Tunable control of insect pheromone biosynthesis inNicotiana benthamiana

Abstract

Synthetic regulatory elements that provide control over the timing and levels of gene expression are useful for maximizing yields from heterologous biosynthetic pathways. Previous work has demonstrated that plants and microbes can be engineered to produce insect sex pheromones, providing a route for low-cost production of these compounds, which are valued for species-specific control of agricultural pests. Strong constitutive expression of pathway genes can lead to toxicity and metabolic loads, preventing normal growth and development and thus limiting biomass and affecting overall yields. In this study we demonstrate the ability to inducibly control the accumulation of Lepidopteran sex pheromones in leaves of Nicotiana benthamiana. Further, we show how construct architecture influences expression and product yields in multigene constructs, applying this to control the relative expression of genes within the pathway, thereby tuning the accumulation of pheromone components. The approaches demonstrated here provide new insights into the heterologous reconstruction of metabolic pathways in plants.

Engineering the Biosynthesis of Late-Stage Vinblastine Precursors Precondylocarpine Acetate, Catharanthine, Tabersonine in Nicotiana benthamiana

“…29,36 However, only a few studies where multi-TU assemblies containing different regulatory elements were used for in planta pathway reconstitution have been reported. 38,39 We built a multitranscriptional unit vector where four biosynthetic genes were expressed under the control of the SlUbq10 promoter/terminator pair (4TU_SGD_GS_GO_R-edox1, Figure 3A), as this allowed rapid and efficient multi-TU assembly, despite the size and repetitiveness of the assembled sequences. To quantify the final product (PCA, 9), we coinfiltrated the two missing biosynthetic enzymes, Redox2 and SAT, on separate vectors, each also under the control of the SlUbq10 promoter/terminator pair along with this 4TU construct.…”

“… 29 , 36 However, only a few studies where multi-TU assemblies containing different regulatory elements were used for in planta pathway reconstitution have been reported. 38 , 39 …”

“…A large number of strains may not be efficiently transformed into all N. benthamiana cells, which would negatively impact product yields. , Developments in modular cloning technologies now allow stacking of multiple transcriptional units onto a single plasmid. ,, Attempting to improve the yield of our MIA products, we used the Golden Braid modular assembly technology to express several of the C. roseus biosynthetic enzymes from a single vector. A vast toolkit of regulatory elements for use in plant synthetic biology was developed in the recent years. , However, only a few studies where multi-TU assemblies containing different regulatory elements were used for in planta pathway reconstitution have been reported. , …”

Transcriptional deregulation of stress-growth balance in Nicotiana benthamiana biofactories producing insect sex pheromones

“…This could be attributed to the fact that in the previous work, first-generation v1.2 plants were used, while in this study, we sampled and analysed their progeny. Despite the much higher production of ( Z )-11-hexadecenyl acetate in SxPv1.2, the pool of the precursor ( Z )-11-hexadecen-1-ol did not seem to be affected, supporting the need for the improvement of the rate-limiting acetyltransferase step ( Kallam et al., 2022 ).…”

“…To better understand the events prompting the transition from the normal to stressed state and its major regulatory players ( Wang and Zhang, 2021 ), sampling after production initiation in a well-regulated and non-leaky inducible expression system (e.g. Garcia-Perez et al., 2022 ; Kallam et al., 2022 ) could give more conclusive insights.…”