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Plants have expanded various biosynthetic enzyme families to produce a wide diversity of natural products; however, most enzymes encoded in plant genomes remain uncharacterized, highlighting the need for new functional genomic approaches. Here, we report a platform enabling the rapid functional characterization of plant family 1 glycosyltransferases, which serve important roles in plant development, defense, and communication. Using substrate-multiplexed reactions, mass spectrometry, and automated analysis, we screen 85 enzymes against a diverse library of 453 natural products, for a total of nearly 40,000 possible reactions. The resulting dataset reveals a widespread promiscuity and a strong preference for planar, hydroxylated aromatic substrates among family 1 glycosyltransferases. We also characterize glycosyltransferases with an unusually wide substrate scope and with a non-canonical Cys-Asp catalytic dyad. This work establishes a widely-applicable enzymatic screening pipeline, reflects the immense glycosylation capability of plants, and has implications in biocatalysis, metabolic engineering, and gene discovery. Most enzymes involved in plant natural products biosynthesis remain uncharacterized. Here, the authors establish a screening pipeline and apply it to family 1 glycosyltransferases to reveal their promiscuity and preference for planar, hydroxylated aromatic substrates.

Due to the rampant rise in obesity and diabetes, consumers are desperately seeking for ways to reduce their sugar intake, but to date there are no options that are both accessible and without sacrifice of palatability. One of the most promising new ingredients in the food system as a non-nutritive sugar substitute with near perfect palatability is D-psicose. D-psicose is currently produced using an in vitro enzymatic isomerization of D-fructose, resulting in low yield and purity, and therefore requiring substantial downstream processing to obtain a high purity product. This has made adoption of D-psicose into products limited and results in significantly higher per unit costs, reducing accessibility to those most in need. Here, we found that Escherichia coli natively possesses a thermodynamically favorable pathway to produce D-psicose from D-glucose through a series of phosphorylation-epimerization-dephosphorylation steps. To increase carbon flux towards D-psicose production, we introduced a series of genetic modifications to pathway enzymes, central carbon metabolism, and competing metabolic pathways. In an attempt to maximize both cellular viability and D-psicose production, we implemented methods for the dynamic regulation of key genes including clustered regularly interspaced short palindromic repeats inhibition (CRISPRi) and stationary-phase promoters. The engineered strains achieved complete consumption of D-glucose and production of D-psicose, at a titer of 15.3 g L -1 , productivity of 2 g L -1 h -1 , and yield of 62% under test tube conditions. These results demonstrate the viability of whole-cell catalysis as a sustainable alternative to in vitro enzymatic synthesis for the accessible production of D-psicose.