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

Abstract

Summary In the natural pesticides known as pyrethrins, which are esters produced in flowers of Tanacetum cinerariifolium (Asteraceae), the monoterpenoid acyl moiety is pyrethric acid or chrysanthemic acid. We show here that pyrethric acid is produced from chrysanthemol in six steps catalyzed by four enzymes, the first five steps occurring in the trichomes covering the ovaries and the last one occurring inside the ovary tissues. Three steps involve the successive oxidation of carbon 10 (C10) to a carboxylic group by TcCHH, a cytochrome P450 oxidoreductase. Two other steps involve the successive oxidation of the hydroxylated carbon 1 to give a carboxylic group by TcADH2 and TcALDH1, the same enzymes that catalyze these reactions in the formation of chrysanthemic acid. The ultimate result of the actions of these three enzymes is the formation of 10‐carboxychrysanthemic acid in the trichomes. Finally, the carboxyl group at C10 is methylated by TcCCMT, a member of the SABATH methyltransferase family, to give pyrethric acid. This reaction occurs mostly in the ovaries. Expression in N. benthamiana plants of all four genes encoding aforementioned enzymes, together with TcCDS, a gene that encodes an enzyme that catalyzes the formation of chrysanthemol, led to the production of pyrethric acid.

Pyrethrin Biosynthesis: The Cytochrome P450 Oxidoreductase CYP82Q3 Converts Jasmolone To Pyrethrolone

“…The details of the enzymatic steps leading to the biosynthesis of the monoterpenoid acids chrysanthemic acid and pyrethric acid were recently elucidated (Rivera et al, 2001;Xu et al, 2018Xu et al, , 2019. Chrysanthemyl diphosphate synthase (TcCDS) condenses two molecules of the universal terpene precursor dimethylallyl diphosphate (DMAPP) to produce chysanthemyl diphosphate.…”

“…Chrysanthemic acid is generated from two molecules of DMAPP in sequential reactions catalyzed by TcCDS, TcADH, and TcALDH (Xu et al, 2018). Pyrethric acid is generated from two molecules of DMAPP by the action of these three enzymes plus TcCHH and TcCCMT (Xu et al, 2019). The pyrethrin esters are formed in a reaction catalyzed by the GDSL lipase-like protein (TcGLIP), which links the acid moiety to the alcohol moiety (Kikuta et al, 2012).…”

“…To obtain pyrethric acid from chrysanthemol, the activities of TcADH2 and TcALDH1, which form the carboxylic group at C1, are combined with the activity of a cytochrome P450 oxidoreductase, named chrysanthemol 10-hydroxylase (TcCHH), which catalyzes three consecutive oxidation reactions of carbon 10 to form 10-carboxychrysanthemic acid; 10-carboxychrysanthemic acid is then converted to pyrethric acid by the methylation of the 10-carboxyl group by the SABATH-class methyltransferase named 10-carboxychrysanthemic acid 10-methyltransferase (TcCCMT; Xu et al, 2019).…”

“…The genes involved in the biosynthesis of chrysanthemic acid, 10-carboxychrysanthemic acid, and jasmolone are active mostly in the trichomes of the ovaries (Ramirez et al, 2012;Li et al, 2018;Xu et al, 2018Xu et al, , 2019. In contrast, TcCCMT, responsible for the final step in the synthesis of pyrethric acid, and TcGLIP, the gene encoding the GDSL lipase-like protein responsible for linking the acid moiety to the alcohol moiety to form the final pyrethrin molecules, are maximally expressed in the pericarp of the ovaries (Kikuta et al, 2012;Xu et al, 2019). Pyrethrins are also constitutively made in the leaves at lower levels than in the flowers, but their synthesis in the leaves is induced by wounding or treatment with methyl jasmonate (MeJA: Ueda and Matsuda, 2011).…”

“…To identify such enzymes involved in pyrethrin biosynthesis, we previously performed Pearson coexpression analysis on the RNAsequencing and metabolic data bases from different stages of pyrethrum flowers, leaves, and stems to identify cytochrome P450 oxidoreductase genes whose expression pattern in pyrethrum flowers positively correlated to some degree with the expression of TcCDS and the synthesis of pyrethrins. A total of 12 gene candidates were selected whose coefficients were higher than 0.75 using TcCDS as a reference (Li et al, 2018;Xu et al, 2018Xu et al, , 2019. We subsequently showed that one of them, designated as CYP71BZ1, encoded Figure 2.…”