| 1. |
- Ding, Baojian, et al.
(författare)
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Sequence variation determining stereochemistry of a delta-11 desaturase active in moth sex pheromone biosynthesis
- 2016
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Ingår i: Insect Biochemistry and Molecular Biology. - : Elsevier BV. - 1879-0240 .- 0965-1748. ; 74, s. 68-75
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Tidskriftsartikel (refereegranskat)abstract
- A Δ11 desaturase from the oblique banded leaf roller moth Choristoneura rosaceana takes the saturated myristic acid and produces a mixture of (E)-11-tetradecenoate and (Z)-11-tetradecenoate with an excess of the Z isomer (35:65). A desaturase from the spotted fireworm moth Choristoneura parallela also operates on myristic acid substrate but produces almost pure (E)-11-tetradecenoate. The two desaturases share 92% amino acid identity and 97% amino acid similarity. There are 24 amino acids differing between these two desaturases. We constructed mutations at all of these positions to pinpoint the sites that determine the product stereochemistry. We demonstrated with a yeast functional assay that one amino acid at the cytosolic carboxyl terminus of the protein (258E) is critical for the Z activity of the C. rosaceana desaturase. Mutating the glutamic acid (E) into aspartic acid (D) transforms the C. rosaceana enzyme into a desaturase with C. parallela-like activity, whereas the reciprocal mutation of the C. parallela desaturase transformed it into an enzyme producing an intermediate 64:36 E/Z product ratio. We discuss the causal link between this amino acid change and the stereochemical properties of the desaturase and the role of desaturase mutations in pheromone evolution.
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| 2. |
- Yuvaraj, Jothi Kumar, et al.
(författare)
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Characterization of odorant receptors from a non-ditrysian moth, Eriocrania semipurpurella sheds light on the origin of the sex pheromone receptors in Lepidoptera
- 2017
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Ingår i: Molecular biology and evolution. - : Oxford University Press (OUP). - 0737-4038 .- 1537-1719. ; 34:11, s. 2733-2746
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Tidskriftsartikel (refereegranskat)abstract
- Pheromone receptors (PRs) are essential in moths to detect sex pheromones for mate finding. However, it remainsunknown from which ancestral proteins these specialized receptors arose. The oldest lineages of moths, so-callednon-ditrysian moths, use short-chain pheromone components, secondary alcohols, or ketones, so called Type 0 pheromonesthat are similar to many common plant volatiles. It is, therefore, possible that receptors for these ancestralpheromones evolved from receptors detecting plant volatiles. Hence, we identified the odorant receptors (ORs) from anon-ditrysian moth, Eriocrania semipurpurella (Eriocraniidae, Lepidoptera), and performed functional characterizationof ORs using HEK293 cells. We report the first receptors that respond to Type 0 pheromone compounds; EsemOR3displayed highest sensitivity toward (2S, 6Z)-6-nonen-2-ol, whereas EsemOR5 was most sensitive to the behavioralantagonist (Z)-6-nonen-2-one. These receptors also respond to plant volatiles of similar chemical structures, but withlower sensitivity. Phylogenetically, EsemOR3 and EsemOR5 group with a plant volatile-responding receptor from thetortricid moth Epiphyas postvittana (EposOR3), which together reside outside the previously defined lepidopteran PRclade that contains the PRs from more derived lepidopteran families. In addition, one receptor (EsemOR1) that falls atthe base of the lepidopteran PR clade, responded specifically to b-caryophyllene and not to any other additional plant orpheromone compounds. Our results suggest that PRs for Type 0 pheromones have evolved from ORs that detectstructurally-related plant volatiles. They are unrelated to PRs detecting pheromones inmore derived Lepidoptera, which,in turn, also independently may have evolved a novel function from ORs detecting plant volatiles.
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| 3. |
- Yuvaraj, Jothi K., et al.
