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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.

Abstract Background The study and application of microbial consortia are topics of interest in the fields of metabolic engineering and synthetic biology. In this study, we report the design and optimisation of Elizabethkingia meningoseptica and Escherichia coli co-culture, which bypass certain limitations found during the molecular modification of E. meningoseptica, such as resistance to many antibiotics and fewer available molecular tools. Results The octaprenyl pyrophosphate synthase from E. meningoseptica sp. F2 (EmOPPS) was expressed, purified, and identified in the present study. Then, owing to the low vitamin K2 production by E. coli or E. meningoseptica sp. F2 monoculture, we introduced the E. meningoseptica and E. coli co-culture strategy to improve vitamin K2 biosynthesis. We achieved production titres of 32 mg/L by introducing vitamin K2 synthesis-related genes from E. meningoseptica sp. F2 into E. coli, which were approximately three-fold more than the titre achieved with E. meningoseptica sp. F2 monoculture. This study establishes a foundation for further engineering of MK-n (n = 4, 5, 6, 7, 8) in a co-cultivation system of E. meningoseptica and E. coli. Finally, we analysed the surface morphology, esterase activity, and membrane permeability of these microbial consortia using scanning electron microscopy, confocal laser scanning microscopy, and flow cytometry, respectively. The results showed that the co-cultured bacteria were closely linked and that lipase activity and membrane permeability improved, which may be conducive to the exchange of substances between bacteria. Conclusions Our results demonstrated that co-culture engineering can be a useful method in the broad field of metabolic engineering of strains with restricted molecular modifications.

Nutritional food supplements and pharmaceutical products produced with vitamin K2 as raw materials a very promising market in the global scope. The main production method of vitamin K2 is microbial fermentation, but approximately 50% of vitamin K2 synthesized by the main production strain Bacillus subtilis natto exists in extracellular form, which is not easy to separate and extract. In order to solve this problem, in this study, we synthesized a novel cellulose flocculant, MCC-g-LMA, by grafting reaction using microcrystalline cellulose (MCC) and lauryl methacrylate (LMA) as monomers, and ammonium persulfate as an initiator to flocculate VK2 from the fermentation supernatant. The flocculant was characterized by Fourier transform infrared spectroscopy (FTIR), elemental analysis, and scanning electron microscopy (SEM), and the grafting reaction was successful. When the flocculant dosage was 48.0 mg/L and pH was 5.0, the flocculation rate of the MCC-g-LMA on the fermentation supernatant reached 85.3%, and the enrichment rate of VK2 reached 90.0%. Furthermore, we explored the flocculation mechanism of VK2 by the MCC-g-LMA and speculated that the flocculation mechanism mainly included adsorption bridging, hydrophobic association and net trapping and sweep effect. In this study, the extraction method for trace high-value biological products in the fermentation supernatant was improved, which provided a method and theoretical basis for the efficient separation and purification of VK2 and other terpenoids.