Toward a monocot SynBio toolkit: assessing regulatory element performance and eudicot compatibility

Synthetic biology, monocots, transgene expression, genetic regulatory elements, protoplasts, fluorometry. Given the preeminent status of monocotyledonous species in global agriculture, efforts toward enhancing their yield, resilience, and quality with synthetic biology (SynBio) approaches are warranted. We therefore sought to assemble and characterize a toolkit of genetic regulatory elements to address the relative paucity of components appropriate for genetic engineering in cereals and grasses. Here, we present an 80-member combinatorial parts library consisting of 17 promoter, 17 5' untranslated region (UTR), 18 promoter-5'UTR fusion, and 28 3'UTR sequences derived from viral, microbial, and plant sources. Evaluation of the effects of 77 combinations of these elements on GFP reporter expression in Zea mays protoplasts revealed a 74.5-fold dynamic range of expression levels. Novel combinations of the CaMV 35S promoter and viral 5'UTR sequences generated reporter expression levels that approximated the strong CsVMV viral promoter-5'UTR fusion. Newly identified 3'UTR sequences from plant small heat shock protein (HSP) species regulated impressive enhancements of reporter expression that exceeded the widely used 35S, ocs, and nos terminators. To assess cross-clade compatibility of the regulatory element combinations, reporter cassettes were also transiently assayed in Nicotiana benthamiana leaves using agroinfiltration. A total of 34 regulatory element combinations exhibited interspecific activity, 14 of which displayed similar impacts on reporter gene expression across the two species. This toolkit will enable tunable transgene expression in monocots suitable for myriad SynBio applications including genome editing, manipulation of metabolic pathways, and genetic circuit construction.