Cofactors are crucial chemicals that maintain cellular redox balance and drive the cell to do synthetic and catabolic reactions. They are involved in practically all enzymatic activities that occur in live cells. It has been a hot research topic in recent years to manage their concentrations and forms in microbial cells by using appropriate techniques to obtain more high-quality target products. In this review, we first summarize the physiological functions of common cofactors, and give a brief overview of common cofactors acetyl coenzyme A, NAD(P)H/NAD(P)+, and ATP/ADP; then we provide a detailed introduction of intracellular cofactor regeneration pathways, review the regulation of cofactor forms and concentrations by molecular biological means, and review the existing regulatory strategies of microbial cellular cofactors and their application progress, to maximize and rapidly direct the metabolic flux to target metabolites. Finally, we speculate on the future of cofactor engineering applications in cell factories. Graphical Abstract

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

Vitamin B6 is an essential nutrient with extensive applications in the medicine, food, animal feed, and cosmetics industries. Pyridoxine (PN), the most common commercial form of vitamin B6, is currently chemically synthesized using expensive and toxic chemicals. However, the low catalytic efficiencies of natural enzymes and the tight regulation of the metabolic pathway have hindered PN production by the microbial fermentation process. Here, we report an engineered Escherichia coli strain for PN production. Parallel pathway engineering is performed to decouple PN production and cell growth. Further, protein engineering is rationally designed including the inefficient enzymes PdxA, PdxJ, and the initial enzymes Epd and Dxs. By the iterative multimodule optimization strategy, the final strain produces 1.4 g/L of PN with productivity of 29.16 mg/L/h by fed-batch fermentation. The strategies reported here will be useful for developing microbial strains for the production of vitamins and other bioproducts having inherently low metabolic fluxes. 

Protein engineering and iterative multimodule optimization for vitamin B6 production in Escherichia coli

Menaquinone-7 (MK-7), a subtype of vitamin K2 (VK2), 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 K3 (VK3) as a structural analog of MK-7. The resulting strain, BS012, exhibited heightened tolerance to high VK3 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 VK2 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. 

Adaptive laboratory evolution for improved tolerance of vitamin K in Bacillus subtilis

Menaquinone-7 (MK-7) is an important class of vitamin K2 that is essential in human health and can prevent osteoporosis and cardiovascular disease. However, due to the complex synthesis pathway, the synthesis efficiency is low. The main objective of this study was to explore the effect of enhanced supply of precursors in Bacillus natto. Three precursors of pyruvate, shikimic acid, and sodium glutamate were chosen to investigate the effect of enhanced supply of precursors on MK-7 synthesis. Then, the optimal concentrations, different combinations, and different adding times were systematically studied, respectively. Results showed that the combination of shikimic acid and sodium glutamate could boost MK-7 production by 2 times, reaching 50 mg/L of MK-7 titer and 0.52 mg/(L·h) of MK-7 productivity. Furthermore, adding shikimic acid and sodium glutamate initially and feeding pyruvate at 48 h and 72 h increased MK-7 production to 58 mg/L. At the same time, the expression of the three related genes was also significantly upregulated. Subsequently, a new fermentation strategy combining the precursors enhancement and product secretion was proposed to enhance MK-7 yield and MK-7 productivity to 63 mg/L and 0.45 mg/(L·h). This study proposed a new fermentation regulation strategy for the enhancement of vitamin K2 biosynthesis. 

Enhancing menaquinone-7 biosynthesis through strengthening precursor supply and product secretion

This study optimized the menaquinone-7 (MK-7) synthetic pathways in Bacillus subtilis (B. subtilis) natto NB205, a strain that originated from natto, to enhance its MK-7 production. Utilizing mutation breeding, we developed NBMK308, a mutant strain that demonstrated a significant 117.23% increase in MK-7 production. A comprehensive transcriptome analysis identified two key genes, ispA and ispE, as being critical in MK-7 synthesis. The dual-sgRNA CRISPRa system was utilized to achieve precise regulation of ispA and ispE in the newly engineered strain, A3E3. This strategic modulation resulted in a significant enhancement of MK-7 production, achieving increases of 20.02% and 201.41% compared to traditional overexpression systems and the original strain NB205, respectively. Furthermore, the fermentation supernatant from A3E3 notably inhibited Salmonella invasion in Caco-2 cells, showcasing its potential for combating such infections. The safety of the dual-sgRNA CRISPRa system was confirmed through cell assays. The utilization of the dual-sgRNA CRISPRa system in this study was crucial for the precise regulation of key genes in MK-7 synthesis, leading to a remarkable increase in production and demonstrating additional therapeutic potential in inhibiting pathogenic infections. This approach effectively combined the advantages of microbial fermentation and biotechnology, addressing health and nutritional challenges.

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

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. 

/pdf/bottom-up-synthetic-biology-approach-for-improving-the-3g5zuyrc.pdf

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

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