Team:Valencia UPV/Project/modules/biosynthesis

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Project > Modules > Pheromone Biosynthesis

Pheromone Biosynthesis

Biosynthesis

This is the core of our project. Sexy Plant is a pest control choice based on the production of sexual pheromones, which will confuse male insects and make them unable to find the female. For that reason, engineering the genome of Nicotiana benthamiana to produce a set of sexual pheromones was our priority.

We wanted to produce pheromones to generate mating disruption in species which cause great damage in crops, so we did some research and consulted experts in the CEQA . We also took into account that they should be species with already identified sexual pheromones as well as their biosynthetic pathways. We found the perfect group of species to fight against, Moths.

Sexual pheromones for a large number of moths have already been identified. Most of these species use type I pheromones. These consist of “straight-chain compounds 10-18 carbons in length with a functional group of a primary alcohol, aldehyde, or acetate ester, and usually with several double bonds” [1]. Moths de novo synthesize these pheromones in the pheromone gland (PG) through modifications of fatty acid biosynthetic pathways” [5] Three of the most commonly found major components compounds are Z11-16:OAc, Z11-16:Ald and Z11-16:OH [1] (see figure 1). In addition, biosynthetic pathways of these pheromones had already been elucidated in some species [2-5], which was a great help to create our biosynthetic pathways.

Figure 1. Structure of moths sexual pheromones. A: Structure of Z11-16:OH. B:Structure of Z11-16:Ald. C: Structure of Z11-16:OAc.

In previous transcriptome analysis from the moth Agrotis ipsilon, specific sets of enzymes were identified to be differentially expressed in the pheromone glands. These enzymes were Acetyl-CoA carboxylases, fatty acid synthases, desaturases, acyl-CoA reductases, alcohol oxidases, aldehyde reductases and acetyltransferases. They were found to be differentially expressed in the moths pheromone glands compared to the rest of the organism [5]. These results mean these enzymes are involved in pheromones biosynthesis.

Previous approaches

Moth sexual pheromones had already been synthesized previously using different approaches: Hagström et al produced Z11-16:OH and Z11-16:Ald in yeast [6]. Ding et al produced Z11-16:OAc in Nicotiana benthamiana, our chassis plant, by transient expression of individual enzymes [7]. However, the production of these three pheromones in plant had never been tested using an in cis multigenic construction … until we arrived.

Our system

We created a plant able to synthesize sexual pheromones Z11-16:OAc, Z11-16:Ald and Z11-16:OH, the most important ones in moths sexual behavior (see the tables 1 to 3 below). Sexy Plant expresses sets of genes which transform Palmitic Acid CoA, an abundant compound in N. benthamiana leaves, into these three pheromones. Depending on the pheromone to be produced, different pathways are introduced in Biobricks standard and expressed in the plant.

As it was done in Ding's work [7], a Δ11 desaturase from Amyelois transitella (accession number JX964774) and HarFAR_KKYR, an improved version of HarFAR-3 fatty acid reductase from Helicoverpa armigera (accession number JF709978) were expressed to produce Z11-16:OH. Additionally, 1,2-diacyl-sn-glycerol:acetyl-CoA acetyltransferase from Euonymus alatus (accession number GU594061) was expressed along with the other two enzymes to produce Z11-16:OAc [7]. Inspired by Hagström approach [6], production of Z11-16:Ald could be accomplished by over expressing a fatty-acid alcohol oxidase from Nicotiana benthamiana. The whole biosynthetic pathway is depicted in figure 2.

Figure 2. Biosynthetic pathway of moth sexual pheromones. Sexual pheromones are bordered in purple. Taking Palmitic Acid CoA (16:CoA) as substrate, the expression of the genes AtrΔ11 (Desaturase) and HarFAR (Reductase) leads to the production of Z11-16:OH. By adding the gene EaDAcT (Acetyltransferase) in the previous system, production displaced to obtaining Z11-16:OAc. If a Alcohol Oxidase (FAO) is included instead of EaDAcT, Z11-16:Ald will be produced.

Finding pests

Complementing our efforts to produce the pheromones, we did a thorough search in the Pherobase database (http://www.pherobase.com) with software we specifically developed for this purpose and checked which insects met the conditions both to have one of the pheromones produced by the Sexy Plant as a major pheromone component and also to be considered a plague. Results from this analysis can be found in the tables below. We can conclude that there is a large number of insects causing plagues that are affected by our pheromones.

