Engineering <i>Escherichia coli</i> for Perillyl Alcohol Production with Reduced Endogenous Dehydrogenation

Perillyl alcohol is a widely utilized antitumor agent in cancer therapy. Due to the limitations associated with chemical synthesis and plant extraction methods, bioengineering microorganisms for the production of perillyl alcohol and its precursor limonene offers a more scalable and industrially compatible approach. In this study, we present the design of an <i>Escherichia coli</i> cell factory capable of de novo synthesis of limonene and perillyl alcohol from glucose. A comprehensive systems engineering approach was employed to increase precursor availability, enhance electron transfer efficiency, and reduce byproduct formation, resulting in efficient perillyl alcohol production. Specifically, ribosome-binding site optimization and increased enzyme expression improved limonene production to 417.04 mg/L. Additionally, the overexpression of cytochrome P450 electron transport proteins, knockdown of endogenous ethanol dehydrogenase to prevent product loss, and implementation of two-phase fermentation led to the production of 309.1 mg/L perillyl alcohol in shake flask cultures, which marks the highest titer reported for shake flask systems. This illustrates the critical role of reducing perillyl aldehyde byproduct formation in enhancing the feasibility of <i>Escherichia coli-</i>based perillyl alcohol production, providing a foundation for its economical large-scale production via microbial bioprocessing.