An enzyme cascade for green synthesis of chrysanthemol and chrysanthemic acid: key precursors for pyrethrins and pyrethroids

Abstract

Chrysanthemic acid is a critical chiral building block for both natural pyrethrins and synthetic pyrethroids. However, its supply is constrained by the limitations of plant extraction and the low atom economy of chemical synthesis. In this study, we engineered an artificial enzymatic cascade—both in vitro and in Escherichia coli—for the biosynthesis of chrysanthemol and chrysanthemic acid via the isopentenol utilization pathway (IUP). We first identified a highly efficient isopentenyl kinase, SmaThiM, which effectively debottlenecked the upstream pathway and enabled the in vitro production of chrysanthemol from the readily available substrate prenol. Through systematic optimization of gene organization, fusion protein design, and the implementation of a biphasic system, we developed a whole-cell biocatalyst capable of producing chrysanthemol at a titer of 510 mg L⁻¹. To extend this approach to chrysanthemic acid production, two plant-derived enzymes were integrated into the cascade, and the resulting optimized whole-cell biocatalyst achieved a titer of 203 mg L⁻¹ of (1R,3R)-chrysanthemic acid. These titers represent the highest microbial production levels reported to date for both compounds, demonstrating a promising, green, and high-efficiency biotechnological route for the manufacture of essential pesticide precursors.