Metabolic Engineering of <i>Escherichia coli</i> for Enhanced Production of Cembratrien-ols via Precursor Supply Optimization and Membrane Engineering

Cembratrien-ols (CBT-ols) are diterpenoid compounds derived from <i>Nicotiana</i> plants, exhibit significant insecticidal activity, and have attracted considerable attention for the development of sustainable biopesticides. In this study, an efficient CBT-ols biosynthesis strain was constructed by integrating an artificial isopentenol utilization pathway into <i>Escherichia coli</i>. Multiple endogenous pyrophosphatase genes were systematically knocked out to enhance precursor supply, increasing CBT-ol production to 211.6 ± 5.74 mg/L. To further promote CBT-ol accumulation, cell membrane engineering was employed to expand membrane storage capacity, resulting in a yield of 475.6 ± 13.73 mg/L. Through fermentation optimization via continuous feeding, the engineered strain produced a final yield of 2.87 g/L in a 5 L bioreactor, with a substrate conversion rate of 44.3%, which represents the highest reported yield to date. These findings underscore the substantial benefits of the isopentenol utilization pathway in optimizing synthesis processes, thereby establishing a more robust foundation for the production of isoprenoid compounds.