@article{paulsel_williams_2023, title={Current State-of-the-Art Toward Chemoenzymatic Synthesis of Polyketide Natural Products}, volume={9}, ISSN={["1439-7633"]}, url={https://doi.org/10.1002/cbic.202300386}, DOI={10.1002/cbic.202300386}, abstractNote={Abstract}, journal={CHEMBIOCHEM}, author={Paulsel, Thaddeus Q. and Williams, Gavin J.}, year={2023}, month={Sep} }
 @article{welch_cossin_paulsel_williams_2022, title={Targeted enzyme modifications enable regioselective biosynthesis of fluorinated polyketides}, volume={2}, ISSN={["2667-1093"]}, DOI={10.1016/j.checat.2022.09.042}, abstractNote={In attempts to enhance natural products as therapeutic agents, fluorination has emerged as a new tool for synthetic biologists and chemists. In recent articles published in Nature Chemistry and Nature Chemical Biology, the Grininger and Chang groups leveraged their expertise in engineering polyketide biosynthesis to incorporate fluorine into polyketide scaffolds.}, number={10}, journal={CHEM CATALYSIS}, author={Welch, Sydney D. and Cossin, Jared and Paulsel, Thaddeus Q. and Williams, Gavin J.}, year={2022}, month={Oct}, pages={2440–2443} }
 @article{kalkreuter_bingham_keeler_lowell_schmidt_sherman_williams_2021, title={Computationally-guided exchange of substrate selectivity motifs in a modular polyketide synthase acyltransferase}, volume={12}, ISSN={["2041-1723"]}, url={https://doi.org/10.1038/s41467-021-22497-2}, DOI={10.1038/s41467-021-22497-2}, abstractNote={Abstract}, number={1}, journal={NATURE COMMUNICATIONS}, publisher={Springer Science and Business Media LLC}, author={Kalkreuter, Edward and Bingham, Kyle S. and Keeler, Aaron M. and Lowell, Andrew N. and Schmidt, Jennifer J. and Sherman, David H. and Williams, Gavin J.}, year={2021}, month={Apr} }
 @article{li_reed_wright_cropp_williams_2021, title={Development of Genetically Encoded Biosensors for Reporting the Methyltransferase-Dependent Biosynthesis of Semisynthetic Macrolide Antibiotics}, volume={10}, ISSN={["2161-5063"]}, url={https://doi.org/10.1021/acssynbio.1c00151}, DOI={10.1021/acssynbio.1c00151}, abstractNote={Clarithromycin is an improved semisynthetic analogue of the naturally occurring macrolide, erythromycin. The subtle modification of a methyl group on the C-6 hydroxyl group endows the molecule with improved acid stability and results in a clinically useful antibiotic. Here, we show that the effector specificity of the biosensor protein, MphR, can be evolved to selectively recognize clarithromycin and therefore report on the production of this molecule in vivo. In addition, a crystal structure of the evolved variant reveals the molecular basis for selectivity and provides a guide for the evolution of a new metabolic function using this biosensor.}, number={10}, journal={ACS SYNTHETIC BIOLOGY}, publisher={American Chemical Society (ACS)}, author={Li, Yiwei and Reed, Megan and Wright, H. Tonie and Cropp, T. Ashton and Williams, Gavin J.}, year={2021}, month={Oct}, pages={2520–2531} }
 @misc{calzini_malico_mitchler_williams_2021, title={Protein engineering for natural product biosynthesis and synthetic biology applications}, volume={34}, ISSN={["1741-0134"]}, DOI={10.1093/protein/gzab015}, abstractNote={Abstract}, journal={PROTEIN ENGINEERING DESIGN & SELECTION}, author={Calzini, Miles A. and Malico, Alexandra A. and Mitchler, Melissa M. and Williams, Gavin J.}, year={2021}, month={Jun} }
 @misc{mitchler_garcia_montero_williams_2021, title={Transcription factor-based biosensors: a molecular-guided approach for natural product engineering}, volume={69}, ISSN={["1879-0429"]}, DOI={10.1016/j.copbio.2021.01.008}, abstractNote={Natural products and their derivatives offer a rich source of chemical and biological diversity; however, traditional engineering of their biosynthetic pathways to improve yields and access to unnatural derivatives requires a precise understanding of their enzymatic processes. High-throughput screening platforms based on allosteric transcription-factor based biosensors can be leveraged to overcome the screening bottleneck to enable searching through large libraries of pathway/strain variants. Herein, the development and application of engineered allosteric transcription factor-based biosensors is described that enable optimization of precursor availability, product titers, and downstream product tailoring for advancing the natural product bioeconomy. We discuss recent successes for tailoring biosensor design, including computationally-based approaches, and present our future outlook with the integration of cell-free technologies and de novo protein design for rapidly generating biosensor tools.}, journal={CURRENT OPINION IN BIOTECHNOLOGY}, author={Mitchler, Melissa M. and Garcia, Jessie M. and Montero, Nichole E. and Williams, Gavin J.}, year={2021}, month={Jun}, pages={172–181} }
