On the way of making plants smell like moths - a synthetic biology approach

Sammanfattning: Abstract: Moth caterpillars are major agricultural pests in many parts of the world. In general female moths attract male mates with their pheromone over long distance. Most of the described moth pheromones belong to the same class of chemical compounds, fatty acid derivatives that are produced de novo in the pheromone gland. The pheromone biosynthesis involves desaturation, chain-shortening by ?-oxidation and functional group modification such as reduction, acetylation or oxidation, etc. These enzymes have evolved to function in the production of the complex chemical signals used for sex attraction, thus contributing to the chemical diversity of moth pheromones. In the current thesis, several desaturases were characterized. Firstly, a terminal fatty-acyl-CoA desaturase from winter moth (Operophtera brumata) was cloned and expressed heterologously in yeast and proved its ability to convert Z11,Z14,Z17-eicosatrienoic acid to Z11,Z14,Z17,19-eicosatetraenoic acid. This is the first report on methyl terminal desaturase ever. Secondly, desaturation steps in Cydia pomonella and Grapholita molesta, which use unsaturated dodecenyl alcohol and/or acetate as sex pheromone component(s), were characterized. We found the desaturases in C. pomonella work consecutively, account for the production of conjugated double-bond in the fatty acyl chain (E8,E10-12:CoA). But in the case of G. molesta, we found ?10 desaturase on myristic acid. The E/Z10-14:Acyl, which after chain-shortening, reduction and acetylation may produce the G. molesta pheromone consisting of Z8-12:OAc, E8-12:OAc and Z8-12:OH. Thirdly, the stereospecificity of two ?11 desaturases was investigated. A ?11 desaturase from Choristoneura rosaceana takes saturated 14 carbon and produce a mixture of (E)-11-tetradecenoate and (Z)-11-tetradecenoate with an excess of the Z isomer. A desaturase from C. parallela also takes saturated 14C but produce almost pure E11-14:Acyl. Reciprocal site-directed mutations on this two desaturases revealed that one amino acid at the C-terminal of the protein is critical for the Z activity of the desaturase (gain or lose of function). This study shed light on cracking the stereospecificity of desaturase. The next study focused on fatty alcohol acetyltransferase that bears great implications in many moth pheromone biosynthesis pathways. It catalyzes the formation of acetate ester by transferring acetate group from the acetyl-CoA to the fatty alcohol. Since no insect-derived pheromone biosynthetic acetyltransferase has been cloned, we heterologously expressed a plant derived acetyltransferase, EaDAcT, in a yeast system, to test the functionality and validity in converting moth pheromone intermediates, fatty alcohols, into final pheromone product, fatty alcohol acetate esters. The results showed EaDAcT could convert various fatty alcohols with chain length range from 10 to 18 carbons, with double bound at varying positions, into their corresponding acetate esters. EaDAcT prefers shorter chain length to the long ones, unsaturated to the saturated ones. The microsome preparations showed an activity pattern similar to the activity observed in the in vivo experiments. Next, through massive sequencing of pheromone producing tissue, we identified genes that might be involved in the pheromone biosynthetic process of the turnip moth (Agrotis segetum), such as: fatty acid synthase, ?-oxidation enzymes, desaturase, fatty acyl reductase, acetyltransferase, etc. The final study was assembling the parts identified previously in a chassis to make moth pheromones. Using Nicotiana benthamiana, as a plant factory, we produced typical moth sex pheromone components by transient expression of up to four genes coding for consecutive biosynthetic steps. We specifically produced biologically active multi-component sex pheromones for two species of small ermine moths. The fatty alcohol fractions from the genetically modified plants were acetylated and mixed to mimic the respective sex pheromones of Yponomeuta evonymella and Y. padella. Although the composition of the plant-derived mixtures was not optimized, these mixtures were very efficient and specific for trapping of male moths, matching the activity of conventionally produced synthetic pheromones. Our long-term vision is to design tailor-made production of any moth pheromone component in genetically modified plants. Such semi-synthetic preparation of sex pheromones may be a novel and cost-effective way of producing moderate to large quantities of pheromones for integrated pest management, with high purity and a minimum of hazardous waste.