Novel family of terpene synthases evolved from trans-isoprenyl diphosphate synthases in a flea beetle Franziska Berana,1, Peter Rahfeldb,2, Katrin Luckc, Raimund Nagelc,3, Heiko Vogeld, Natalie Wielsche, Sandra Irmischc,4, Srinivasan Ramasamyf, Jonathan Gershenzonc, David G. Heckeld, and Tobias G. Köllnerc aResearch Group Sequestration and Detoxification in Insects, Max Planck Institute for Chemical Ecology, 07745 Jena, Germany; bDepartment of Bioorganic Chemistry, Max Planck Institute for Chemical Ecology, 07745 Jena, Germany; cDepartment of Biochemistry, Max Planck Institute for Chemical Ecology, 07745 Jena, Germany; dDepartment of Entomology, Max Planck Institute for Chemical Ecology, 07745 Jena, Germany; eDepartment of Mass Spectrometry, Max Planck Institute for Chemical Ecology, 07745 Jena, Germany; and fEntomology, AVRDC–The World Vegetable Center, Shanhua, Tainan 74151, Taiwan Edited by Jerrold Meinwald, Cornell University, Ithaca, NY, and approved February 5, 2016 (received for review November 27, 2015) Sesquiterpenes play important roles in insect communication, for monoterpene myrcene in the bark beetle Ips pini represents the example as pheromones. However, no sesquiterpene synthases, only insect terpene synthase known to date (14). the enzymes involved in construction of the basic carbon skeleton, The reactions catalyzed by TPSs involve the generation of a have been identified in insects to date. We investigated the highly reactive carbocation intermediate, which can undergo a wide biosynthesis of the sesquiterpene (6R,7S)-himachala-9,11-diene in array of different cyclizations, hydride shifts, and other rearrange- the crucifer flea beetle Phyllotreta striolata, a compound previously ments. The reaction cascade is either initiated by a metal ion- identified as a male-produced aggregation pheromone in several dependent ionization of the diphosphate moiety or a protonation of Phyllotreta species. A (6R,7S)-himachala-9,11-diene–producing ses- the substrate, and can be terminated by proton abstraction or water quiterpene synthase activity was detected in crude beetle protein addition (11, 13, 15). Because the reaction cascade may be Z E Z E extracts, but only when ( , )-farnesyl diphosphate [( , )-FPP] was branched and termination may occur at multiple levels, many TPSs offered as a substrate. No sequences resembling sesquiterpene syn- are multiproduct enzymes forming complex mixtures of compounds P. striolata thases from plants, fungi, or bacteria were found in the (16–19). Moreover, some TPSs also accept multiple substrates to trans transcriptome, but we identified nine divergent putative -iso- produce monoterpenes, sesquiterpenes, and diterpenes (11). prenyl diphosphate synthase (trans-IDS) transcripts. Four of these BIOCHEMISTRY trans Among sesquiterpene-producing insects are several genera in the putative -IDSs exhibited terpene synthase (TPS) activity when leaf beetle subfamily Galerucinae in which males emit species-spe- heterologously expressed. Recombinant PsTPS1 converted (Z,E)-FPP cific volatile sesquiterpene blends comprised mainly of himachalene- to (6R,7S)-himachala-9,11-diene and other sesquiterpenes observed type compounds (20–23). In the flea beetles Phyllotreta cruciferae, in beetle extracts. RNAi-mediated knockdown of PsTPS1 mRNA in P. striolata males led to reduced emission of aggregation pheromone, Phyllotreta striolata,andPhyllotreta vittula,(6R,7S)-himachala-9,11- Ps diene is a major male-produced sesquiterpene, and this compound confirming a significant role of TPS1 in pheromone biosynthesis. – Two expressed enzymes showed genuine IDS activity, with PsIDS1 was shown to be a key aggregation pheromone component (23 25). synthesizing (E,E)-FPP, whereas PsIDS3 produced neryl diphosphate, (Z,Z)-FPP, and (Z,E)-FPP. In a phylogenetic analysis, the PsTPS en- Significance zymes and PsIDS3 were clearly separated from a clade of known coleopteran trans-IDS enzymes including PsIDS1 and PsIDS2. How- Whether insect sesquiterpenes are synthesized de novo, derived ever, the exon–intron structures of IDS and TPS genes in P. striolata from plant precursors, or produced by symbionts is often un- are conserved, suggesting that this TPS gene family evolved from known. We identified an evolutionarily novel terpene synthase trans-IDS ancestors. gene family in the striped flea beetle, a notorious pest of Brassica crops in North America and Asia, and one of these genes was isoprenyl diphosphate synthase | sesquiterpene synthase | shown to be directly involved in the biosynthesis of the male- (6R,7S)-himachala-9,11-diene | aggregation pheromone | flea beetle specific sesquiterpene aggregation pheromone. Phylogenetic and gene structure analyses indicate that an expansion of the trans- erpenes