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.

Vitamin B₆ is an essential nutrient, which is widely used in food products, feed additives, pharmaceuticals, disease diagnosis, and other fields. Pyridoxal‐5′‐phosphate (PLP), the active form of vitamin B₆, is an important coenzyme participating in a variety of enzyme reactions. At present, the oxazole method is mainly used for the production of vitamin B₆, but toxic and corrosive intermediates produced in the synthesis process, which does not conform to the green manufacturing. Therefore, there is considerable interest in shifting from chemical processes to sustainable fermentation techniques and research on PLP metabolism of other valuable compounds. In this review, we will briefly describe the enzymes that PLP participated and focus on the advances in vitamin B₆ biosynthesis and discuss its application to engineering bacteria that overproduce other commercially valuable substances including cadaverine, 3,4‐dihydroxyphenylacetate‐l‐alanine, γ‐aminobutyric acid, and l‐phosphinothricin. It will provide a reference for the biosynthesis of vitamin B₆ or other valuable compounds in which PLP participates in the reaction, and we also look forward to the future development prospect of PLP.

Pyridoxine, also known as vitamin B₆, is an essential cofactor in numerous cellular processes. Its importance in various applications has led to a growing interest in optimizing its production through microbial biosynthesis. However, an imbalance in the net production of NADH disrupts intracellular cofactor levels, thereby limiting the efficient synthesis of pyridoxine. In our study, we focused on multiple cofactor engineering strategies, including the enzyme design involved in NAD⁺-dependent enzymes and NAD⁺ regeneration through the introduction of heterologous NADH oxidase (Nox) coupled with the reduction in NADH production during glycolysis. Finally, the engineered E. coli achieved a pyridoxine titer of 676 mg/L in a shake flask within 48 h by enhancing the driving force. Overall, the multiple cofactor engineering strategies utilized in this study serve as a reference for enhancing the efficient biosynthesis of other target products.

Vitamin B₆ refers to a family of water-soluble B vitamin, which is essential for various physiological functions, including amino acid metabolism, neurotransmitter synthesis, and hemoglobin synthesis. The biosynthesis of pyridoxine (PN), a commercial form of vitamin B₆, through microbial fermentation has garnered widespread attention owing to its environmentally friendly and safe production methods, as well as its mild reaction conditions. However, the low yield of natural strains limits their application. This study focused on constructing a high-yielding strain of PN through pathway engineering and process optimization. Firstly, five key deoxyxylulose-5-phosphate-dependent pathway genes (epd, pdxB, serC, pdxA, and pdxJ) were overexpressed in Bacillus subtilis ARTP, which improved the PN titer by 3.2-fold to 2.9 mg/L. Subsequently, pdxST genes involved in the DXP-independent pathway were screened from various strains. Ribosome binding site (RBS) sequences were optimized to regulate their expression, which further increased the PN titer to 24.6 mg/L. Finally, systematic medium optimization was identified as a critical strategy for enhancing PN biosynthesis, leading to a remarkable 1.8-fold improvement in PN production. Under optimized fermentation conditions, the engineered strain achieved a PN titer of 174.6 mg/L in fed-batch fermentation, which represents the highest level reported to date in B. subtilis. Overall, this study presents an effective strategy combining pathway engineering and medium optimization for significantly improving PN production, offering valuable insights for the industrial development of PN biosynthesis.

Metabolic engineering has a wide range of applications, spanning key sectors such as energy, pharmaceuticals, agriculture, chemicals, and environmental sustainability. Its core focus is on precisely modulating metabolic pathways to achieve efficient, sustainable, and environmentally friendly biomanufacturing processes, offering new possibilities for societal sustainable development. Gene attenuation is a critical technique within metabolic engineering, pivotal in optimizing metabolic fluxes and improving target metabolite yields. This review article discusses gene attenuation mechanisms, the applications across various biological systems, and implementation strategies. Additionally, we address potential future challenges and explore its potential to drive further advancements in the field.

Pyridoxine (PN), a major commercial form of vitamin B₆, is mainly produced via chemical synthesis, raising environmental concerns. Microbial fermentation offers a greener alternative, but low biomass and titer limit industrial application. In this study, we developed an integrated strategy combining DO-stat restricted mix-carbon feeding with two-stage pressure-controlled fermentation conditions, and medium optimization to enhance cell growth and PN production in Escherichia coli. DO-stat feeding enabled dynamic regulation of oxygen and carbon sources, while a two-stage pressure-controlled (0.1 bar for 0–24 h, 0.2 bar thereafter) improved both growth and PN biosynthesis. Meanwhile, a novel medium (CRS-67), optimized via Taguchi design, increased shake flask PN titer by 275 mg/L, 75.5 % over CS medium. Under systematic fermentation optimization in a 5-L bioreactor, OD₆₀₀ reached 142.8 and PN titer reached 3.33 g/L—the highest reported level to date. This study presents a robust and scalable process for microbial PN production and offers insights for industrial biomanufacturing of vitamin B₆.