Team:Valencia UPV/Project/modules/biosynthesis

Project > Modules > 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.

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.

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

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

Insects attracted by Z11-16:OH

References