Insects attracted by Z11-16:Ald and Z11-16:OH

Importance	Insect	Plague
High	Helicoverpa armigera	Many crops, cotton, ornamentals, fruit trees...
High	Helicoverpa zea	Many crops, cotton, linum...
High	Heliothis peltigera	Cotton
High	Heliothis virescens	Many crops, cotton, tobacco, fruit trees...
Medium	Gortyna xanthenes	Artichoke (Comunidad Valenciana)
Medium	Platyptilia carduidactyla	Artichoke
Medium	Diatraea considerata	Sugar cane

Insects attracted by Z11-16:OAc and Z11-16:OH

Importance	Insect	Plague
Medium	Crocidolomia binotalis	Cabbage
Medium	Mamestra brassicae	Coliflower
Medium	Feltia jaculifera	Maize, sorghum
Medium	Euxoa messoria	Apple, cultivated vegetables, flowers...
Medium	Sesamia calamistis	Sugar cane
Medium	Platyptilia carduidactyla	Maize

Insects attracted by Z11-16:OH

Importance	Insect	Plague
Medium	Chilo zacconius	Rice

← Go to Moths Behaviour Go to Modules Go to Release →

References

Matsumoto S (2010) Molecular Mechanisms Underlying Sex Pheromone Production in Moths. Bioscience, Biotechnology, and Biochemistry 74: 223-231.
Choi M-Y, Han KS, Boo KS, Jurenka RA (2002) Pheromone biosynthetic pathways in the moths Helicoverpa zea and Helicoverpa assulta. Insect Biochemistry and Molecular Biology 32: 1353-1359.
Wang H-L, Zhao C-H, Wang C-Z (2005) Comparative study of sex pheromone composition and biosynthesis in Helicoverpa armigera, H. assulta and their hybrid. Insect Biochemistry and Molecular Biology 35: 575-583.
Fu X, Fukuzawa M, Tabata J, Tatsuki S, Ishikawa Y (2005) Sex pheromone biosynthesis in Ostrinia zaguliaevi, a congener of the European corn borer moth O. nubilalis. Insect Biochemistry and Molecular Biology 35: 621-626.
Gu S-H, Wu K-M, Guo Y-Y, Pickett J, Field L, et al. (2013) Identification of genes expressed in the sex pheromone gland of the black cutworm Agrotis ipsilon with putative roles in sex pheromone biosynthesis and transport. BMC Genomics 14: 636.
Hagström A, Wang H-L, Lienard M, Lassance J-M, Johansson T, et al. (2013) A moth pheromone brewery: production of (Z)-11-hexadecenol by heterologous co-expression of two biosynthetic genes from a noctuid moth in a yeast cell factory. Microbial Cell Factories 12: 125.
Ding BJ, Hofvander P, Wang HL, Durrett TP, Stymne S, et al. (2014) A plant factory for moth pheromone production. Nat Commun 5: 3353.

