Tunable control of insect pheromone biosynthesis in Nicotiana benthamiana

Abstract

Previous work has demonstrated that plants can be used as production platforms for molecules used in health, medicine, and agriculture. Production has been exemplified in both stable transgenic plants and using transient expression strategies. In particular, species of Nicotiana have been engineered to produce a range of useful molecules, including insect sex pheromones, which are valued for species-specific control of agricultural pests. To date, most studies have relied on strong constitutive expression of all pathway genes. However, work in microbes has demonstrated that yields can be improved by controlling and balancing gene expression. Synthetic regulatory elements that provide control over the timing and levels of gene expression are therefore useful for maximizing yields from heterologous biosynthetic pathways. In this study, we demonstrate the use of pathway engineering and synthetic genetic elements for controlling the timing and levels of production of Lepidopteran sex pheromones in Nicotiana benthamiana. We demonstrate that copper can be used as a low-cost molecule for tightly regulated inducible expression. Further, we show how construct architecture influences relative gene expression and, consequently, product yields in multigene constructs. We compare a number of synthetic orthogonal regulatory elements and demonstrate maximal yields from constructs in which expression is mediated by dCas9-based synthetic transcriptional activators. The approaches demonstrated here provide new insights into the heterologous reconstruction of metabolic pathways in plants.

Keywords: Nicotiana benthamiana; metabolic engineering; pheromones; synthetic promoters; transcriptional activation.

Figure 1

Heterologous production of Lepidopteran sex pheromones. (a) Plant production of the two main volatile components in many Lepidopteran sex pheromones (Z)‐11‐hexadecenol (Z11‐16OH) and (Z)‐11‐hexadecenyl acetate (Z11‐16OAc) from endogenous 16C fatty acyl CoA (Z11‐16CoA) was previously achieved by heterologous expression of a Δ11 desaturase, a fatty acid reductase and a diacylglycerol acetyltransferase. The accumulation of (Z)‐11‐hexadecenal (Z11‐16:Ald) was also observed, presumably catalysed by an endogenous alcohol oxidase (Mateos‐Fernández et al., 2021). (b) Differences in the quantities and ratios of Z11‐16OAc and Z11‐16OH obtained by co‐expression of diacylglycerol transferases from Euonymus alatus (EaDAct), E. fortunei (EfDAct), Saccharomyces cerevisiae (ScATF1) and S. pastorianus (SpATF1‐2) with a fatty acid reductase from Helicoverpa armigera (HarFAR) and a Δ11 desaturase from Amyelois transitella (AtrΔ11). Values shown are the mean and standard error of n = 3 biological replicates (independent infiltrations). Means annotated with a common Greek letter (α, β) are not significantly different by a one‐way ANOVA with post‐hoc Tukey HSD at the 5% level of significance.

Figure 2

Copper‐inducible expression of Lepidopteran pheromones. (a) Schematic of a plant expression construct containing synthetic genes encoding the copper‐responsive transcription factor CUP2 in translational fusion with the Gal4 activation domain and the coding sequences of AtrΔ11, HarFAR and SpATF1‐2 under control of a minimal 35s promoter preceded by four copies of the CUP2 binding site (CBS). (b) Total ion chromatogram showing the accumulation of Z11‐16:OH and Z11‐16:OAc in leaves of N. benthamiana co‐infiltrated with Agrobacterium strains containing the expression construct (678) and a construct expressing the P19 suppressor of silencing only after application of 2.5 mm copper sulphate (CuSO₄).

Figure 3

Construct architecture influences expression and product yield. (a) The level of expression of firefly luciferase (LucF) and nanoluciferase (LucN) in a multigene construct is dependent on the position in which the gene is assembled. Values shown are the mean and standard error of n = 6 biological replicates (independent infiltrations) and differences were analysed using a Kruskal–Wallis test followed by pairwise Wilcoxon rank sum test with Benjamini‐Hochberg correction. Bars annotated with a common Greek letter (α, β, γ, δ) are not significantly different. (b) Schematics of plant expression constructs containing synthetic genes for copper‐inducible expression of lepidopteran sex pheromones. (c) The relative positions of pathway genes influenced the overall yield and the relative ratios of pheromone products. Values shown are the mean and standard error of n = 3 biological replicates (independent infiltrations). Means annotated with common Greek letters (α, β) are not significantly different by a one‐way ANOVA followed by Post‐hoc Tukey test at the 5% level of significance.

Figure 4

Comparison of synthetic transcriptional activators. Normalized luminescence from reporter constructs activated by copper‐inducible (a) GAL4:𝚽C31 (b) activator‐like effector (TALE) and (c) dCasEV2.1 synthetic transcriptional activators. In all systems, expression levels increase with copper and with the number of transcriptional activator binding sites in the promoter. Copper‐inducible expression of dCasEV2.1 maintains tight control (low background) of gene expression. Values shown are the mean and standard error of n = 3 biological replicates (independent infiltrations). P‐values were calculated using Welch two sample t‐test: *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.

Figure 5

Copper‐inducible, CRISPR/Cas9‐mediated control of pheromone biosynthesis. (a) Schematic of plant expression constructs containing elements for copper‐inducible expression of the dCasEV2.1 transcriptional activator (above) and multigene constructs containing coding sequences for AtrΔ11, HarFAR and SpATF1‐2. The latter are assembled with promoters consisting of a minimal DFR core promoter fused to unique sequences containing the conserved gRNA target sites (b) Application of CuSO₄ results in dCasEV2.1‐mediated production of the pheromone components (Z11‐16OH and Z11‐16OAc). Values shown are the mean and standard error of n = 3 biological replicates (independent infiltrations). Means followed by a common Greek letter (α, β) are not significantly different (one‐way ANOVA with post‐hoc Tukey HSD at the 5% level of significance).

Figure 6

Functionality of the copper sensing dCasEV2.1 module in stable Nicotiana benthamiana transgenics. (a) Transgenic plants expressing the CBS:dCasEV2.1 (construct GB4068) were agroinfiltrated with a luciferase reporter module (construct GB3324) and sgRNA module (construct 1838). (b) Normalized expression levels of luciferase in T₀ CBS:dCas transgenic plants after copper induction. Values are the mean and standard deviation of n = 3 independent infiltrations. P‐values were calculated using Student's t‐test; *P ≤ 0.05, ***P ≤ 0.001; ns = not significant. The figure includes images from Biorender (biorender.com).

Figure 7

CRISPR/Cas9‐mediated control of pheromone biosynthesis. (a) Schematic of constructs for constitutive or dCasEV2.1 activated expression of AtrΔ11, HarFAR and SpATF1‐2 using promoters consisting of a minimal DFR core promoter fused to unique sequences containing the conserved gRNA target sites. (b) Yields of pheromone components (Z11‐16OH and Z11‐16OAc) obtained following transient agroinfiltration. (c) Schematics of constructs encoding the guided pathway (sgRNA integrated) or non‐guided pathway (sgRNA infiltrated) used to produce transgenic lines and transiently expressed dCasEV2.1 activating elements (d) Pheromone levels obtained from T₁ progeny of four independent T₀ lines encoding either the guided (GB2618) or non‐guided (GB3898) pathway infiltrated with constructs expressing regulatory elements. Values represent the mean and standard deviation of n = 3 biological replicates (independent infiltrations). Values annotated with a common Greek letter (α) are not significantly different (one‐way ANOVA with post‐hoc Tukey HSD at the 5% level of significance). This figure includes images from Biorender (biorender.com).