Targeted membrane permeability alterations in Enterobacter hormaechei ZS01 for high-level production of 3-succinyl-pyridine from the tobacco waste.

Pyridine derivatives exhibit notable bioactivity and therapeutic potential, yet their chemical synthesis remains challenging. Nicotine, as a readily available precursor, offers a promising route for the biosynthesis of high-value pyridine compounds from tobacco waste. In this study, a comprehensive valorization strategy was developed based on Enterobacter hormaechei ZS01, a highly efficient nicotine-degrading strain previously isolated. ZS01 converts nicotine into the antihypertensive precursor 3-succinoyl-pyridine (SP) via the pyrrolidine pathway. Initially, the molybdenum cofactor biosynthesis gene, moaE, was deleted in ZS01, yielding the mutant strain ZS01ΔmoaE, which exhibited a 2.78 fold increase in extracellular SP concentration compared to the wild type. To relieve the limitation imposed by cell membrane permeability, the lipopolysaccharide synthesis genes rfaF and rfaC were further deleted, resulting in a final extracellular SP concentration of 1.29 g/L, representing a 1.33 fold increase over ZS01ΔmoaE. Subsequent optimization of reaction conditions was conducted, and the optimal parameters were determined to be 30 °C, pH 8.0, 1 mM Mg²⁺. In a 5 L bioreactor, with an initial nicotine concentration of 2 g/L, an SP titer of 4.11 g/L and a conversion rate of 68.82% were achieved. This work presents the construction of an engineered microbial catalyst capable of efficient SP biosynthesis, offering a scalable route for the bioconversion of tobacco waste into high-value pyridine derivatives, with strong potential for industrial application.