@@ Line 2: / Line 2: @@
 <html>
-<div align="center"><div id="cn-box" align="justify"></br>
+<div align="center"><div id="cn-box" align="justify">
+<p><h3 class="hook" align="left"><a>Project</a> > <a href="https://2014.igem.org/Team:Valencia_UPV/Project/modules">Modules</a> > <a>Pheromone Biosynthesis</a> </h3></p><br/></br>
+<div align="center"><span class="coda"><roja>P</roja>heromone <roja>B</roja>iosynthesis</span> </div><br/><br/>
 <p><strong>Biosynthesis</strong></p><br/>
-<p>The Sexy Plant project aims at engineering pheromone-emitting plants for pest control in agriculture by means of mating disruption. To this end, it was necessary to build a pheromone biosynthetic pathway in the genome of a plant.</p><br/><br/>
+<p>This is the core of our project. <span class="red-bold">Sexy Plant</span> is a pest control choice based on the production of sexual pheromones, which will confuse male insects and make them unable to find the female. For that reason, engineering the genome of <span class="italic">Nicotiana benthamiana</span> to produce a set of sexual pheromones was our priority.</p><br/><br/>
+<p>We wanted to produce pheromones to generate mating disruption in species which cause great damage in crops, so we did some research and consulted experts in the CEQA . We also took into account that they should be species with already identified sexual pheromones as well as their biosynthetic pathways. We found the perfect group of species to fight against, Moths.</p><br/><br/>
+<p>Sexual pheromones for a large number of moths have already been identified. Most of these species use type I pheromones. These consist of “straight-chain compounds 10-18 carbons in length with a functional group of a primary alcohol, aldehyde, or acetate ester, and usually with several double bonds” [1].  Moths de novo synthesize these pheromones in the pheromone gland (PG) through modifications of fatty acid biosynthetic pathways” [5] Three of the most commonly found major components compounds are <span class="blue-bold">Z11-16:OAc</span>, <span class="green-bold">Z11-16:Ald</span> and <span class="red-bold">Z11-16:OH</span> [1] (see figure 1). In addition, biosynthetic pathways of these pheromones had already been elucidated in some species [2-5], which was a great help to create our biosynthetic pathways.</p><br/><br/><br/>
+<div align="center"><img width="320px" src="https://static.igem.org/mediawiki/2014/8/80/VUPVBiosynthesis_figure_1.png" alt="pheromone_structures" title="Moths Pheromones Structure"></img></div><br/>
+			<div align="center"><p style="text-align: justify; font-style: italic; font-size: 0.8em; width: 700px;"><span class="black-bold">Figure 1. Structure of moths sexual pheromones</span>. A: Structure of <span class="red-bold">Z11-16:OH</span>. B:Structure of <span class="green-bold">Z11-16:Ald</span>. C: Structure of <span class="blue-bold">Z11-16:OAc</span>.</p></div><br/><br/>
+<p>In previous transcriptome analysis from the moth <span class="italic">Agrotis ipsilon</span>, specific sets of enzymes were identified to be differentially expressed in the pheromone glands. These enzymes were Acetyl-CoA carboxylases, fatty acid synthases, desaturases, acyl-CoA reductases, alcohol oxidases, aldehyde reductases and acetyltransferases. They were found to be differentially expressed in the moths pheromone glands compared to the rest of the organism [5]. These results mean these enzymes are involved in pheromones biosynthesis.</p><br/><br/>
+<p><strong>Previous approaches</strong></p><br/>
+<p>Moth sexual pheromones had already been synthesized previously using different approaches: Hagström et al produced <span class="red-bold">Z11-16:OH</span> and <span class="green-bold">Z11-16:Ald</span> in yeast [6]. Ding et al produced <span class="blue-bold">Z11-16:OAc</span> in <span class="italic">Nicotiana benthamiana</span>, our chassis plant, by transient expression of individual enzymes [7]. However, the production of these three pheromones in plant had never been tested using an in cis multigenic construction … until we arrived.</p><br/><br/>
-<p>Moths, species of the order Lepidoptera, are often considered important pest in crops all over the world. The biosynthetic pathways involved in the production of sexual pheromones of some moth species considered pests have previously been described [1-3]. Some of these main pheromone components of a large amount of moth species are Z11-16:OAc, Z11-16:Ald and Z11-16:OH.</p><br/><br/>
+<p><strong>Our system</strong></p><br/>
-<p>The production of Z11-16:OAc in Nicotiana benthamiana was early described by Ding et al. [4], who demonstrated the production of Z11-16:OAc by mixing individual elements of the pathway. Therefore we decided to build this complete pathway in cis, in addition to the pathways to produce Z11-16:Ald and Z11-16:OH, using Biobricks standard.</p><br/><br/>