(författare)
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Putative ligand binding sites of two functionally characterized bark beetle odorant receptors
- 2021
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Ingår i: BMC Biology. - : Springer Science and Business Media LLC. - 1741-7007. ; 19
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Tidskriftsartikel (refereegranskat)abstract
- Background: Bark beetles are major pests of conifer forests, and their behavior is primarily mediated via olfaction. Targeting the odorant receptors (ORs) may thus provide avenues towards improved pest control. Such an approach requires information on the function of ORs and their interactions with ligands, which is also essential for understanding the functional evolution of these receptors. Hence, we aimed to identify a high-quality complement of ORs from the destructive spruce bark beetle Ips typographus (Coleoptera, Curculionidae, Scolytinae) and analyze their antennal expression and phylogenetic relationships with ORs from other beetles. Using 68 biologically relevant test compounds, we next aimed to functionally characterize ecologically important ORs, using two systems for heterologous expression. Our final aim was to gain insight into the ligand-OR interaction of the functionally characterized ORs, using a combination of computational and experimental methods. Results: We annotated 73 ORs from an antennal transcriptome of I. typographus and report the functional characterization of two ORs (ItypOR46 and ItypOR49), which are responsive to single enantiomers of the common bark beetle pheromone compounds ipsenol and ipsdienol, respectively. Their responses and antennal expression correlate with the specificities, localizations, and/or abundances of olfactory sensory neurons detecting these enantiomers. We use homology modeling and molecular docking to predict their binding sites. Our models reveal a likely binding cleft lined with residues that previously have been shown to affect the responses of insect ORs. Within this cleft, the active ligands are predicted to specifically interact with residues Tyr84 and Thr205 in ItypOR46. The suggested importance of these residues in the activation by ipsenol is experimentally supported through site-directed mutagenesis and functional testing, and hydrogen bonding appears key in pheromone binding. Conclusions: The emerging insight into ligand binding in the two characterized ItypORs has a general importance for our understanding of the molecular and functional evolution of the insect OR gene family. Due to the ecological importance of the characterized receptors and widespread use of ipsenol and ipsdienol in bark beetle chemical communication, these ORs should be evaluated for their potential use in pest control and biosensors to detect bark beetle infestations.
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| 4. |
- Zhang, Dan-Dan, et al.
(författare)
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Receptor for detection of a Type II sex pheromone in the winter moth Operophtera brumata
- 2016
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 6
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Tidskriftsartikel (refereegranskat)abstract
- How signal diversity evolves under stabilizing selection in a pheromone-based mate recognitionsystem is a conundrum. Female moths produce two major types of sex pheromones, i.e., long-chainacetates, alcohols and aldehydes (Type I) and polyenic hydrocarbons and epoxides (Type II), alongdifferent biosynthetic pathways. Little is known on how male pheromone receptor (PR) genes evolvedto perceive the different pheromones. We report the identification of the first PR tuned to Type IIpheromones, namely ObruOR1 from the winter moth, Operophtera brumata (Geometridae). ObruOR1clusters together with previously ligand-unknown orthologues in the PR subfamily for the ancestralType I pheromones, suggesting that O. brumata did not evolve a new type of PR to match the novel TypeII signal but recruited receptors within an existing PR subfamily. AsegOR3, the ObruOR1 orthologuepreviously cloned from the noctuid Agrotis segetum that has Type I acetate pheromone components,responded significantly to another Type II hydrocarbon, suggesting that a common ancestor with TypeI pheromones had receptors for both types of pheromones, a preadaptation for detection of Type II sexpheromone.
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| 5. |
- Da Silva, Maycon R., et al.
