Gavin J. Williams

Probing the Aglycon Promiscuity of an Engineered Glycosyltransferase

Sugars appended to pharmaceutically important natural products influence key pharmacological properties and/or molecular mechanism of action.[1] However, studies designed to systematically understand and/or exploit the role of carbohydrates in drug discovery are often limited by the availability of practical synthetic and/or biosynthetic tools.[2] Among the contemporary options to address this limitation,[3–4] chemoenzymatic glycorandomization utilizes a set of flexible enzymes consisting of an anomeric kinase, sugar-1-phosphate nucleotidylytransferase, and natural product glycosyltransferase (GT).[4–6] While chemoenzymatic glycorandomization has been successfully applied to alter the natural sugar moieties of numerous natural products,[4–8] the process remains primarily restricted by enzyme specificity and availability of suitable GTs for the target of interest. Thus, although there is precedent for improving non-glycosylated therapeutics via glycoconjugation, including colchicine,[9] mitomycin,[10] podophyllotoxin,[11] rapamycin,[12] isophosphoramide mustards,[13] or taxol,[14] such targets remain beyond chemoenzymatic strategies. Recent studies on OleD, the oleandomycin (1) GT from Streptomyces antibioticus (Scheme 1a), revealed an enhanced triple mutant (A242V/S132F/P67T, referred to herein as ‘ASP’) that displayed marked improvement in proficiency and substrate promiscuity.[4] To probe the synthetic utility of this enhanced catalyst and expand upon previous reports of acceptor promiscuity for wild-type (WT) OleD,[15] we report a comparison of the aglycon specificities of the WT and ‘ASP’ OleD variants toward 137 drug-like acceptors. This study highlights the ability of OleD variants to glucosylate a total of 71 diverse acceptors, catalyze iterative glycosylation with numerous substrates, and establishes OleD as the first multifunctional GT capable of generating O-, S- and N-glycosides.

Modifying the stereochemistry of an enzyme-catalyzed reaction by directed evolution

Aldolases have potential as tools for the synthesis of stereochemically complex carbohydrates. Here, we show that directed evolution can be used to alter the stereochemical course of the reaction catalyzed by tagatose-1,6-bisphosphate aldolase. After three rounds of DNA shuffling and screening, the evolved aldolase showed an 80-fold improvement in kcat/Km toward the non-natural substrate fructose 1,6-bisphosphate, resulting in a 100-fold change in stereospecificity. 31P NMR spectroscopy was used to show that, in the synthetic direction, the evolved aldolase catalyzes the formation of carbon—carbon bonds with unnatural diastereoselectivity, where the >99:<1 preference for the formation of tagatose 1,6-bisphosphate was switched to a 4:1 preference for the diastereoisomer, fructose 1,6-bisphosphate. This demonstration is of considerable significance to synthetic chemists requiring efficient syntheses of complex stereoisomeric products, such as carbohydrate mimetics.

The impact of enzyme engineering upon natural product glycodiversification

Glycodiversification of natural products is an effective strategy for small molecule drug development. Recently, improved methods for chemo-enzymatic synthesis of glycosyl donors has spurred the characterization of natural product glycosyltransferases (GTs), revealing that the substrate specificity of many naturally occurring GTs as too stringent for use in glycodiversification. Protein engineering of natural product GTs has emerged as an attractive approach to overcome this limitation. This review highlights recent progress in the engineering/evolution of enzymes relevant to natural product glycodiversification with a particular focus upon GTs.

Directed evolution of enzymes for biocatalysis and the life sciences

Abstract.Engineering the specificity and properties of enzymes and proteins within rapid time frames has become feasible with the advent of directed evolution. In the absence of detailed structural and mechanistic information, new functions can be engineered by introducing and recombining mutations, followed by subsequent testing of each variant for the desired new function. A range of methods are available for mutagenesis, and these can be used to introduce mutations at single sites, targeted regions within a gene or randomly throughout the entire gene. In addition, a number of different methods are available to allow recombination of point mutations or blocks of sequence space with little or no homology. Currently, enzyme engineers are still learning which combinations of selection methods and techniques for mutagenesis and DNA recombination are most efficient. Moreover, deciding where to introduce mutations or where to allow recombination is actively being investigated by combining experimental and computational methods. These techniques are already being successfully used for the creation of novel proteins for biocatalysis and the life sciences.

