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 sequencing-based 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 trans-chrysanthemic 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.

Figure 1.

A, Structures of pyrethrins. B, Proposed pathway for the biosynthesis of trans-chrysanthemic acid.

Figure 2.

GC-MS analysis of pyrethrins and terpenoids from T. cinerariifolium leaves and flowers at different stages of development. A, Flowers of different stages of development and a leaf of T. cinerariifolium. B, Changes in relative concentrations of pyrethrin I during floral development. Pyrethrin I is the most abundant pyrethrin in the flower, and changes in the concentrations of other pyrethrins follow the same pattern as those of pyrethrin I. C, GC-MS chromatogram of the total ion mode of MTBE extracts from leaves and flowers harvested at stage 4. In each flower/leaf comparison, samples are shown with the same relative y axis scale, but the 7.2- to 15.2-min section is shown at a smaller scale to magnify the peaks. Peaks identified as terpenoids and internal standard (tetradecane) are labeled. D, GC-MS chromatogram (total ion mode) of MTBE extracts from leaves and flowers of different stages of development, showing the trans-chrysanthemic acid levels in each sample. E, Concentrations of trans-chrysanthemic acid in the leaf and in different stages of flowers. Quantification was achieved by normalization of the peaks in D to the tetradecane internal standard and comparison with a standard curve of authentic trans-chrysanthemic acid (n = 3; means ± sd). FW, Fresh weight.

Figure 3.

Identification of candidate ADH and ALDH genes for trans-chrysanthemic acid biosynthesis. A, Images of T. cinerariifolium flowers of different stages and of leaves from which RNA samples were obtained for RNAseq analysis. B, Average-linkage hierarchical clustering of relative transcript abundance of putative ADHs and ALDHs with TcCDS and TcGLIP based on the number of reads of each transcript in each RNAseq library. The tree and heat map were generated by Cluster 3.0 software (see “Materials and Methods”). C, Verification of levels of expression of TcCDS, TcGLIP, TcADH1, TcADH2, and TcALDH1 by qRT-PCR. Transcript levels are expressed relative to that of GAPDH (glyceraldehyde-3-phosphate dehydrogenase) in each sample (n = 4; means ± sd). **, P < 0.01 and *, P < 0.05. The differences between leaf, stem, and root data points and any flower data points are all significant at P < 0.001.

Figure 4.

Phylogenetic analysis of candidate T. cinerariifolium dehydrogenases for trans-chrysanthemic acid biosynthesis based on protein sequences. A, Phylogenetic tree for TcADH1 and TcADH2. B, Phylogenetic tree for TcALDH1. The protein sequences from other species are of functionally characterized enzymes whose sequences were identified by BLAST search to be most closely related to the T. cinerariifolium sequences. Phylogenetic analyses were conducted in MEGA7 (Kumar et al., 2016) with the following parameters: multiple sequence alignment with ClustalW, phylogenetic construction with the maximum likelihood method, and bootstrap tests of 1,000 replicates.

Figure 5.

Gas chromatography analyses of products obtained in in vitro biochemical assays of TcADH2 and TcALDH1. For all assays analyzed, reaction products were extracted with 100 µL of MTBE and run on an Rxi-5Sil column. Tetradecane was used as an internal standard. A, Synthesis of trans-chrysanthemol substrate. Top trace, 1 mm of a commercially available standard of a trans- and cis-chrysanthemol mixture; middle trace, reaction products obtained by incubating 30 μg of recombinant TcCDS with 2.5 mm DMAPP in a 50-μL reaction for 24 h at 30°C; bottom trace, reaction products obtained by incubating the products of the TcCDS-catalyzed condensation of DMAPP with 5 units of alkaline phosphatase (ALP) for 1 h at 37°C. B, In vitro production of trans-chrysanthemol. Reaction products obtained by incubating 0.64 mm trans-chrysanthemol and 1.5 mm NAD⁺ with 5 µL of eluted protein from empty vector (top trace) or 1.25 μg of purified TcADH2 (bottom trace) in a 60-μL reaction volume for 5 min. C, Production of trans-chrysanthemic acid from trans-chrysanthemol in a coupled assay containing 0.64 mm trans-chrysanthemol and 1.5 mm NAD⁺ with 1.25 μg of purified TcADH2 and 6 μg of purified TcALDH1 in a 60-μL reaction volume for 5, 10, 15, 25, and 45 min. A control reaction was performed using 5 µL of eluted protein from the empty vector. The bottom trace shows 0.3 mm of a commercial trans-chrysanthemic acid.

Figure 6.

Production of trans-chrysanthemol, trans-chrysanthemic acid, and related compounds in N. benthamiana leaves transiently expressing TcCDS, TcADH2, and TcALDH1 proteins. A, GC-MS chromatograms of MTBE extracts of N. benthamiana leaves expressing EGFP (control), TcCDS, TcCDS and TcADH2, and TcCDS with TcADH2 and TcALDH1. For terpenes, m/z = 123 was monitored, and for the internal control tetradecane, m/z = 198 was monitored. Peaks related to trans-chrysanthemol, trans-chrysanthemic acid, and internal standard are labeled. B and C, Concentrations of free trans-chrysanthemol (B) and free trans-chrysanthemic acid (C) in N. benthamiana leaves expressing the indicated constructs were determined by comparison with an authentic standard. FW, Fresh weight. D to F, Relative levels of Unknown 1 (D), Unknown 2 (E), and Unknown 3 (F) in N. benthamiana leaves expressing the indicated constructs. For each compound, the plant material expressing a specific construct that showed the highest levels (average of three biological replicates) was set at 100%. The data in B to F represent means ± sd from triplicate biological replicates. N.D., Not detected.

Figure 7.

LC-MS analysis of N. benthamiana leaves simultaneously expressing the three enzymes TcCDS, TcADH2, and TcALDH1. A to D, Extracted ion chromatograms of m/z 803.37 and 831.33 are shown for EGFP single expression control (A), TcCDS (B), TcCDS + TcADH2 (C), and TcCDS + TcADH2 + TcALDH1 (D). Chromatograms are all scaled the same, as indicated by the ion current (1.01e7) in the top right corner of each chromatogram. The sample for D was diluted 10-fold compared with the other three samples due to the high concentration of m/z 831.33 in the sample. E and F, MS/MS for ions m/z 803.37 (E) and m/z 831.33 (F) along with the proposed compound structures based on exact mass, fragmentation pattern, and similarity to previously published data. G, Relative levels of trans-chrysanthemic acid in N. benthamiana leaves expressing all TcCDS, TcADH2, and TcALDH1 with or without sodium hydroxide treatment.