 @article{gayen_nichols_williams_2020, title={An artificial pathway for polyketide biosynthesis}, volume={3}, ISSN={2520-1158}, url={http://dx.doi.org/10.1038/s41929-020-0483-4}, DOI={10.1038/s41929-020-0483-4}, number={7}, journal={Nature Catalysis}, publisher={Springer Science and Business Media LLC}, author={Gayen, Anuran K. and Nichols, Lindsay and Williams, Gavin J.}, year={2020}, month={Jul}, pages={536–538} }
 @article{zin_williams_ekins_2020, title={Cheminformatics Analysis and Modeling with MacrolactoneDB}, volume={10}, ISSN={2045-2322}, url={http://dx.doi.org/10.1038/s41598-020-63192-4}, DOI={10.1038/s41598-020-63192-4}, abstractNote={Abstract}, number={1}, journal={Scientific Reports}, publisher={Springer Science and Business Media LLC}, author={Zin, Phyo Phyo Kyaw and Williams, Gavin J. and Ekins, Sean}, year={2020}, month={Apr} }
 @article{kalkreuter_bingham_keeler_lowell_schmidt_sherman_williams_2020, title={Computationally-guided exchange of substrate selectivity motifs in a modular polyketide synthase acyltransferase}, volume={4}, url={https://doi.org/10.1101/2020.04.23.058214}, DOI={10.1101/2020.04.23.058214}, abstractNote={ABSTRACT}, publisher={Cold Spring Harbor Laboratory}, author={Kalkreuter, Edward and Bingham, Kyle S and Keeler, Aaron M and Lowell, Andrew N and Schmidt, Jennifer J. and Sherman, David H and Williams, Gavin J}, year={2020}, month={Apr} }
 @article{zin_williams_fourches_2020, title={SIME: synthetic insight-based macrolide enumerator to generate the V1B library of 1 billion macrolides}, volume={12}, ISSN={1758-2946}, url={http://dx.doi.org/10.1186/s13321-020-00427-6}, DOI={10.1186/s13321-020-00427-6}, abstractNote={Abstract}, number={1}, journal={Journal of Cheminformatics}, publisher={Springer Science and Business Media LLC}, author={Zin, Phyo Phyo Kyaw and Williams, Gavin and Fourches, Denis}, year={2020}, month={Apr} }
 @article{malico_nichols_williams_2020, title={Synthetic biology enabling access to designer polyketides}, volume={58}, ISSN={1367-5931}, url={http://dx.doi.org/10.1016/j.cbpa.2020.06.003}, DOI={10.1016/j.cbpa.2020.06.003}, abstractNote={The full potential of polyketide discovery has yet to be reached owing to a lack of suitable technologies and knowledge required to advance engineering of polyketide biosynthesis. Recent investigations on the discovery, enhancement, and non-natural use of these biosynthetic gene clusters via computational biology, metabolic engineering, structural biology, and enzymology-guided approaches have facilitated improved access to designer polyketides. Here, we discuss recent successes in gene cluster discovery, host strain engineering, precursor-directed biosynthesis, combinatorial biosynthesis, polyketide tailoring, and high-throughput synthetic biology, as well as challenges and outlooks for rapidly generating useful target polyketides.}, journal={Current Opinion in Chemical Biology}, publisher={Elsevier BV}, author={Malico, Alexandra A. and Nichols, Lindsay and Williams, Gavin J.}, year={2020}, month={Oct}, pages={45–53} }
 @article{malico_calzini_gayen_williams_2020, title={Synthetic biology, combinatorial biosynthesis, and chemo-enzymatic synthesis of isoprenoids (September, 10.1007/s10295-020-02306-3, 2020)}, volume={47}, ISSN={["1476-5535"]}, url={https://doi.org/10.1007/s10295-020-02327-y}, DOI={10.1007/s10295-020-02327-y}, abstractNote={Abstract}, number={12}, journal={JOURNAL OF INDUSTRIAL MICROBIOLOGY & BIOTECHNOLOGY}, publisher={Springer Science and Business Media LLC}, author={Malico, Alexandra A. and Calzini, Miles A. and Gayen, Anuran K. and Williams, Gavin J.}, year={2020}, month={Dec}, pages={1181–1181} }