Poly Specific trans-Acyltransferase Machinery Revealed via Engineered Acyl-CoA Synthetases

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.

Engineering polyketide synthases and nonribosomal peptide synthetases

Naturally occurring polyketides and nonribosomal peptides with broad and potent biological activities continue to inspire the discovery of new and improved analogs. The biosynthetic apparatus responsible for the construction of these natural products has been the target of intensive protein engineering efforts. Traditionally, engineering has focused on substituting individual enzymatic domains or entire modules with those of different building block specificity, or by deleting various enzymatic functions, in an attempt to generate analogs. This review highlights strategies based on site-directed mutagenesis of substrate binding pockets, semi-rational mutagenesis, and whole-gene random mutagenesis to engineer the substrate specificity, activity, and protein interactions of polyketide and nonribosomal peptide biosynthetic machinery.

A high-throughput fluorescence-based glycosyltransferase screen and its application in directed evolution

This protocol details the application of a high-throughput fluorescence-based screen, in conjunction with error-prone PCR/saturation mutagenesis, for altering the proficiency and/or promiscuity of a secondary metabolite glycosyltransferase (GT) via directed evolution. Given the structural and mechanistic similarities among secondary metabolite-associated GTs, this approach may provide a template for engineering other members of the GT-B superfamily.

Mutant Malonyl-CoA Synthetases with Altered Specificity for Polyketide Synthase Extender Unit Generation

Tailoring guide: We have used structure-guided saturation mutagenesis followed by colorimetric screening to identify mutant malonyl-CoA synthetases with altered substrate specificity. One particular mutant displayed a 240-fold shift in specificity (see graphic). These mutant enzymes will be useful tools for providing extender units to probe the activity of polyketide synthases.

Reprogramming acyl carrier protein interactions of an acyl-CoA promiscuous trans-acyltransferase

Protein interactions between acyl carrier proteins (ACPs) and trans-acting acyltransferase domains (trans-ATs) are critical for regioselective extender unit installation by many polyketide synthases, yet little is known regarding the specificity of these interactions, particularly for trans-ATs with unusual extender unit specificities. Currently, the best-studied trans-AT with nonmalonyl specificity is KirCII from kirromycin biosynthesis. Here, we developed an assay to probe ACP interactions based on leveraging the extender unit promiscuity of KirCII. The assay allows us to identify residues on the ACP surface that contribute to specific recognition by KirCII. This information proved sufficient to modify a noncognate ACP from a different biosynthetic system to be a substrate for KirCII. The findings form a foundation for further understanding the specificity of trans-AT:ACP protein interactions and for engineering modular polyketide synthases to produce analogs.

Inversion of Extender Unit Selectivity in the Erythromycin Polyketide Synthase by Acyltransferase Domain Engineering

Acyltransferase (AT) domains of polyketide synthases (PKSs) select extender units for incorporation into polyketides and dictate large portions of the structures of clinically relevant natural products. Accordingly, there is significant interest in engineering the substrate specificity of PKS ATs in order to site-selectively manipulate polyketide structure. However, previous attempts to engineer ATs have yielded mutant PKSs with relaxed extender unit specificity, rather than an inversion of selectivity from one substrate to another. Here, by directly screening the extender unit selectivity of mutants from active site saturation libraries of an AT from the prototypical PKS, 6-deoxyerythronolide B synthase, a set of single amino acid substitutions was discovered that dramatically impact the selectivity of the PKS with only modest reductions of product yields. One particular substitution (Tyr189Arg) inverted the selectivity of the wild-type PKS from its natural substrate toward a non-natural alkynyl-modified extender unit while maintaining more than twice the activity of the wild-type PKS with its natural substrate. The strategy and mutations described herein form a platform for combinatorial biosynthesis of site-selectively modified polyketide analogues that are modified with non-natural and non-native chemical functionality.