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Team:Valencia UPV/Project/modules/biosynthesis
From 2014.igem.org
Biosynthesis
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.
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.
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.
Pheromones
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.
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.
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.
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]
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.
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.
Insects attracted by Z11-16:Ald and Z11-16:OH
Insects attracted by Z11-16:OAc and Z11-16:OH
Insects attracted by Z11-16:OH
