This article highlights previous and prospective uses for plant glycosyltransferases in generating and diversifying bioactive glycosides, with implications in drug discovery applications.

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.

Agrobacterium tumefaciens is an important tool in plant biotechnology due to its natural ability to transfer DNA into the genomes of host plants. Genetic manipulations of A. tumefaciens have yielded considerable advances in increasing transformational efficiency in a number of plant species and cultivars. Moreover, there is overwhelming evidence that modulating the expression of various mediators of A. tumefaciens virulence can lead to more successful plant transformation; thus, the application of synthetic biology to enable targeted engineering of the bacterium may enable new opportunities for advancing plant biotechnology. In this review, we highlight engineering targets in both A. tumefaciens and plant hosts that could be exploited more effectively through precision genetic control to generate high-quality transformation events in a wider range of host plants. We then further discuss the current state of A. tumefaciens and plant engineering with regard to plant transformation and describe how future work may incorporate a rigorous synthetic biology approach to tailor strains of A. tumefaciens used in plant transformation.

Plants offer a vast source of bioactive chemicals with the potential to improve human health through the prevention and treatment of disease. However, many potential therapeutics are produced in small amounts or in species that are difficult to cultivate. The rapidly evolving field of plant synthetic biology provides tools to capitalize on the inventive chemistry of plants by transferring metabolic pathways for therapeutics into far more tenable plants, increasing our ability to produce complex pharmaceuticals in well-studied plant systems. Plant synthetic biology also provides methods to enhance the ability to fortify crops with nutrients and nutraceuticals. In this review, we discuss (1) the potential of plant synthetic biology to improve human health by generating plants that produce pharmaceuticals, nutrients, and nutraceuticals and (2) the technological challenges hindering our ability to generate plants producing health-promoting small molecules.

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.

It is estimated that 38.4% of adults in the United States will be diagnosed with cancer (National Cancer Institute). With this high rate of incidence, the exploration of new options for cancer prevention and treatment is of high importance. Plant secondary metabolites are strong candidates for study due to their potential to be used in anti‐cancer dietary supplements. Isothiocyanates are products of the glucoraphanin biosynthesis pathway that have been demonstrated to display potent anti‐cancer and anti‐inflammatory properties. Previous studies have identified the genes in the core glucoraphanin biosynthesis pathway (CGBP); however, heterologous expression of the CGBP in tobacco has produced glucoraphanin at only 0.35 μmol g‐1 dry weight (Mikkelsen et al. 2010). In order to increase our titer, we used co‐expression analysis to identify a total of forty‐two gene candidates for optimizing the CGBP. We tested these candidates for their effect on glucoraphanin biosynthesis by transiently expressing the CGBP with candidate genes in tobacco. LC‐MS analysis of the transformed leaf tissue for glucoraphanin content revealed that six of the forty‐two genes increased glucoraphanin production. We also found several genes that produced significantly less glucoraphanin per gram dry weight when combined with the CGBP than the CGBP alone. The increases in heterologous glucoraphanin production implicate the six genes as important factors in the glucoraphanin biosynthetic pathway with the potential to raise concentrations closer to recommended supplement quantities. Support or Funding Information This project is funded by NIH Grant R00AT009573