Engineering Bienzymatic Cascade for Efficient Biosynthesis of Citronellal and Citronellol

ABSTRACT Citronellal and citronellol are monoterpenoids that serve as key ingredients in the flavor and fragrance industry and hold significant potential for pharmaceutical applications. Currently, these compounds are primarily obtained through chemical synthesis or extraction from plant essential oils. However, these methods raise concerns regarding environmental sustainability, carbon emissions, and the growing attentions for natural production. A promising alternative is microbial biosynthesis, which enables the natural and carbon‐neutral production of these compounds in an environmentally friendly manner. In this study, we developed bienzymatic cascades for the efficient production of citronellal and citronellol from geraniol, utilizing E. coli 's endogenous glycerol dehydrogenase (GLDA), NADPH‐dependent aldehyde reductase (AHR), and Catharanthus roseus iridoid synthase (CrIS). The highest in vitro production reached 92 mg/L of citronellal and 170 mg/L of citronellol in a 5‐h enzymatic reaction. We also engineered these enzymatic cascades into E. coli strains for in vivo production, achieving up to 714 mg/L of citronellol. This study establishes new bienzymatic cascades for the efficient biosynthesis of citronellal and citronellol, demonstrating a possible bio‐based production for natural flavor and fragrance compounds.