Production of Volatile Moth Sex Pheromones in TransgenicNicotiana benthamianaPlants

Abstract

Plant-based bio-production of insect sex pheromones has been proposed as an innovative strategy to increase the sustainability of pest control in agriculture. Here we describe the engineering of transgenic plants producing (Z)-11-hexadecen-1-ol (Z11-16OH) and (Z)-11-hexadecenyl acetate (Z11-16OAc), two main volatile components in many Lepidoptera sex pheromone blends. We assembled multiple multigene DNA constructs encoding the pheromone biosynthetic pathway and stably transformed them in Nicotiana benthamiana plants. The constructs comprised the Amyelois transitella AtrΔ11 desaturase gene, the Helicoverpa armigera farnesyl reductase HarFAR gene, and the Euonymus alatus diacylglycerol acetyltransferase EaDAct gene in different configurations. All the pheromone-producing plants showed dwarf phenotypes, whose severity correlated with pheromone levels. All but one of the recovered lines produced high levels of Z11-16OH but very low levels of Z11-16OAc, probably as a result of recurrent truncations at the level of the EaDAct gene. Only one plant line (SxPv1.2) was recovered harbouring an intact pheromone pathway and producing moderate levels of Z11-16OAc (11.8 μg g-1 FW), next to high levels of Z11-16OH (111.4 μg g-1). Z11-16OAc production was accompanied in SxPv1.2 by a partial recovery of the dwarf phenotype. SxPv1.2 was used to estimate the rates of volatile pheromone release, which resulted in 8.48 ng g-1 FW per day for Z11-16OH and 9.44 ng g-1 FW per day for Z11-16OAc. Our results suggest that pheromone release acts as a limiting factor in pheromone bio-dispenser strategies and establish a roadmap for biotechnological improvements.

Insect pest management in the age of synthetic biology

“…While living biodispensers would not be useful for attract and kill strategies where the pheromone and an insecticide are combined within a trap, they could be used in mating disruption strategies in which the aim is to impede the ability of males to locate a female mate. In 2020, live plant dispensers of chemical signals were included in a shortlist of emerging products of bioengineering (Kemp et al ., 2020 ) and, recently, transgenic N. benthamiana plants transformed with a three‐gene pathway resulted in the production of high levels of the (Z) ‐11‐hexadecen‐1‐ol (Z11‐16OH) and (Z) ‐11‐hexadecenyl acetate (Z11‐16OAc) components of moth pheromones (Mateos‐Fernández et al ., 2021 ). Although pheromone production was shown to negatively influence plant growth and only a small fraction of the pheromones were released to the environment in volatile forms, this first report of a living biodispenser could pave the way for future iterations.…”

Tunable control of insect pheromone biosynthesis inNicotiana benthamiana

“…Heterologous production of moth sex pheromones from endogenous 16C fatty acyl CoA is achieved by constitutive expression of Δ11 desaturase, fatty acid reductase and diacylglycerol acetyltransferase ( Figure 1A ). The genes encoding moth acetyltransferases involved in pheromone biosynthesis have not yet been identified and previous attempts at heterologous biosynthesis have employed either an enzyme from the plant, Euonymus alatus (EaDAct) (Ding et al ., 2014; Mateos-Fernández et al ., 2021), or from the yeast, Saccharomyces cerevisae (ScATF1) (Ding et al ., 2016; Xia et al ., 2022), with the latter producing more acetate. We first compared EaDAct and ScATF1 with two further diacylglycerol acetyltransferases from Euonymus fortunii (EfDAct) (Tran et al, 2017) and Saccharomyces pastorianus (SpATF1-2) (Yoshimoto et al ., 1999).…”

“…Indeed, a major advantage of biological manufacturing is the ability to produce complex molecules, including those for which chemical synthesis has proven difficult or commercially non-viable due to the requirement of multiple stereoselective steps (Cravens et al, 2019). For example, there is growing interest in the biological production of insect sex pheromones, many of which are stereochemically complex, for species-specific control of agricultural pests (Ding et al, 2014; Holkenbrink et al, 2020; Mateos Fernández et al, 2022; Mateos-Fernández et al, 2021; Xia et al, 2021).…”

“…They also produce many metabolic precursors and cofactors allowing the facile reconstruction of metabolic pathways often without the need to engineer host genes and pathways (Patron, 2020; Stephenson et al, 2020). For example, precursors of Lepidopteran sex pheromones have been produced in field-grown transgenic Camelina sativa (false flax) (Wang et al, 2022)), while biosynthesis of the pheromone molecules has been demonstrated in other plant species (Ding et al, 2014; Mateos-Fernández et al, 2021; Xia et al, 2022).…”

“…Further, high-level transgenic expression of some molecules can impact growth and development. For example, we recently showed that growth was compromised in transgenic lines N. benthamiana producing the highest yields (per gram fresh weight) of the Lepidopteran sex pheromone components, (Z)-11-hexadecenyl acetate (Z11-16OAc) and (Z)-11-hexadecenol (Z11-16OH) (Mateos-Fernández et al, 2021). Analysis of transcriptional changes revealed stress-like responses, including the downregulation of photosynthesis-related genes (Juteršek et al, 2022).…”