 @article{malico_calzini_gayen_williams_2020, title={Synthetic biology, combinatorial biosynthesis, and chemo‑enzymatic synthesis of isoprenoids}, volume={47}, ISSN={1367-5435 1476-5535}, url={http://dx.doi.org/10.1007/s10295-020-02306-3}, DOI={10.1007/s10295-020-02306-3}, abstractNote={Abstract}, number={9-10}, journal={Journal of Industrial Microbiology & Biotechnology}, publisher={Springer Science and Business Media LLC}, author={Malico, Alexandra A. and Calzini, Miles A. and Gayen, Anuran K. and Williams, Gavin J.}, year={2020}, month={Sep}, pages={675–702} }
 @article{lund_hall_williams_2019, title={An Artificial Pathway for Isoprenoid Biosynthesis Decoupled from Native Hemiterpene Metabolism}, volume={8}, ISSN={2161-5063 2161-5063}, url={http://dx.doi.org/10.1021/acssynbio.8b00383}, DOI={10.1021/acssynbio.8b00383}, abstractNote={Isoprenoids are constructed in nature using hemiterpene building blocks that are biosynthesized from lengthy enzymatic pathways with little opportunity to deploy precursor-directed biosynthesis. Here, an artificial alcohol-dependent hemiterpene biosynthetic pathway was designed and coupled to several isoprenoid biosynthetic systems, affording lycopene and a prenylated tryptophan in robust yields. This approach affords a potential route to diverse non-natural hemiterpenes and by extension isoprenoids modified with non-natural chemical functionality. Accordingly, the prototype chemo-enzymatic pathway is a critical first step toward the construction of engineered microbial strains for bioconversion of simple scalable building blocks into complex isoprenoid scaffolds.}, number={2}, journal={ACS Synthetic Biology}, publisher={American Chemical Society (ACS)}, author={Lund, Sean and Hall, Rachael and Williams, Gavin J}, year={2019}, month={Jan}, pages={232–238} }
 @article{lund_courtney_williams_2019, title={Cover Feature: Probing the Substrate Promiscuity of Isopentenyl Phosphate Kinase as a Platform for Hemiterpene Analogue Production (ChemBioChem 17/2019)}, volume={8}, url={https://doi.org/10.1002/cbic.201900494}, DOI={10.1002/cbic.201900494}, abstractNote={The cover feature picture shows the substrate promiscuity of isopentenyl monophosphate kinase—a key enzyme in a recently described artificial metabolic pathway for hemiterpene biosynthesis. The remarkable broad substrate tolerance of this enzyme sets the stage to produce unnatural isoprenoids by coupling the activity of the kinase to downstream isoprenoid biosynthesis. More information can be found in the communication by G. J. Williams et al. on page 2217 in Issue 17, 2019 (DOI: 10.1002/cbic.201900135).}, journal={ChemBioChem}, publisher={Wiley}, author={Lund, Sean and Courtney, Taylor and Williams, Gavin J.}, year={2019}, month={Sep} }
 @article{kalkreuter_keeler_malico_bingham_gayen_williams_2019, title={Development of a Genetically Encoded Biosensor for Detection of Polyketide Synthase Extender Units in Escherichia coli}, volume={8}, ISSN={2161-5063 2161-5063}, url={http://dx.doi.org/10.1021/acssynbio.9b00078}, DOI={10.1021/acssynbio.9b00078}, abstractNote={The scaffolds of polyketides are constructed via assembly of extender units based on malonyl-CoA and its derivatives that are substituted at the C2-position with diverse chemical functionality. Subsequently, a transcription-factor-based biosensor for malonyl-CoA has proven to be a powerful tool for detecting malonyl-CoA, facilitating the dynamic regulation of malonyl-CoA biosynthesis and guiding high-throughput engineering of malonyl-CoA-dependent processes. Yet, a biosensor for the detection of malonyl-CoA derivatives has yet to be reported, severely restricting the application of high-throughput synthetic biology approaches to engineering extender unit biosynthesis and limiting the ability to dynamically regulate the biosynthesis of polyketide products that are dependent on such α-carboxyacyl-CoAs. Herein, the FapR biosensor was re-engineered and optimized for a range of mCoA concentrations across a panel of E. coli strains. The effector specificity of FapR was probed by cell-free transcription-translation, revealing that a variety of non-native and non-natural acyl-thioesters are FapR effectors. This FapR promiscuity proved sufficient for the detection of the polyketide extender unit methylmalonyl-CoA in E. coli, providing the first reported genetically encoded biosensor for this important metabolite. As such, the previously unknown broad effector promiscuity of FapR provides a platform to develop new tools and approaches that can be leveraged to overcome limitations of pathways that construct diverse α-carboxyacyl-CoAs and those that are dependent on them, including biofuels, antibiotics, anticancer drugs, and other value-added products.