play important roles in insect communication and isoprenyl diphosphate synthase gene family in the ancestor of the subfamily Galerucinae enabled functional diversification toward Tdefense, especially the C15 sesquiterpenes, which often act as sex, alarm, or aggregation pheromones or protection against this terpene synthase gene family. These insights into how flea beetles synthesize their aggregation pheromones may lead to enemies (1–3). To understand more about the biological func- new approaches for pest management. tion and evolution of sesquiterpenes in insects, it would be helpful to have more knowledge of their biosynthetic origins. Author contributions: F.B., P.R., R.N., S.I., J.G., D.G.H., and T.G.K. designed research; F.B., P.R., Sesquiterpenes are biosynthesized from three C5 isopentenoid K.L., R.N., H.V., N.W., and T.G.K. performed research; S.R. contributed new reagents/ units supplied by the mevalonate pathway, which are then joined analytic tools; F.B., P.R., R.N., H.V., N.W., and T.G.K. analyzed data; and F.B. and T.G.K. wrote the paper. sequentially via the action of enzymes known as trans-isoprenyl The authors declare no conflict of interest. diphosphate synthases (trans-IDS) to produce the linear C 15 This article is a PNAS Direct Submission. intermediate (E,E)-farnesyl diphosphate (FPP). Trans-IDS Data deposition: The sequences reported in this paper have been deposited in the Gen- enzymes have been identified and characterized in a number of Bank database (accession nos. KT959237–KT959261). insect species (4–10). The huge diversity of sesquiterpene carbon 1To whom correspondence should be addressed. Email: [email protected]. skeletons are formed from FPP in the next step by the catalysis of 2Present address: Department of Chemistry, The University of British Columbia, Vancouver, terpene synthases (TPSs). Numerous TPSs have been identified BC V6T 1Z1, Canada. in plants, fungi, and bacteria based on sequence similarity (11– 3Present address: Department of Biochemistry, Biophysics and Molecular Biology, Iowa 13). However, no homologs of known terpene synthases have State University, Ames, IA 50011. 4Present address: Michael Smith Laboratories, The University of British Columbia, Vancouver, been reported from available insect genomic and transcriptomic BC V6T 1Z4, Canada. sequences (3, 7). A unique bifunctional enzyme producing the This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. C10 intermediate geranyl diphosphate (GPP) as well as the linear 1073/pnas.1523468113/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1523468113 PNAS Early Edition | 1of6 Downloaded by guest on September 24, 2021 We investigated the biosynthesis of the sesquiterpene aggregation Identification and Functional Characterization of IDS-like Genes from pheromone in P. striolata, an important pest of crucifer crops in P. striolata. We searched for candidate genes encoding TPS enzymes North America and Southeast Asia (26). Here, we report the in a P. striolata transcriptome database (27) based on amino acid identification of an evolutionarily novel family of terpene syn- sequence similarity, but no sequences homologous to known plant, thases in this flea beetle. No transcripts with similarity to plant or fungal, or bacterial TPS were found. However, nine transcripts were microbial terpene synthases were evident in a transcriptome of predictedtoencodetrans-IDS enzymes (i.e., GPPS or FPPS). Full- this species. However, a remarkably high number of genes were length ORFs of these genes were obtained using rapid amplification present that were predicted to encode enzymes similar to insect of cDNA ends–PCR (RACE-PCR) (Dataset S1). An alignment of trans-IDSs, which produce geranyl diphosphate [(E)-GPP] and/ the corresponding amino acid sequences (Fig. S2) revealed consid- or farnesyl diphosphate [(E,E)-FPP] from the C5 precursors erable sequence divergence as aminoacididentitiesrangedfrom isopentenyl diphosphate (IPP) and dimethylallyl diphosphate 13.5% to 72.8%. The active site of trans-IDS enzymes generally (DMAPP) (5, 9, 10). Functional characterization of the recombi- contains two aspartate-rich motifs (DDxxD), which are critical for nant IDS-like enzymes led to the discovery of four TPS enzymes catalytic activity (28), but only three out of the nine proteins pos- which formed a separate clade in a phylogenetic analysis of co- sessed both motifs. In five proteins, the second aspartate-rich motif leopteran trans-IDS and trans–IDS-like enzymes. We investigated was altered, and in one protein both motifs were modified (Fig. S2). the role of these TPS in the aggregation pheromone biosynthesis To determine the enzymatic activities of the P. striolata IDS-like of P. striolata as well as their putative evolutionary