+<p>We created a plant able to synthesize sexual pheromones <span class="blue-bold">Z11-16:OAc</span>, <span class="green-bold">Z11-16:Ald</span> and <span class="red-bold">Z11-16:OH</span>, the most important ones in moths sexual behavior (see the tables 1 to 3 below). Sexy Plant expresses sets of genes which transform Palmitic Acid CoA, an abundant compound in <span class="italic">N. benthamiana</span> leaves, into these three pheromones. Depending on the pheromone to be produced, different pathways are introduced in Biobricks standard and expressed in the plant.</p><br/><br/>
-<p><strong>Pheromones</strong></p><br/>
+<img width="250px" style="float:right;" src="https://static.igem.org/mediawiki/2014/3/3c/VUPVBiosynthesis_side.png" alt="plants_moths"></img><br/>
-<p>Pheromones to be produced in the Sexy Plant are pheromones affecting the sexual behaviour in a broad variety of moth species. “Most moth species utilize Type I pheromones that consist of straight-chain compounds 10-18 carbons in length with a functional group of a primary alcohol, aldehyde, or acetate ester, and usually with several double bonds, de novo synthesized in the pheromone gland (PG) through modifications of fatty acid biosynthetic pathways” [5]. Due to this fact, Type I pheromones were selected to be synthesized by the Sexy Plant.</p><br/><br/>
+<p><br/>As it was done in Ding's work [7], a Δ11 desaturase from <span class="italic">Amyelois transitella</span> (accession number JX964774) and HarFAR_KKYR, an improved version of HarFAR-3 fatty acid reductase from <span class="italic">Helicoverpa armigera</span> (accession number JF709978) were expressed to produce <span class="red-bold">Z11-16:OH</span>. Additionally, 1,2-diacyl-sn-glycerol:acetyl-CoA acetyltransferase from <span class="italic">Euonymus alatus</span> (accession number GU594061) was expressed along with the other two enzymes to produce <span class="blue-bold">Z11-16:OAc</span> [7]. Inspired by Hagström approach [6], production of <span class="green-bold">Z11-16:Ald</span> could be accomplished by over expressing a fatty-acid alcohol oxidase from <span class="italic">Nicotiana benthamiana</span>. The whole biosynthetic pathway is depicted in figure 2.</p><br/><br/><br/><br/>
-<p>Pheromones to be produced in the Sexy Plant are pheromones affecting the sexual behaviour in a broad variety of moth species. “Most moth species utilize Type I pheromones that consist of straight-chain compounds 10-18 carbons in length with a functional group of a primary alcohol, aldehyde, or acetate ester, and usually with several double bonds, de novo synthesized in the pheromone gland (PG) through modifications of fatty acid biosynthetic pathways” [5]. Due to this fact, Type I pheromones were selected to be synthesized by the Sexy Plant.</p><br/><br/>
-<p>In previous transcriptome analysis from Agrotis ipsilon, specific sets of enzymes were identified to be differentially expressed in the pheromone glands.  These enzymes were Acetyl-CoA carboxylases, fatty acid synthases, desaturases, acyl-CoA reductases, alcohol oxidases, aldehyde reductases and acetyltransferases were found to be differentially expressed in the pheromone glands compared to the rest of the organism [6]. These results mean these enzymes are involved in pheromones biosynthesis. </p><br/><br/>
+<div align="center"><img width="450px" src="https://static.igem.org/mediawiki/2014/f/fd/VUPVBiosynthesis_figure_2.png" alt="pheromone_pathway" title="Pheromones Pathway"></img></div><br/>
+			<div align="center"><p style="text-align: justify; font-style: italic; font-size: 0.8em; width: 700px;"><span class="black-bold">Figure 2. Biosynthetic pathway of moth sexual pheromones</span>. Sexual pheromones are bordered in purple. Taking Palmitic Acid CoA (16:CoA) as substrate, the expression of the genes AtrΔ11 (Desaturase) and HarFAR (Reductase) leads to the production of <span class="red-bold">Z11-16:OH</span>. By adding the gene EaDAcT (Acetyltransferase) in the previous system, production displaced to obtaining <span class="blue-bold">Z11-16:OAc</span>. If a Alcohol Oxidase (FAO) is included instead of EaDAcT, <span class="green-bold">Z11-16:Ald</span> will be produced.</p></div><br/><br/>
-<p>Hagström et al produced moth sexual pheromone Z-11:160H in yeast from palmitic acid by introducing a Δ11 fatty-acyl desaturase and a fatty-acyl reductase from Agrotis segetum. This pathway was first taken into account to be implemented in the Sexy Plant, however, different subcellular location of the substrate arose doubts about this pathway.</p><br/><br/>
-<p>The pathways developed by Ding et al were incorporated in cis in the Sexy Plant to produce successfully synthesized Z11-16:Ald and Z11-16:OH. In Ding's work a Δ11 desaturase from Amyelois transitella (accession number JX964774)and HarFAR_KKYR, an improved version of HarFAR-3 fatty acid reductase from Helicoverpa armigera (accession number JF709978), and 1,2-diacyl-sn-glycerol:acetyl-CoA acetyltransferase from Euonymus alatus (accession number GU594061) were expressed to obtain these pheromones.[4]</p><br/><br/>
+<p><strong>Finding pests</strong></p><br/>