}, number={6}, journal={ACS Synthetic Biology}, publisher={American Chemical Society (ACS)}, author={Kalkreuter, Edward and Keeler, Aaron M. and Malico, Alexandra A. and Bingham, Kyle S. and Gayen, Anuran K. and Williams, Gavin J.}, year={2019}, month={May}, pages={1391–1400} }
 @article{kalkreuter_crowetipton_lowell_sherman_williams_2019, title={Engineering the Substrate Specificity of a Modular Polyketide Synthase for Installation of Consecutive Non-Natural Extender Units}, volume={141}, ISSN={0002-7863 1520-5126}, url={http://dx.doi.org/10.1021/jacs.8b10521}, DOI={10.1021/jacs.8b10521}, abstractNote={There is significant interest in diversifying the structures of polyketides to create new analogues of these bioactive molecules. This has traditionally been done by focusing on engineering the acyltransferase (AT) domains of polyketide synthases (PKSs) responsible for the incorporation of malonyl-CoA extender units. Non-natural extender units have been utilized by engineered PKSs previously; however, most of the work to date has been accomplished with ATs that are either naturally promiscuous and/or located in terminal modules lacking downstream bottlenecks. These limitations have prevented the engineering of ATs with low native promiscuity and the study of any potential gatekeeping effects by domains downstream of an engineered AT. In an effort to address this gap in PKS engineering knowledge, the substrate preferences of the final two modules of the pikromycin PKS were compared for several non-natural extender units and through active site mutagenesis. This led to engineering of the methylmalonyl-CoA specificity of both modules and inversion of their selectivity to prefer consecutive non-natural derivatives. Analysis of the product distributions of these bimodular reactions revealed unexpected metabolites resulting from gatekeeping by the downstream ketoreductase and ketosynthase domains. Despite these new bottlenecks, AT engineering provided the first full-length polyketide products incorporating two non-natural extender units. Together, this combination of tandem AT engineering and the identification of previously poorly characterized bottlenecks provides a platform for future advancements in the field.}, number={5}, journal={Journal of the American Chemical Society}, publisher={American Chemical Society (ACS)}, author={Kalkreuter, Edward and CroweTipton, Jared M. and Lowell, Andrew N. and Sherman, David H. and Williams, Gavin J.}, year={2019}, month={Jan}, pages={1961–1969} }
 @article{lund_courtney_williams_2019, title={Probing the Substrate Promiscuity of Isopentenyl Phosphate Kinase as a Platform for Hemiterpene Analogue Production}, volume={20}, ISSN={1439-4227 1439-7633}, url={http://dx.doi.org/10.1002/cbic.201900135}, DOI={10.1002/cbic.201900135}, abstractNote={Abstract}, number={17}, journal={ChemBioChem}, publisher={Wiley}, author={Lund, Sean and Courtney, Taylor and Williams, Gavin J.}, year={2019}, month={Jul}, pages={2217–2221} }
 @article{lund_courtney_williams_2019, title={Probing the Substrate Promiscuity of Isopentenyl Phosphate Kinase as a Platform for Hemiterpene Analogue Production}, volume={2}, url={https://doi.org/10.26434/chemrxiv.7765241.v1}, DOI={10.26434/chemrxiv.7765241.v1}, abstractNote={Isoprenoids are a large class of natural products with wide-ranging applications. Synthetic biology approaches to the manufacture of isoprenoids and their new-to-nature derivatives are limited due to the provision in Nature of just two hemiterpene building blocks for isoprenoid biosynthesis. To address this limitation, artificial chemo-enzymatic pathways such as the alcohol-dependent hemiterpene pathway (ADH) serve to leverage consecutive kinases to convert exogenous alcohols to pyrophosphates that could be coupled to downstream isoprenoid biosynthesis. To be successful, each kinase in this pathway should be permissive of a broad range of substrates. For the first time, we have probed the promiscuity of the second enzyme in the ADH pathway, isopentenyl phosphate kinase from Thermoplasma acidophilum, towards a broad range of acceptor monophosphates. Subsequently, we evaluate the suitability of this enzyme to provide non-natural pyrophosphates and provide a critical first step in characterizing the rate limiting steps in the artificial ADH pathway.