Chrysanthemyl Diphosphate Synthase Operates in Planta as a Bifunctional Enzyme with Chrysanthemol Synthase Activity

Abstract

Background: Chrysanthemyl diphosphate synthase (CDS) is known to catalyze the formation of the irregular terpenoid, chrysanthemyl diphosphate (CPP). Results: CDS also catalyzes the next step from CPP to chrysanthemol. Conclusion: CDS is actually a chrysanthemol synthase (CHS) with bifunctional enzyme activity. Significance: Identification of CHS increases our understanding in terpene biosynthesis and paves the way for engineering biosynthesis of the most widely used natural pesticide, pyrethrins.

Coexpression Analysis Identifies Two Oxidoreductases Involved in the Biosynthesis of the Monoterpene Acid Moiety of Natural Pyrethrin Insecticides in Tanacetum cinerariifolium

“…5A). As reported previously (Rivera et al, 2001;Yang et al, 2014), the CDS-catalyzed reaction also produces small amounts of lavandulyl diphosphate, causing the transchrysanthemol preparation to contain a small amount of lavandulol (Fig. 5A).…”

“…The mass spectrum-mass spectrum (MS/MS) of this metabolite (Fig. 7E) was identical to that of a compound produced in tobacco (Nicotiana tabacum) expressing TcCDS (Yang et al, 2014) that these workers putatively identified as trans-chrysanthemyl malonylglucoside. In samples expressing TcCDS + TcADH2, a peak with m/z 831.3328 was detected with an MS/MS that closely matched the spectrum of another monoterpene acid glucoside, geranyl-6-O-malonyl-b-D-glucopyranoside (Yang et al, 2011).…”

“…For controls, we infiltrated N. benthamiana leaves with TcCDS alone, TcCDS and TcADH2, or EGFP (Enhanced Green Fluorescent Protein; see "Materials and Methods") as a control. In these experiments, the complete open reading frame of TcCDS was used, including the transit peptide that was shown to direct the protein to the plastids (Yang et al, 2014). Transformed leaves were harvested 10 d after infiltration, and the products were extracted and analyzed by GC-MS. As expected, transient expression of TcCDS alone resulted in the production of trans-chrysanthemol at 18.3 nmol g 21 fresh weight and trace amounts of transchrysanthemic acid (Fig.…”

“…The transcriptomic profiling data (assembled transcripts and the number of reads from each transcript) were used to perform coexpression cluster analysis (Eisen et al, 1998) to identify candidate ADH and ALDH genes whose expression patterns most resembled that of TcCDS, the gene encoding the key enzyme in the pathway for trans-chrysanthemic acid (Rivera et al, 2001;Yang et al, 2014), as well as that of TcGLIP, encoding the enzyme that forms the final pyrethrin product (Kikuta et al, 2012). Both genes were shown to have peak transcript levels at the earliest developmental stage (stage 2) f Kinetic parameters were determined with 0.04 mM trans-chrysanthemal.…”

“…However, no such enzymatic activities were reported thus far in T. cinerariifolium. A recent study by Yang et al (2014) reported that prolonged incubation of Escherichia coli-produced recombinant CDS protein with low concentrations of DMAPP led to the detection of trans-chrysanthemol in addition to CDP, suggesting that CDS can produce trans-chrysanthemol in vivo. Furthermore, the enzymes responsible for the subsequent oxidation reactions of trans-chrysanthemol to transchrysanthemal and then to trans-chrysanthemic acid (Fig.…”