Plants as Factories for Insect Pheromone Production : Deciphering and Reconstructing Sex Pheromone Biosynthetic Pathways of Female Moths

Abstract: Compared to other organisms in which the fatty acyl desaturases (FADs) are mostly involved in normal cellular lipid metabolism, moth FADs have evolved extent functions in the biosynthesis of sex pheromones. Female moths releasespecies-specific sex pheromones to attract conspecific males over a long distance for mating. Moth FADs are key enzymes producing the great diversity of moth sex pheromones. They introduce double bonds in specific positions and with specific geometry in the fatty acyl pheromone precursors. In this thesis, I use a variety of experimental approaches including isotope labelling experiments and heterologous expression of gene candidates to characterize several novel FADs involved in pheromone production: The multi-functional SexiDes5 from the beet armyworm Spodoptera exigua and SlitDes5 from the congeneric Spodoptera litura were found to have Δ12 desaturase activities. They use palmitic acid to produce (Z)-11-hexadecenoic acid and the subsequently chainshortened product (Z)-9-tetradecenoic acid to produce (Z,E)-9,12-tetradecadienoic acid. The European grapevine moth, Lobesia botrana was shown to produce its major pheromone precursor (E,Z)-7,9-dodecanoic acid by an Δ7 FAD. A pheromone gland-specific CsupYPAQ from the rice stem borer Chilo suppressalis was proven to have high activity on palmitic acid to produce (Z)-11-hexadecenoic acid. The highly evolved moth FADs can be used for production of customized pheromone precursors in transformed organisms for a variety of purposes. Compared to the current conventional synthetic approach which producing thehazardous waste during the production process, using semi-synthetic method to produce moth pheromones based on plant-derived pheromone precursors are environmentally friendly I investigated the use of several plant platforms to express a suite of biosynthetic enzymes for moth pheromone precursor production. By employing the Agrobacteriummediated transformation, I constructed transgenic Nicotiana spp. and Camelina lines for production of C12 to C16 chain length pheromone precursors. The transformed Nicotiana spp. can produce (Z)-11-hexadecenoic acid, (E)-11- tetradecenoic acid, (Z)-11-tetradecenoic acid. The best line from N. benthamiana produced 17.6% (weight%) of (Z)-11-hexadecenoic acid of total fatty acid in vegetative tissue. Also, 7.6% of (E)-9-dodecenoic acid and 6.3% of doubly unsaturated (E,E)-8,10-dodecenoic acid of total fatty acids were produced in seeds of engineered Camelina plants,implying that a significant amount of pheromone precursors might be produced by cultivating these transgenic plants under field conditions. Knowledge of additional pheromone biosynthetic gene functions can improve the possibility and feasibility of synthesizing customized moth pheromones in plant factories. A fatty acyl elongase (ELO) combined with a Δ11 FAD is thought to provide the fatty acyl pheromone precursors in C. suppressalis. I functionally characterized an ELO gene CsupELO4 encoding a protein elongating the major pheromone precursor (Z)-11-hexadecenoic acid into (Z)-13-octadecenoic acid, the precursor of a minor pheromone component. This is the first ELO gene that has been functionally characterized in Lepidoptera. The fatty acyl-CoA pheromone precursors are postulated to be reduced and reoxidized toproduce the aldehyde pheromone components. I characterized CsupFAR2 from C. suppressalis that encodes a fatty acyl reductase (FAR) reducing the major fatty acyl precursors into corresponding fatty alcohols, which are convert into the fattyaldehyde pheromones by followed-up oxidation. Genetically modified plants actually releasing moth pheromones may be used as part of a push-pull strategy. I made an attempt to engineer Nicotiana spp. plants that would release (Z)-11-hexadecenol and (Z)-11-hexadecenyl acetate. Icloned the promoter CYP71D16, which is a trichome-specific promoter from tobacco Nicotiana tabacum, driving the pheromone biosynthetic genes. I surprisingly found that the production of (Z)-11-hexadecenol increased from 18 to 70 μg per gram fresh leaf when the gene of HarFAR was expressed under CYP71D16 promoter compared to a constitutive promoter CaMV35S. However, no pheromone compounds were found in the plant headspace volatiles.