}, publisher={American Chemical Society (ACS)}, author={Lund, Sean and Courtney, Taylor and Williams, Gavin}, year={2019}, month={Feb} }
 @article{zin_williams_fourches_2018, title={Cheminformatics-based enumeration and analysis of large libraries of macrolide scaffolds}, volume={10}, ISSN={1758-2946}, url={http://dx.doi.org/10.1186/s13321-018-0307-6}, DOI={10.1186/s13321-018-0307-6}, abstractNote={We report on the development of a cheminformatics enumeration technology and the analysis of a resulting large dataset of virtual macrolide scaffolds. Although macrolides have been shown to have valuable biological properties, there is no ready-to-screen virtual library of diverse macrolides in the public domain. Conducting molecular modeling (especially virtual screening) of these complex molecules is highly relevant as the organic synthesis of these compounds, when feasible, typically requires many synthetic steps, and thus dramatically slows the discovery of new bioactive macrolides. Herein, we introduce a cheminformatics approach and associated software that allows for designing and generating libraries of virtual macrocycle/macrolide scaffolds with user-defined constitutional and structural constraints (e.g., types and numbers of structural motifs to be included in the macrocycle, ring size, maximum number of compounds generated). To study the chemical diversity of such generated molecules, we enumerated V1M (Virtual 1 million Macrolide scaffolds) library, each containing twelve common structural motifs. For each macrolide scaffold, we calculated several key properties, such as molecular weight, hydrogen bond donors/acceptors, topological polar surface area. In this study, we discuss (1) the initial concept and current features of our PKS (polyketides) Enumerator software, (2) the chemical diversity and distribution of structural motifs in V1M library, and (3) the unique opportunities for future virtual screening of such enumerated ensembles of macrolides. Importantly, V1M is provided in the Supplementary Material of this paper allowing other researchers to conduct any type of molecular modeling and virtual screening studies. Therefore, this technology for enumerating extremely large libraries of macrolide scaffolds could hold a unique potential in the field of computational chemistry and drug discovery for rational designing of new antibiotics and anti-cancer agents.}, number={1}, journal={Journal of Cheminformatics}, publisher={Springer Science and Business Media LLC}, author={Zin, Phyo Phyo Kyaw and Williams, Gavin and Fourches, Denis}, year={2018}, month={Nov} }
 @article{kasey_zerrad_li_cropp_williams_2018, title={Development of Transcription Factor-Based Designer Macrolide Biosensors for Metabolic Engineering and Synthetic Biology}, volume={7}, ISSN={2161-5063 2161-5063}, url={http://dx.doi.org/10.1021/ACSSYNBIO.7B00287}, DOI={10.1021/ACSSYNBIO.7B00287}, abstractNote={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.}, number={1}, journal={ACS Synthetic Biology}, publisher={American Chemical Society (ACS)}, author={Kasey, Christian M. and Zerrad, Mounir and Li, Yiwei and Cropp, T. Ashton and Williams, Gavin J.}, year={2018}, month={Jan}, pages={227–239} }
