Natural products, many of which are decorated with essential sugar residues, continue to serve as a key platform for drug development. Adding or changing sugars attached to such natural products can improve the parent compound's pharmacological properties, specificity at multiple levels, and/or even the molecular mechanism of action. Though some natural-product glycosyltransferases (GTs) are sufficiently promiscuous for use in altering these glycosylation patterns, the stringent specificity of others remains a limiting factor in natural-product diversification and highlights a need for general GT engineering and evolution platforms. Herein we report the use of a simple high-throughput screen based on a fluorescent surrogate acceptor substrate to expand the promiscuity of a natural-product GT via directed evolution. Cumulatively, this study presents variant GTs for the glycorandomization of a range of therapeutically important acceptors, including aminocoumarins, flavonoids and macrolides, and a potential template for engineering other natural-product GTs.

Polyketide synthases construct polyketides with diverse structures and biological activities via the condensation of extender units and acyl thioesters. Although a growing body of evidence suggests that polyketide synthases might be tolerant to non-natural extender units, in vitro and in vivo studies aimed at probing and utilizing polyketide synthase specificity are severely limited to only a small number of extender units, owing to the lack of synthetic routes to a broad variety of acyl-CoA extender units. Here, we report the construction of promiscuous malonyl-CoA synthetase variants that can be used to synthesize a broad range of malonyl-CoA extender units substituted at the C2-position, several of which contain handles for chemoselective ligation and are not found in natural biosynthetic systems. We highlighted utility of these enzymes by probing the acyl-CoA specificity of several trans-acyltransferases, leading to the unprecedented discovery of poly specificity toward non-natural extender units, several of which are not found in naturally occurring biosynthetic pathways. These results reveal that polyketide biosynthetic machinery might be more tolerant to non-natural substrates than previously established, and that mutant synthetases are valuable tools for probing the specificity of biosynthetic machinery. Our data suggest new synthetic biology strategies for harnessing this promiscuity and enabling the regioselective modification of polyketides.

A sweet library: Two variants (wild‐type (WT) and a triple mutant) of glycosyltransferase (GT) OleD have been shown to catalyze glycosylation of over 70 substrates, formation of O‐, S‐ and N‐glycosidic bonds, and iterative glycosylation (see scheme). Identified substrates include nucleophiles not previously known to act in GT reactions and span numerous natural product and therapeutic drug classes.

A comprehensive two-phase "hot spot" saturation mutagenesis strategy for the rapid evolution of glycosyltransferase (GT) specificity for nonnatural acceptors is described. Specifically, the application of a high-throughput screen (based on the fluorescent acceptor umbelliferone) was used to identify key amino acid hot spots that contribute to GT proficiency and/or promiscuity. Saturation mutagenesis of the corresponding hot spots facilitated the utilization of a lower-throughput screen to provide OleD prodigy capable of efficiently glycosylating the nonnatural acceptor novobiocic acid with an array of unique sugars. Incredibly, even in the absence of a high-throughput screen for novobiocic acid glycosylation, this approach rapidly led to improvements in the desired catalytic activity of several hundred-fold.

Macrolides are a large group of natural products that display broad and potent biological activities and are biosynthesized by type I polyketide synthases (PKSs) and associated enzymatic machinery. There is an urgent need to access macrolides and unnatural macrolide derivatives for drug discovery, drug manufacture, and probe development. Typically, efforts to engineer the biosynthesis of macrolides and macrolide analogues in various microbial hosts are hampered by the complexity of macrolide biosynthetic pathways and our limited ability to rationally reprogram type I PKSs and post-PKS machinery. High-throughput approaches based on synthetic biology and directed evolution could overcome this problem by testing the function of large libraries of variants. Yet, methods that can identify mutant enzymes, pathways, and strains that produce the desired macrolide target are not generally available. Here we show that the promiscuous macrolide sensing transcription factor MphR is a powerful platform for engineering variants with tailored properties. We identified variants that displayed improved sensitivity toward erythromycin, tailored the inducer specificity, and significantly improved sensitivity to macrolides that were very poor inducers of the wild-type MphR biosensor. Designer macrolide biosensors should find broad utility and enable applications related to high-throughput synthetic biology and directed evolution of macrolide biosynthesis.