Development of a suite of activatable plant synthetic promoter systems using a bacterial LysR-type transcriptional regulator FdeR

Advancing plant synthetic biology requires an abundant supply of orthogonal and tunable genetic parts to express multiple genes simultaneously. Current genetic parts, particularly promoters, used in plants are still limited and often suffer from tissue specificity and endogenous regulation <i>in planta</i>. Synthetic promoter systems that combine engineered plant-compatible transcription factors (TFs) with synthetic promoters provide a promising alternative approach to enrich current plant synthetic biology toolkits. Leveraging the feature that LysR-type TFs usually bind to the operators regardless of the presence or absence of ligands, we present a systematic approach to develop and characterize a large suite of synthetic promoter systems based on a single LysR-type bacterial TF. Using FdeR from the soil bacterium <i>Herbaspirillum seropedicae</i> and its corresponding operator, <i>fdeO</i>, we developed 52 synthetic promoter systems regulated by four FdeR-derived synthetic activators. Transient expression assays in <i>Nicotiana benthamiana</i> showed that the synthetic promoters were constitutively activated by synthetic activators in a ligand-independent manner, and the resulting promoters spanned a wide range of expression activities, with the lowest equivalent to minimal CaMV 35S promoter and the highest with ∼65% of CaMV 35S activity. We also developed modular workflows and pipelines to accelerate the development of the synthetic transcription platform, including a MoClo-based plug-and-play assembly system for plant-compatible promoter construction and a yeast-based prescreening platform for rapid TF evaluation in eukaryotic cells. This work has demonstrated a scalable framework for developing a large set of synthetic promoter systems from minimal genetic components, leveraging bacterial TF diversity to expand the plant synthetic biology toolkit for robust, orthogonal gene expression.