Production of moth sex pheromone precursors in Nicotiana spp.: a worthwhile new approach to pest control

Abstract

Pheromones are environmentally friendly alternatives to traditional pesticides for pest control. They are widely applied for insect monitoring, mating disruption and mass trapping. Nicotiana benthamiana and N. tabacum are potential green biomass production platforms of moth sex pheromones. Using these two Nicotiana species as plant factories, we expressed biosynthetic genes of plant and insect origin in leaf tissue. Moth sex pheromone precursors (E)-11-tetradecenoic acid, (Z)-11-tetradecenoic acid and (Z)-11-hexadecenoic acid were produced by introducing the acyl-ACP thioesterases CpuFatB1 from Cuphea pulcherrima or CpaFatB2 from C. palustris and the fatty acyl desaturases Ave∆11 from Argyrotaenia velutinana, CpaE11 from Choristoneura parallela or Atr∆11 from Amyelois transitella, under the control of CaMV-35S promoter. Among the Nicotiana spp. transformants, the best line produced (Z)-11-hexadecenoic acid at 17.6% of total fatty acids in leaves, during flowering stage, corresponding to 335 µg of (Z)-11-hexadecenoic acid per gram of fresh leaf. The (Z)-11-hexadecenoic acid production lines from N. benthamiana were selected for further propagation to obtain homozygous lines. In the N. benthamiana T2 generation, the production quantity of (Z)-11-hexadecenoic acid was stable. Our study demonstrates the feasibility of stable transformation of N. benthamiana for production of moth pheromone precursors in vegetative tissue.

Production of Volatile Moth Sex Pheromones in TransgenicNicotiana benthamianaPlants

“…The maximum pheromone accumulation levels measured in SxPv1.2 reached 174.5 µg g -1 FW (totalling both alcohol and acetate forms). This is about half of the levels of precursors reported by Ding et al, (2014) in transient experiments (381 µg g -1 ) or by Xia et al, (2020) in stable plants (335 µg g -1 ), and may indicate a partial conversion into biologically active forms, or upper limit for toxicity, especially in the case of Z11-16OH. However, only a small portion of the plant pheromone content can be detected in the environment after a 48h incubation.…”

“…Such toxic effects can hamper plant viability and result in a negative selection pressure against the genotypes with higher pheromone production levels. Interestingly, whereas Xia et al, (2020) found strong deleterious effects associated to the production of (E)-11-tetradecenoic acid, the same authors regenerated normal plants that accumulated Z11-16CoA, the direct precursor of the volatile pheromones produced here. The fact that the simple addition of a desaturase activity leads to deleterious effects may indicate that Z11-16OH itself is responsible for the toxic effects observed when accumulated in leaves.…”

“…As results of our analysis, two main bottlenecks were identified: the associated growth penalty and the poor release rates of the pheromones to the environment. As highlighted also by Reynolds et al, (2017) and later by Xia et al, (2020), one of the most significant downsides to the constitutive overexpression of medium-chain fatty acid biosynthesis pathways in plants is the associated developmental abnormalities. These may result from an imbalance caused by diverting metabolic resources from fatty acid metabolism towards the products of interest, and possibly from the toxicity of the end-products.…”

“…This research was initiated as a Synthetic Biology project in the frame of the iGEM competition, where undergraduate students proposed the use of genetically engineered plants as dispensers of insect sex pheromones. The manufacturing of semiochemicals and their precursors employing biological factories such as microbial bioreactors (Hagström et al, 2013;Holkenbrink et al, 2020) or plant biofactories (Ding et al, 2014;Ortiz et al, 2020;Xia et al, 2020) has become an intensively pursued objective, fuelled by the expected gains in sustainability. Beyond the general biofactory concept, our envisioned long-term approach consists in the design of plants that function as autonomous bio-dispensers of semiochemicals.…”

“…Fatty acids are then modified (by desaturation, chain length modification, isomerization or substitution), and precursors are exported to the cytosol, where the final steps of pheromone biosynthesis take place. Nešněrová et al, (2004), Ding et al, (2014) and Xia et al, (2020) have employed species from the genus Nicotiana (N. tabacum and N. benthamiana) to set up plant biofactories to produce lepidopteran pheromones or their precursors. These two plant species are excellent biofactory candidates for metabolic engineering, thanks to their large leaf biomass (especially for plastid-derived products) and amenability to genetic manipulation, both through stable transformation and agroinfiltration.…”