 @article{kalkreuter_williams_2018, title={Engineering enzymatic assembly lines for the production of new antimicrobials}, volume={45}, ISSN={1369-5274}, url={http://dx.doi.org/10.1016/j.mib.2018.04.005}, DOI={10.1016/j.mib.2018.04.005}, abstractNote={A large portion of natural products are biosynthesized by the polyketide synthase and non-ribosomal peptide synthetase enzymatic assembly lines. Recent advancements in the study of these megasynthases has led to many new examples that demonstrate the production of non-natural natural products. The field is likely nearing the ability to design and build new biosynthetic pathways de novo. We discuss the various recent approaches taken towards this goal, focusing on the installation of new substrates, the swapping of enzymatic domains and modules, and the impact of metabolic engineering and synthetic biology. We also address the challenges remaining alongside the many successes in this area.}, journal={Current Opinion in Microbiology}, publisher={Elsevier BV}, author={Kalkreuter, Edward and Williams, Gavin J}, year={2018}, month={Oct}, pages={140–148} }
 @article{carpenter_williams_2018, title={Extender Unit Promiscuity and Orthogonal Protein Interactions of an Aminomalonyl-ACP Utilizing Trans-Acyltransferase from Zwittermicin Biosynthesis}, volume={13}, ISSN={1554-8929 1554-8937}, url={http://dx.doi.org/10.1021/acschembio.8b00867}, DOI={10.1021/acschembio.8b00867}, abstractNote={Trans-acting acyltransferases (trans-ATs) are standalone enzymes that select and deliver extender units to polyketide synthase assembly lines. Accordingly, there is interest in leveraging trans-ATs as tools to regioselectively diversify polyketide structures. Yet, little is known regarding the extender unit and acyl carrier protein (ACP) specificity of trans-ATs, particularly those that utilize unusual ACP-linked extender units. For example, the biosynthesis of the antibiotic zwittermicin involves the trans-AT ZmaF, which is responsible for installing a rare ACP-linked aminomalonyl extender unit. Here, we developed a method to access a panel of non-natural and non-native ACP-linked extender units and used it to probe the promiscuity of ZmaF, revealing one of the most promiscuous ATs characterized to date. Furthermore, we demonstrated that ZmaF is highly orthogonal with respect to its ACP specificity, and the ability of ZmaF to trans-complement noncognate PKS modules was also explored. Together, these results set the stage for further engineering ZmaF as a tool for polyketide diversification.}, number={12}, journal={ACS Chemical Biology}, publisher={American Chemical Society (ACS)}, author={Carpenter, Samantha M. and Williams, Gavin J.}, year={2018}, month={Nov}, pages={3361–3373} }
 @article{nazari_malico_ekelöf_lund_williams_muddiman_2017, title={Direct analysis of terpenes from biological buffer systems using SESI and IR-MALDESI}, volume={410}, ISSN={1618-2642 1618-2650}, url={http://dx.doi.org/10.1007/s00216-017-0570-9}, DOI={10.1007/s00216-017-0570-9}, abstractNote={Terpenes are the largest class of natural products with a wide range of applications including use as pharmaceuticals, fragrances, flavorings, and agricultural products. Terpenes are biosynthesized by the condensation of a variable number of isoprene units resulting in linear polyisoprene diphosphate units, which can then be cyclized by terpene synthases into a range of complex structures. While these cyclic structures have immense diversity and potential in different applications, their direct analysis in biological buffer systems requires intensive sample preparation steps such as salt cleanup, extraction with organic solvents, and chromatographic separations. Electrospray post-ionization can be used to circumvent many sample cleanup and desalting steps. SESI and IR-MALDESI are two examples of ionization methods that employ electrospray post-ionization at atmospheric pressure and temperature. By coupling the two techniques and doping the electrospray solvent with silver ions, olefinic terpenes of different classes and varying degrees of volatility were directly analyzed from a biological buffer system with no sample workup steps.}, number={3}, journal={Analytical and Bioanalytical Chemistry}, publisher={Springer Science and Business Media LLC}, author={Nazari, Milad and Malico, Alexandra A. and Ekelöf, Måns and Lund, Sean and Williams, Gavin J. and Muddiman, David C.}, year={2017}, month={Aug}, pages={953–962} }
