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

Abstract

Flowers of Tanacetum cinerariifolium produce a set of compounds known collectively as pyrethrins, which are commercially important pesticides that are strongly toxic to flying insects but not to most vertebrates. A pyrethrin molecule is an ester consisting of either trans-chrysanthemic acid or its modified form, pyrethric acid, and one of three alcohols, jasmolone, pyrethrolone, and cinerolone, that appear to be derived from jasmonic acid. Chrysanthemyl diphosphate synthase (CDS), the first enzyme involved in the synthesis of trans-chrysanthemic acid, was characterized previously and its gene isolated. TcCDS produces free trans-chrysanthemol in addition to trans-chrysanthemyl diphosphate, but the enzymes responsible for the conversion of trans-chrysanthemol to the corresponding aldehyde and then to the acid have not been reported. We used an RNA sequencingbased approach and coexpression correlation analysis to identify several candidate genes encoding putative trans-chrysanthemol and trans-chrysanthemal dehydrogenases. We functionally characterized the proteins encoded by these genes using a combination of in vitro biochemical assays and heterologous expression in planta to demonstrate that TcADH2 encodes an enzyme that oxidizes trans-chrysanthemol to trans-chrysanthemal, while TcALDH1 encodes an enzyme that oxidizes trans-chrysanthemal into transchrysanthemic acid. Transient coexpression of TcADH2 and TcALDH1 together with TcCDS in Nicotiana benthamiana leaves results in the production of trans-chrysanthemic acid as well as several other side products. The majority (58%) of trans-chrysanthemic acid was glycosylated or otherwise modified. Overall, these data identify key steps in the biosynthesis of pyrethrins and demonstrate the feasibility of metabolic engineering to produce components of these defense compounds in a heterologous host.

Pyrethric acid of natural pyrethrin insecticide: complete pathway elucidation and reconstitution in Nicotiana benthamiana

“…Pyrethrum ( Tanacetum cinerariifolium ) seeds were germinated in soil at 30°C and the plants were grown under the condition as described previously (Xu et al ., 2018b). Various pyrethrum tissues, including flowers, leaves and stems, were collected and flash frozen in liquid nitrogen and stored in −80°C until use.…”

“…All of these genes were spliced into pEAQ‐HT binary vector using the same method above to express them under the control of 35S promoter. The constructs of TcCDS , TcADH2 , TcALDH1 and GFP for transient expression in N. benthamiana leaves were obtained from our previous work (Xu et al ., 2018b). The constructs of TcGLIP encoding a His 6 tag in the N‐terminus was made as described previously (Li et al ., ).…”

“…The procedure used for transient expression in N. benthamiana leaves was performed as described previously (Xu et al ., 2018b) with minor modification. Briefly, A. tumefaciens strain GV3101 cells harboring pEAQ‐HT constructs carrying specific genes was grown in Luria–Bertani medium at 28°C at 200 rpm for overnight to OD 600 value of 1.0 and then centrifuged at 3000 g for 15 min to remove medium culture.…”

“…The GC‐MS analysis was performed as described previously (Xu et al ., 2018b). Aliquots (1 μl) of sample were injected into a Shimadzu Qp‐2010 GC‐MS system equipped with the Rxi‐5Sil column (30 m × 0.25 mm × 0.25 μm film thickness; Restek, PA, USA).…”

“…). We recently showed that chrysanthemol is converted to chrysanthemic acid by the consecutive actions of two enzymes present in pyrethrum flowers, T. cinerariifolium alcohol dehydrogenase 2 (TcADH2) and aldehyde dehydrogenase 1 (TcALDH1; Xu et al ., 2018b; Fig. ).…”