(författare)
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Identification of two additional behaviorally active gland constituents of female Diatraea saccharalis (Fabricius) (Lepidoptera Crambidae)
- 2021
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Ingår i: Journal of the Brazilian Chemical Society. - : Sociedade Brasileira de Quimica (SBQ). - 0103-5053. ; 32:1, s. 225-230
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Tidskriftsartikel (refereegranskat)abstract
- The sugarcane borer, Diatraea saccharalis (Fabricius), is the major pest insect attacking sugarcane crops in Brazil. Population control by insecticide treatment is not efficient due to simultaneous presence of all developmental stages of the insect throughout the year, and alternative control methods are needed. Two female-produced sex pheromone components, (Z,E)-hexadeca-9,11-dienal and (Z)- hexadec-11-enal, have previously been reported to elicit antennal activity and behavioral response of males in flight tunnel experiments. However, the attractiveness of these compounds in field tests has been very low. In this study, two additional female-produced compounds in D. saccharalis eliciting consistent antennal response in males were identified as (Z)-hexadec-9-enal and hexadecanal. In flight tunnel assays, the behavioral response to a quaternary blend was significantly higher when compared with the previously identified binary blend. Subtracting (Z)-hexadec-9-enal or hexadecanal from the full blend did not reduce attraction, indicating redundancy in the communication channel. We conclude that additional compounds are part of the sex pheromone of D. saccharalis, which may improve the efficiency of trap lures for monitoring of this pest.
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| 6. |
- Ding, Bao Jian, et al.
(författare)
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Biosynthesis of the sex pheromone component (E,Z)-7,9-Dodecadienyl acetate in the European Grapevine Moth, Lobesia botrana, involving ∆11 desaturation and an elusive ∆7 desaturase
- 2021
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Ingår i: Journal of Chemical Ecology. - : Springer Science and Business Media LLC. - 0098-0331 .- 1573-1561. ; 47:3, s. 248-264
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Tidskriftsartikel (refereegranskat)abstract
- The European grapevine moth, Lobesia botrana, uses (E,Z)-7,9-dodecadienyl acetate as its major sex pheromone component. Through in vivo labeling experiments we demonstrated that the doubly unsaturated pheromone component is produced by ∆11 desaturation of tetradecanoic acid, followed by chain shortening of (Z)-11-tetradecenoic acid to (Z)-9-dodecenoic acid, and subsequently introduction of the second double bond by an unknown ∆7 desaturase, before final reduction and acetylation. By sequencing and analyzing the transcriptome of female pheromone glands of L. botrana, we obtained 41 candidate genes that may be involved in sex pheromone production, including the genes encoding 17 fatty acyl desaturases, 13 fatty acyl reductases, 1 fatty acid synthase, 3 acyl-CoA oxidases, 1 acetyl-CoA carboxylase, 4 fatty acid transport proteins and 2 acyl-CoA binding proteins. A functional assay of desaturase and acyl-CoA oxidase gene candidates in yeast and insect cell (Sf9) heterologous expression systems revealed that Lbo_PPTQ encodes a ∆11 desaturase producing (Z)-11-tetradecenoic acid from tetradecanoic acid. Further, Lbo_31670 and Lbo_49602 encode two acyl-CoA oxidases that may produce (Z)-9-dodecenoic acid by chain shortening (Z)-11-tetradecenoic acid. The gene encoding the enzyme introducing the E7 double bond into (Z)-9-dodecenoic acid remains elusive even though we assayed 17 candidate desaturases in the two heterologous systems.
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| 7. |
- Ding, Bao-Jian, et al.
(författare)
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The Yeast ATF1 Acetyltransferase Efficiently Acetylates Insect Pheromone Alcohols: Implications for the Biological Production of Moth Pheromones.
