Glucoraphanin is a plant specialized metabolite found in cruciferous vegetables that has long been a target for production in a heterologous host because it can subsequently be hydrolyzed to form the chemopreventive compound sulforaphane before and during consumption. However, previous studies have only been able to produce small amounts of glucoraphanin in heterologous plant and microbial systems compared to the levels found in glucoraphanin-producing plants, suggesting that there may be missing auxiliary genes that play a role in improving production in planta. In an effort to identify auxiliary genes required for high glucoraphanin production, we leveraged transient expression in Nicotiana benthamiana to screen a combination of previously uncharacterized coexpressed genes and rationally selected genes alongside the glucoraphanin biosynthetic pathway. This strategy alleviated metabolic bottlenecks, which improved glucoraphanin production by 4.74-fold. Our optimized glucoraphanin biosynthetic pathway provides a pathway amenable for high glucoraphanin production.

The transcriptional effector domains of transcription factors play a key role in controlling gene expression; however, their functional nature is poorly understood, hampering our ability to explore this fundamental dimension of gene regulatory networks. To map the trans-regulatory landscape in a complex eukaryote, we systematically characterized the putative transcriptional effector domains of over 400 Arabidopsis thaliana transcription factors for their capacity to modulate transcription. We demonstrate that transcriptional effector activity can be integrated into gene regulatory networks capable of elucidating the functional dynamics underlying gene expression patterns. We further show how our characterized domains can enhance genome engineering efforts and reveal how plant transcriptional activators share regulatory features conserved across distantly related eukaryotes. Our results provide a framework to systematically characterize the regulatory role of transcription factors at a genome-scale in order to understand the transcriptional wiring of biological systems.

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.