 @article{koryakina_kasey_mcarthur_lowell_chemler_li_hansen_sherman_williams_2017, title={Inversion of Extender Unit Selectivity in the Erythromycin Polyketide Synthase by Acyltransferase Domain Engineering}, volume={12}, ISSN={1554-8929 1554-8937}, url={http://dx.doi.org/10.1021/ACSCHEMBIO.6B00732}, DOI={10.1021/acschembio.6b00732}, abstractNote={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.}, number={1}, journal={ACS Chemical Biology}, publisher={American Chemical Society (ACS)}, author={Koryakina, Irina and Kasey, Christian and McArthur, John B. and Lowell, Andrew N. and Chemler, Joseph A. and Li, Shasha and Hansen, Douglas A. and Sherman, David H. and Williams, Gavin J.}, year={2017}, month={Jan}, pages={114–123} }
 @article{musiol-kroll_zubeil_schafhauser_härtner_kulik_mcarthur_koryakina_wohlleben_grond_williams_et al._2017, title={Polyketide Bioderivatization Using the Promiscuous Acyltransferase KirCII}, volume={6}, ISSN={2161-5063 2161-5063}, url={http://dx.doi.org/10.1021/acssynbio.6b00341}, DOI={10.1021/acssynbio.6b00341}, abstractNote={During polyketide biosynthesis, acyltransferases (ATs) are the essential gatekeepers which provide the assembly lines with precursors and thus contribute greatly to structural diversity. Previously, we demonstrated that the discrete AT KirCII from the kirromycin antibiotic pathway accesses nonmalonate extender units. Here, we exploit the promiscuity of KirCII to generate new kirromycins with allyl- and propargyl-side chains in vivo, the latter were utilized as educts for further modification by "click" chemistry.}, number={3}, journal={ACS Synthetic Biology}, publisher={American Chemical Society (ACS)}, author={Musiol-Kroll, Ewa M. and Zubeil, Florian and Schafhauser, Thomas and Härtner, Thomas and Kulik, Andreas and McArthur, John and Koryakina, Irina and Wohlleben, Wolfgang and Grond, Stephanie and Williams, Gavin J. and et al.}, year={2017}, month={Feb}, pages={421–427} }
 @article{ladner_williams_2015, title={Harnessing natural product assembly lines: structure, promiscuity, and engineering}, volume={43}, ISSN={1367-5435 1476-5535}, url={http://dx.doi.org/10.1007/s10295-015-1704-8}, DOI={10.1007/s10295-015-1704-8}, abstractNote={Abstract}, number={2-3}, journal={Journal of Industrial Microbiology & Biotechnology}, publisher={Springer Science and Business Media LLC}, author={Ladner, Christopher C. and Williams, Gavin J.}, year={2015}, month={Nov}, pages={371–387} }
 @article{williams_2015, title={Harnessing the promiscuity of natural product biosynthesis: A platform for engineering pathways with new specificities}, volume={81}, ISSN={0032-0943 1439-0221}, url={http://dx.doi.org/10.1055/S-0035-1556155}, DOI={10.1055/S-0035-1556155}, abstractNote={Many natural products are biosynthesized in a modular fashion by the selection and condensation of small molecule building blocks. Chimeric biosynthetic apparatus can be constructed in an attempt to produce analogues for drug discovery. Yet, the scope and utility of such approaches is limited by the inherent substrate specificity and poor functional modularity of most biosynthetic components. Here, we show that several types of biosynthetic machinery are more tolerant towards non-natural building blocks than has been previously recognized. Such promiscuity forms a platform for constructing new biosynthetic parts with substrate specificities orthogonal to those found in Nature. Accordingly, we describe a comprehensive program of enzyme engineering, directed evolution, and synthetic biology aimed at constructing artificial bacterial strains capable of producing complex natural products that are regioselectively modified with non-natural chemical functionality. Our synthetic biology approach expands the synthetic capabilities of natural product diversification strategies, and provides an improved understanding of the molecular basis for specificity in complex molecular assemblies.}, number={11}, journal={Planta Medica}, publisher={Georg Thieme Verlag KG}, author={Williams, G}, year={2015}, month={Jun} }