- 2016
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Ingår i: Lipids. - : Wiley. - 0024-4201 .- 1558-9307. ; 51, s. 469-475
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Tidskriftsartikel (refereegranskat)abstract
- Many moth pheromones are composed of mixtures of acetates of long-chain (≥10 carbon) fatty alcohols. Moth pheromone precursors such as fatty acids and fatty alcohols can be produced in yeast by the heterologous expression of genes involved in insect pheromone production. Acetyltransferases that subsequently catalyze the formation of acetates by transfer of the acetate unit from acetyl-CoA to a fatty alcohol have been postulated in pheromone biosynthesis. However, so far no fatty alcohol acetyltransferases responsible for the production of straight chain alkyl acetate pheromone components in insects have been identified. In search for a non-insect acetyltransferase alternative, we expressed a plant-derived diacylglycerol acetyltransferase (EaDAcT) (EC 2.3.1.20) cloned from the seed of the burning bush (Euonymus alatus) in a yeast system. EaDAcT transformed various fatty alcohol insect pheromone precursors into acetates but we also found high background acetylation activities. Only one enzyme in yeast was shown to be responsible for the majority of that background activity, the acetyltransferase ATF1 (EC 2.3.1.84). We further investigated the usefulness of ATF1 for the conversion of moth pheromone alcohols into acetates in comparison with Ea DAcT. Overexpression of ATF1 revealed that it was capable of acetylating these fatty alcohols with chain lengths from 10 to 18 carbons with up to 27- and 10-fold higher in vivo and in vitro efficiency, respectively, compared to Ea DAcT. The ATF1 enzyme thus has the potential to serve as the missing enzyme in the reconstruction of the biosynthetic pathway of insect acetate pheromones from precursor fatty acids in yeast.
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| 8. |
- Hou, Xiao-Qing, et al.
(författare)
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Functional Evolution of a Bark Beetle Odorant Receptor Clade Detecting Monoterpenoids of Different Ecological Origins
- 2021
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Ingår i: Molecular biology and evolution. - : Oxford University Press. - 0737-4038 .- 1537-1719. ; 38:11, s. 4934-4947
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Tidskriftsartikel (refereegranskat)abstract
- Insects detect odors using an array of odorant receptors (ORs), which may expand through gene duplication. How and which new functions may evolve among related ORs within a species remain poorly investigated. We addressed this question by functionally characterizing ORs from the Eurasian spruce bark beetle Ips typographus, in which physiological and behavioral responses to pheromones, volatiles from host and nonhost trees, and fungal symbionts are well described. In contrast, knowledge of OR function is restricted to two receptors detecting the pheromone compounds (S)-(-)-ipsenol (ItypOR46) and (R)-(-)-ipsdienol (ItypOR49). These receptors belong to an Ips-specific OR-lineage comprising seven ItypORs. To gain insight into the functional evolution of related ORs, we characterized the five remaining ORs in this Glade using Xenopus oocytes. Two receptors responded primarily to the host tree monoterpenes (+)-3-carene (ItypOR25) and p-cymene (ItypOR27). Two receptors responded to oxygenated monoterpenoids produced in larger relative amounts by the beetle-associated fungi, with ItypOR23 specific for (+)-trans-(1R, 4S)-4-thujanol, and ItypOR29 responding to (+)-isopinocamphone and similar ketones. ItypOR28 responded to the pheromone E-myrcenol from the competitor Ips duplicatus. Overall, the OR responses match well with those of previously characterized olfactory sensory neuron classes except that neurons detecting E-myrcenol have not been identified. The characterized ORs are under strong purifying selection and demonstrate a shared functional property in that they all primarily respond to monoterpenoids. The variation in functional groups among OR ligands and their diverse ecological origins suggest that neofunctionalization has occurred early in the evolution of this OR-lineage following gene duplication.
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| 9. |
- Lassance, Jean Marc, et al.
