A Suite of Constitutive Promoters for Tuning Gene Expression in Plants

Abstract

The need for convenient tools to express transgenes over a large dynamic range is pervasive throughout plant synthetic biology; however, current efforts are largely limited by the heavy reliance on a small set of strong promoters, precluding more nuanced and refined engineering endeavors in planta. To address this technical gap, we characterize a suite of constitutive promoters that span a wide range of transcriptional levels and develop a GoldenGate-based plasmid toolkit named PCONS, optimized for versatile cloning and rapid testing of transgene expression at varying strengths. We demonstrate how easy access to a stepwise gradient of expression levels can be used for optimizing synthetic transcriptional systems and the production of small molecules in planta. We also systematically investigate the potential of using PCONS as an internal standard in plant biology experimental design, establishing the best practices for signal normalization in experiments. Although our library has primarily been developed for optimizing expression in N. benthamiana, we demonstrate the translatability of our promoters across distantly related species using a multiplexed reporter assay with barcoded transcripts. Our findings showcase the advantages of the PCONS library as an invaluable toolkit for plant synthetic biology.

Genetically refactoredAgrobacterium-mediated transformation

“…Previous work using inducible promoters showed that transformation of tobacco was highly sensitive to virE12 expression 39 . Using transiently expressed GFP from a medium strength plant promoter in N. benthamiana as a measure of transformation 41 , we observed that all promoters stronger than the weakest, P J23114 , were able to complement leaf transformation back to wild-type levels ( Figure 1B ) . Proteomics analysis of the Δ virE12 complementation strains confirmed that expression of VirE12 correlated with RFP expression from the same promoters ( Figure 1C ) .…”

Plant Synthetic Promoters

“…However, larger promoter domains with known regulatory properties could be exchanged in the process of domain swapping [215][216][217][218][219][220]. Alternatively, completely novel cis-elements or domain variants identified by searches of available promoter databases or obtained through artificial intelligence support could be experimentally validated in plant material and then employed to construct synthetic promoters [221]. An example of cis-element shuffling involves the placement of the ciselements of known functions in a novel or synthetic stretch of DNA.…”

A comparative analysis of stably expressed genes across diverse angiosperms exposes flexibility in underlying promoter architecture

“…Constitutive promoters, defined here as promoters that are expressing in all tissues at all times, are versatile tools in synthetic biology due to their desirable expression pattern ( Yang and Nemhauser 2022 ; Zhou et al 2023 ). They are often used to drive expression of components used in synthetic circuits or metabolic engineering ( Wu et al 2014 ; South et al 2019 ; Patron 2020 ; Brophy et al 2022 ).…”