wang et al 2019) achieved a maximum yield of 18.9mg/L of MK-7 by knocking out aprN and optimizing fermentation conditions. However, the PhrG-RapG-DegU quorum sensing regulatory system we constructed increased the yield of MK-7 by 4.66 times in the ask, reaching a maximum yield of 102.47 mg/L (Fig.…”

Application of cofactors in the regulation of microbial metabolism: A state of the art review

“… Gu et al (2019) reconstructed the metabolic pathway in GlcNAc-producing strains by introducing the pyruvate iron oxidoreductase PorAB, malate dehydrogenase BmqO, and glyceraldehyde-3-phosphate dehydrogenase GoR to achieve the catalytic processes of pyruvate to acetyl-CoA, malate to oxaloacetate and glyceraldehyde-3-phosphate to 3-phosphoglycerate, respectively, avoiding the excess of NADH and thus achieving intracellular redox homeostasis, ultimately increasing the fermentation yield of GlcNAc by 4.06-fold. The yield of Menaquinone-7 (MK-7) in Bacillus subtilis is low, and Ding et al (2022) made MK-7 production of 39.01 mg/L by overexpressing key rate-limiting enzymes such as 1-deoxyxylulose-5-phosphate synthase, isopentenyl-diphosphate delta-isomerase, 1-deoxyxylulose-5-phosphate reductase. Next, after stoichiometric calculation and optimization of the cofactor regeneration pathway, two NADPH regeneration systems were constructed to enhance the endogenous cofactor regeneration pathway, and heterologous NADH kinase was introduced to increase the availability of NADPH in MK-7 biosynthesis.…”

Dual-sgRNA CRISPRa System for Enhanced MK-7 Production and Salmonella Infection Mitigation in Bacillus subtilis natto Applied to Caco-2 Cells

“…In B. subtilis , MK-7 is a critical intermediate in the electron transport chain, essential for bacterial survival . As a result, B. subtilis is widely used as a host organism for producing enzymes and various chemicals, − including MK-7.…”