(författare)
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Evolution of the codling moth pheromone via an ancient gene duplication
- 2021
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Ingår i: BMC Biology. - : Springer Science and Business Media LLC. - 1741-7007. ; 19:1
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Tidskriftsartikel (refereegranskat)abstract
- Background: Defining the origin of genetic novelty is central to our understanding of the evolution of novel traits. Diversification among fatty acid desaturase (FAD) genes has played a fundamental role in the introduction of structural variation in fatty acyl derivatives. Because of its central role in generating diversity in insect semiochemicals, the FAD gene family has become a model to study how gene family expansions can contribute to the evolution of lineage-specific innovations. Here we used the codling moth (Cydia pomonella) as a study system to decipher the proximate mechanism underlying the production of the ∆8∆10 signature structure of olethreutine moths. Biosynthesis of the codling moth sex pheromone, (E8,E10)-dodecadienol (codlemone), involves two consecutive desaturation steps, the first of which is unusual in that it generates an E9 unsaturation. The second step is also atypical: it generates a conjugated diene system from the E9 monoene C12 intermediate via 1,4-desaturation. Results: Here we describe the characterization of the FAD gene acting in codlemone biosynthesis. We identify 27 FAD genes corresponding to the various functional classes identified in insects and Lepidoptera. These genes are distributed across the C. pomonella genome in tandem arrays or isolated genes, indicating that the FAD repertoire consists of both ancient and recent duplications and expansions. Using transcriptomics, we show large divergence in expression domains: some genes appear ubiquitously expressed across tissue and developmental stages; others appear more restricted in their expression pattern. Functional assays using heterologous expression systems reveal that one gene, Cpo_CPRQ, which is prominently and exclusively expressed in the female pheromone gland, encodes an FAD that possesses both E9 and ∆8∆10 desaturation activities. Phylogenetically, Cpo_CPRQ clusters within the Lepidoptera-specific ∆10/∆11 clade of FADs, a classic reservoir of unusual desaturase activities in moths. Conclusions: Our integrative approach shows that the evolution of the signature pheromone structure of olethreutine moths relied on a gene belonging to an ancient gene expansion. Members of other expanded FAD subfamilies do not appear to play a role in chemical communication. This advises for caution when postulating the consequences of lineage-specific expansions based on genomics alone.
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| 10. |
- Petkevicius, Karolis, et al.
(författare)
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Biotechnological production of the European corn borer sex pheromone in the yeast Yarrowia lipolytica
- 2021
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Ingår i: Biotechnology Journal. - : Wiley. - 1860-6768 .- 1860-7314. ; 16:6
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Tidskriftsartikel (refereegranskat)abstract
- The European corn borer (ECB) Ostrinia nubilalis is a widespread pest of cereals, particularly maize. Mating disruption with the sex pheromone is a potentially attractive method for managing this pest; however, chemical synthesis of pheromones requires expensive starting materials and catalysts and generates hazardous waste. The goal of this study was to develop a biotechnological method for the production of ECB sex pheromone. Our approach was to engineer the oleaginous yeast Yarrowia lipolytica to produce (Z)-11-tetradecenol (Z11-14:OH), which can then be chemically acetylated to (Z)-11-tetradecenyl acetate (Z11-14:OAc), the main pheromone component of the Z-race of O. nubilalis. First, a C14 platform strain with increased biosynthesis of myristoyl-CoA was obtained by introducing a point mutation into the α-subunit of fatty acid synthase, replacing isoleucine 1220 with phenylalanine (Fas2pI1220F). The intracellular accumulation of myristic acid increased 8.4-fold. Next, fatty acyl-CoA desaturases (FAD) and fatty acyl-CoA reductases (FAR) from nine different species of Lepidoptera were screened in the C14 platform strain, individually and in combinations. A titer of 29.2 ± 1.6 mg L-1 Z11-14:OH was reached in small-scale cultivation with an optimal combination of a FAD (Lbo_PPTQ) from Lobesia botrana and FAR (HarFAR) from Helicoverpa armigera. When the second copies of FAD and FAR genes were introduced, the titer improved 2.1-fold. The native FAS1 gene's overexpression led to a further 1.5-fold titer increase, reaching 93.9 ± 11.7 mg L-1 in small-scale cultivation. When the same engineered strain was cultivated in controlled 1 L bioreactors in fed-batch mode, 188.1 ± 13.4 mg L-1 of Z11-14:OH was obtained. Fatty alcohols were extracted from the biomass and chemically acetylated to obtain Z11-14:OAc. Electroantennogram experiments showed that males of the Z-race of O. nubilalis were responsive to biologically-derived pheromone blend. Behavioral bioassays in a wind tunnel revealed attraction of male O. nubilalis, although full precopulatory behavior was observed less often than for the chemically synthesized pheromone blend. The study paves the way for the production of ECB pheromone by fermentation.
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