Design, Evaluation, and Implementation of Synthetic Isopentyldiol Pathways in Escherichia coli

Isopentyldiol (IPDO) is an important raw material in the cosmetic industry. So far, IPDO is exclusively produced through chemical synthesis. Growing interest in natural personal care products has inspired the quest to develop a biobased process. We previously reported a biosynthetic route that produces IPDO via extending the leucine catabolism (route A), the efficiency of which, however, is not satisfactory. To address this issue, we computationally designed a novel non-natural IPDO synthesis pathway (route B) using RetroPath RL, the state-of-the-art tool for bioretrosynthesis based on artificial intelligence methods. We compared this new pathway with route A and two other intuitively designed routes for IPDO biosynthesis from various perspectives. Route B, which exhibits the highest thermodynamic driving force, least non-native reaction steps, and lowest energy requirements, appeared to hold the greatest potential for IPDO production. All three newly designed routes were then implemented in the Escherichia coli BL21(DE3) strain. Results show that the computationally designed route B can produce 2.2 mg/L IPDO from glucose but no IPDO production from routes C and D. These results highlight the importance and usefulness of in silico design and comprehensive evaluation of the potential efficiencies of candidate pathways in constructing novel non-natural pathways for the production of biochemicals.