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

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 β-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, β-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

Release of moth pheromone compounds from Nicotiana benthamiana upon transient expression of heterologous biosynthetic genes

Background Using genetically modified plants as natural dispensers of insect pheromones may eventually become part of a novel strategy for integrated pest management. Results In the present study, we first characterized essential functional genes for sex pheromone biosynthesis in the rice stem borer Chilo suppressalis (Walker) by heterologous expression in Saccharomyces cerevisiae and Nicotiana benthamiana, including two desaturase genes CsupYPAQ and CsupKPSE and a reductase gene CsupFAR2. Subsequently, we co-expressed CsupYPAQ and CsupFAR2 together with the previously characterized moth desaturase Atr increment 11 in N. benthamiana. This resulted in the production of (Z)-11-hexadecenol together with (Z)-11-hexadecenal, the major pheromone component of C. suppressalis. Both compounds were collected from the transformed N. benthamiana headspace volatiles using solid-phase microextraction. We finally added the expression of a yeast acetyltransferase gene ATF1 and could then confirm also (Z)-11-hexadecenyl acetate release from the plant. Conclusions Our results pave the way for stable transformation of plants to be used as biological pheromone sources in different pest control strategies

Release of moth pheromone compounds from Nicotiana benthamiana upon transient expression of heterologous biosynthetic genes

Background: Using genetically modified plants as natural dispensers of insect pheromones may eventually become part of a novel strategy for integrated pest management. Results: In the present study, we first characterized essential functional genes for sex pheromone biosynthesis in the rice stem borer Chilo suppressalis (Walker) by heterologous expression in Saccharomyces cerevisiae and Nicotiana benthamiana, including two desaturase genes CsupYPAQ and CsupKPSE and a reductase gene CsupFAR2. Subsequently, we co-expressed CsupYPAQ and CsupFAR2 together with the previously characterized moth desaturase Atr∆11 in N. benthamiana. This resulted in the production of (Z)-11-hexadecenol together with (Z)-11-hexadecenal, the major pheromone component of C. suppressalis. Both compounds were collected from the transformed N. benthamiana headspace volatiles using solid-phase microextraction. We finally added the expression of a yeast acetyltransferase gene ATF1 and could then confirm also (Z)-11-hexadecenyl acetate release from the plant. Conclusions: Our results pave the way for stable transformation of plants to be used as biological pheromone sources in different pest control strategies

Identify submitochondria and subchloroplast locations with pseudo amino acid composition: Approach from the strategy of discrete wavelet transform feature extraction

AbstractIt is very challenging and complicated to predict protein locations at the sub-subcellular level. The key to enhancing the prediction quality for protein sub-subcellular locations is to grasp the core features of a protein that can discriminate among proteins with different subcompartment locations. In this study, a different formulation of pseudoamino acid composition by the approach of discrete wavelet transform feature extraction was developed to predict submitochondria and subchloroplast locations. As a result of jackknife cross-validation, with our method, it can efficiently distinguish mitochondrial proteins from chloroplast proteins with total accuracy of 98.8% and obtained a promising total accuracy of 93.38% for predicting submitochondria locations. Especially the predictive accuracy for mitochondrial outer membrane and chloroplast thylakoid lumen were 82.93% and 82.22%, respectively, showing an improvement of 4.88% and 27.22% when other existing methods were compared. The results indicated that the proposed method might be employed as a useful assistant technique for identifying sub-subcellular locations. We have implemented our algorithm as an online service called SubIdent (http://bioinfo.ncu.edu.cn/services.aspx)

Insect pest management with sex pheromone precursors from engineered oilseed plants

Pheromones have become an environmentally friendly alternative to conventional insecticides for pest control. Most current pheromone-based pest control products target lepidopteran pests of high-value crops, as today’s manufacturing processes cannot yet produce pheromones at low enough costs to enable their use for lower-value crops, especially commodity crops. Camelina sativa seeds genetically modified to express (Z)-11-hexadecenoic acid, a sex pheromone precursor of several moth species, provided the oil from which the precursor was isolated, purified and transformed into the final pheromone. Trap lures containing this pheromone were then assessed for their capacity to manage moth pests in the field. Plant-derived pheromone lures proved equally effective as synthetic pheromone lures in monitoring the diamondback moth, Plutella xylostella, in cabbage and disrupting mating of cotton bollworm, Helicoverpa armigera, in common bean fields. Our study demonstrates the biological efficacy and economic feasibility of pheromone production in plant factories by metabolic engineering of an oilseed crop

MAS: A versatile Landau-fluid eigenvalue code for plasma stability analysis in general geometry

We have developed a new global eigenvalue code, Multiscale Analysis for plasma Stabilities (MAS), for studying plasma problems with wave toroidal mode number n and frequency omega in a broad range of interest in general tokamak geometry, based on a five-field Landau-fluid description of thermal plasmas. Beyond keeping the necessary plasma fluid response, we further retain the important kinetic effects including diamagnetic drift, ion finite Larmor radius, finite parallel electric field, ion and electron Landau resonances in a self-consistent and non-perturbative manner without sacrificing the attractive efficiency in computation. The physical capabilities of the code are evaluated and examined in the aspects of both theory and simulation. In theory, the comprehensive Landau-fluid model implemented in MAS can be reduced to the well-known ideal MHD model, electrostatic ion-fluid model, and drift-kinetic model in various limits, which clearly delineates the physics validity regime. In simulation, MAS has been well benchmarked with theory and other gyrokinetic and kinetic-MHD hybrid codes in a manner of adopting the unified physical and numerical framework, which covers the kinetic Alfven wave, ion sound wave, low-n kink, high-n ion temperature gradient mode and kinetic ballooning mode. Moreover, MAS is successfully applied to model the Alfven eigenmode (AE) activities in DIII-D discharge #159243, which faithfully captures the frequency sweeping of RSAE, the tunneling damping of TAE, as well as the polarization characteristics of KBAE and BAAE being consistent with former gyrokinetic theory and simulation. With respect to the key progress contributed to the community, MAS has the advantage of combining rich physics ingredients, realistic global geometry and high computation efficiency together for plasma stability analysis in linear regime.Comment: 40 pages, 21 figure