Engineering Precursor and NADPH Supply to Enhance 1,4-Butanediol Production in Plasmid-Free Escherichia coli.

1,4-Butanediol (1,4-BDO) is an important non-natural platform chemical for biodegradable plastics, yet its microbial production is constrained by inadequate NADPH availability. In this study, five heterologous enzymes were introduced into Escherichia coli for the biosynthesis of 1,4-BDO. Through coordinated regulation of pathway enzyme expression and enhancement of key rate-limiting steps, a 1,4-BDO titer of 0.93 g/L was achieved from glucose. Precursor engineering to increase succinyl-CoA availability and pathway intermediate accumulation further improved the titer to 1.87 g/L. Strengthening endogenous NADPH-generating pathways, combined with the development of an intracellular biohybrid system, increased intracellular NADPH levels by 73%, resulting in a 1,4-BDO titer of 2.54 g/L (35.8% improvement). After optimization of fermentation conditions, the optimal strain BDO-25 produced 3.1 g/L and 7.5 g/L of 1,4-BDO in shake-flask and 5-L bioreactor fermentations, respectively, without antibiotics or inducers. This study offers a highly efficient and scale-up feasible strategy for the biosynthesis of NADPH-dependent diols.