Engineering Precursor and NADPH Supply to Enhance 1,4-Butanediol Production in Plasmid-Free <i>Escherichia coli</i>

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.