-<p>Inspired by Hagström's discussion, we expressed an alcohol oxidase in order to try obtain pheromone Z11-16:Ald from Z11-16:OH [7]. To produce Z11-16:OH, a fatty-acid alcohol oxidase, already present in Nicotiana benthamiana, was re-introduced in the system under the regulation of a strong constitutive promoter to ensure transcriptional levels were high enough.</p><br/><br/>
+<p>Complementing our efforts to produce the pheromones, we did a thorough search in the Pherobase database (http://www.pherobase.com) with software we specifically developed for this purpose and checked which insects met the conditions both to have one of the pheromones produced by the Sexy Plant as a major pheromone component and also to be considered a plague. Results from this analysis can be found in the tables below.  We can conclude that there is a large number of insects causing plagues that are affected by our pheromones.</p><br/><br/>
-<p>Complementing our efforts to produce the pheromones, we did a thorough search in the Pherobase database (http://www.pherobase.com) with software we specifically developed for this purpose and checked which insects met the conditions both to have one of the pheromones produced by the Sexy Plant as a major pheromone component and also to be considered a plague. Results from this analysis can be found in the tables below.</p><br/><br/><br/><br/>
+<br/><br/><br/>
-<p>Insects attracted by Z11-16:Ald and Z11-16:OH</p><hr/><br/><br/>
+<p>Insects attracted by <span class="green-bold">Z11-16:Ald</span> and <span class="red-bold">Z11-16:OH</span></p><hr/><br/><br/>
 <div align="center"><table class="normal-table">
@@ Line 82: / Line 100: @@
 </table></div><br/><br/><br/><br/>
-<p>Insects attracted by Z11-16:OAc and Z11-16:OH</p><hr/><br/><br/>
+<p>Insects attracted by <span class="blue-bold">Z11-16:OAc</span> and <span class="red-bold">Z11-16:OH</span></p><hr/><br/><br/>
 <div align="center"><table class="normal-table">
@@ Line 130: / Line 148: @@
 </table></div><br/><br/><br/><br/>
-<p>Insects attracted by Z11-16:OH</p><hr/><br/><br/>
+<p>Insects attracted by <span class="red-bold">Z11-16:OH</span></p><hr/><br/><br/>
 <div align="center"><table class="normal-table">
@@ Line 147: / Line 165: @@
 </table></div><br/><br/><br/><br/>
+<div align="center">
+<a class="button-content" id="goto-left" align="center" href="https://2014.igem.org/Team:Valencia_UPV/Project/modules/moths_behavior"><strong>&larr; Go to Moths Behaviour</strong></a>
+<a class="button-content" id="goto-middle" align="center" href="https://2014.igem.org/Team:Valencia_UPV/Project/modules"><strong>Go to Modules</strong></a>
+<a class="button-content" id="goto-right" align="center" href="https://2014.igem.org/Team:Valencia_UPV/Project/modules/release"><strong>Go to Release &rarr;</strong></a></div></br></br></br><br/>
 <p align="center"><strong>References</strong></p><br/>
 				<div style="position: relative; left: 3%; width: 96%;"><ol>
+<li>Matsumoto S (2010) Molecular Mechanisms Underlying Sex Pheromone Production in Moths. Bioscience, Biotechnology, and Biochemistry 74: 223-231.</li>
 					<li>Choi M-Y, Han KS, Boo KS, Jurenka RA (2002) Pheromone biosynthetic pathways in the moths Helicoverpa zea and Helicoverpa assulta. Insect Biochemistry and Molecular Biology 32: 1353-1359.</li>
 					<li>Wang H-L, Zhao C-H, Wang C-Z (2005) Comparative study of sex pheromone composition and biosynthesis in Helicoverpa armigera, H. assulta and their hybrid. Insect Biochemistry and Molecular Biology 35: 575-583.</li>
 					<li>Fu X, Fukuzawa M, Tabata J, Tatsuki S, Ishikawa Y (2005) Sex pheromone biosynthesis in Ostrinia zaguliaevi, a congener of the European corn borer moth O. nubilalis. Insect Biochemistry and Molecular Biology 35: 621-626.</li>
-					<li>Ding BJ, Hofvander P, Wang HL, Durrett TP, Stymne S, et al. (2014) A plant factory for moth pheromone production. Nat Commun 5: 3353.</li>
-					<li>Matsumoto S (2010) Molecular Mechanisms Underlying Sex Pheromone Production in Moths. Bioscience, Biotechnology, and Biochemistry 74: 223-231.</li>
 					<li>Gu S-H, Wu K-M, Guo Y-Y, Pickett J, Field L, et al. (2013) Identification of genes expressed in the sex pheromone gland of the black cutworm Agrotis ipsilon with putative roles in sex pheromone biosynthesis and transport. BMC Genomics 14: 636.</li>
-					<li>Hagstrom A, Wang H-L, Lienard M, Lassance J-M, Johansson T, et al. (2013) A moth pheromone brewery: production of (Z)-11-hexadecenol by heterologous co-expression of two biosynthetic genes from a noctuid moth in a yeast cell factory. Microbial Cell Factories 12: 125.</li>
+					<li>Hagström A, Wang H-L, Lienard M, Lassance J-M, Johansson T, et al. (2013) A moth pheromone brewery: production of (Z)-11-hexadecenol by heterologous co-expression of two biosynthetic genes from a noctuid moth in a yeast cell factory. Microbial Cell Factories 12: 125.</li>
+					<li>Ding BJ, Hofvander P, Wang HL, Durrett TP, Stymne S, et al. (2014) A plant factory for moth pheromone production. Nat Commun 5: 3353.</li>
 				</ol>