Efficient production of 1,6-hexanediol from adipic acid by engineering of carboxylate reductase coupled with genetically modified Escherichia coli

1,6-hexanediol (1,6-HDO) is an important C6 material for synthesis of polyesters and polyurethanes. The development of a green, and cost-effective synthesis process for 1,6-HDO production is highly desirable. Recent work has demonstrated the biological synthesis of 1,6-HDO from adipic acid (AA) or cyclohexane. However, the 1,6-HDO yields have remained extremely low. In this study, we developed an efficient whole-cell catalytic system for 1,6-HDO production from adipic acid, utilizing carboxylate reductases (CAR) and aldehyde-keto reductase (AKR) in Escherichia coli. Enzyme screening, followed by structure-guided semi-rational engineering was first performed to address the rate-limiting enzyme of CAR. A variant, MabCAR^W283K/L306K, was obtained with a 4-fold increase in specific activity (2.03 U mg⁻¹), and a 2.87-fold enhancement in catalytic efficiency (156.75 s⁻¹· mM⁻¹). Then, the NADPH supply in E. coli was also improved by overexpressing icd and deleting pgi for a further improvement on 1,6-HDO titer. Finally, three genes of eutG, ygiQ, and yiaY involved in 1,6-HDO degradation were identified by comprehensive screening of 52 single gene knockout strains encoding the putative alcohol dehydrogenase in E. coli. Combined deletions of these three genes significantly increased the 1,6-HDO titer by 35.4 %. Under the optimized conditions, the engineered strain was capable of producing 14.5 g/L 1,6-HDO from AA, achieving a yield of 89.6 %, the highest titer reported to date. This work successfully provided an efficient and feasible biosynthetic method for the biotransformation of AA into 1,6-HDO.