 @article{randall_koryakina_williams_muddiman_2014, title={Evaluating nonpolar surface area and liquid chromatography/mass spectrometry response: an application for site occupancy measurements for enzyme intermediates in polyketide biosynthesis}, volume={28}, ISSN={0951-4198}, url={http://dx.doi.org/10.1002/rcm.7051}, DOI={10.1002/rcm.7051}, abstractNote={RATIONALESite occupancy measurements using liquid chromatography/mass spectrometry (LC/MS) are reported throughout the literature. However, site occupancy quantification suffers from ionization bias between modified and unmodified peptides containing the active site. In this study, we explore the MS signal as a function of nonpolar surface area (NPSA) in order to better understand this bias in electrospray response. The correlation between hydrophobicity and LC/MS response was evaluated and applied to study enzyme intermediates in polyketide synthases.}, number={23}, journal={Rapid Communications in Mass Spectrometry}, publisher={Wiley}, author={Randall, Shan M. and Koryakina, Irina and Williams, Gavin J. and Muddiman, David C.}, year={2014}, month={Oct}, pages={2511–2522} }
 @article{walsh_gardner_deiters_williams_2014, title={Intracellular Light-Activation of Riboswitch Activity}, volume={15}, ISSN={1439-4227}, url={http://dx.doi.org/10.1002/cbic.201400024}, DOI={10.1002/cbic.201400024}, abstractNote={Abstract}, number={9}, journal={ChemBioChem}, publisher={Wiley}, author={Walsh, Steven and Gardner, Laura and Deiters, Alexander and Williams, Gavin J.}, year={2014}, month={May}, pages={1346–1351} }
 @article{ye_williams_2014, title={Mapping a Ketosynthase:Acyl Carrier Protein Binding Interface via Unnatural Amino Acid-Mediated Photo-Cross-Linking}, volume={53}, ISSN={0006-2960 1520-4995}, url={http://dx.doi.org/10.1021/bi500936u}, DOI={10.1021/bi500936u}, abstractNote={Probing and interrogating protein interactions that involve acyl carrier proteins (ACP's) in fatty acid synthases and polyketide synthases are critical to understanding the molecular basis for the programmed assembly of complex natural products. Here, we have used unnatural amino acid mutagenesis to site specifically install photo-cross-linking functionality into acyl carrier proteins from diverse systems and the ketosynthase FabF from the Escherichia coli type II fatty acid synthase. Subsequently, a photo-cross-linking assay was employed to systematically probe the ability of FabF to interact with a broad panel of ACP's, illustrating the expected orthogonality of ACP:FabF interactions and the role of charged residues in helix II of the ACP. In addition, FabF residues involved in the binding interaction with the cognate carrier protein were identified via surface scanning mutagenesis and photo-cross-linking. Furthermore, the ability to install the photo-cross-linking amino acid at virtually any position allowed interrogation of the role that carrier protein acylation plays in determining the binding interface with FabF. A conserved carrier protein motif that includes the phosphopantetheinylation site was also shown to play an integral role in maintenance of the AcpP:FabF binding interaction. Our results provide unprecedented insight into the molecular details that describe the AcpP:FabF binding interface and demonstrate that unnatural amino acid based photo-cross-linking is a powerful tool for probing and interrogating protein interactions in complex biosynthetic systems.}, number={48}, journal={Biochemistry}, publisher={American Chemical Society (ACS)}, author={Ye, Zhixia and Williams, Gavin J.}, year={2014}, month={Nov}, pages={7494–7502} }
 @article{ye_musiol_weber_williams_2014, title={Reprogramming Acyl Carrier Protein Interactions of an Acyl-CoA Promiscuous trans-Acyltransferase}, volume={21}, ISSN={1074-5521}, url={http://dx.doi.org/10.1016/j.chembiol.2014.02.019}, DOI={10.1016/j.chembiol.2014.02.019}, abstractNote={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.}, number={5}, journal={Chemistry & Biology}, publisher={Elsevier BV}, author={Ye, Zhixia and Musiol, Ewa M. and Weber, Tilmann and Williams, Gavin J.}, year={2014}, month={May}, pages={636–646} }
 @inproceedings{koryakina_williams_2014, place={Washington, DC}, title={Synthetic Biology Approaches For Combinatorial Biosynthesis Of Polyketide Natural Products.}, booktitle={Abstracts of Papers for the American Chemical Society}, publisher={American Chemical Society}, author={Koryakina, I. and Williams, G.J.}, year={2014}, month={Mar} }
 @article{williams_2013, title={Engineering polyketide synthases and nonribosomal peptide synthetases}, volume={23}, ISSN={0959-440X}, url={http://dx.doi.org/10.1016/j.sbi.2013.06.012}, DOI={10.1016/j.sbi.2013.06.012}, abstractNote={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.}, number={4}, journal={Current Opinion in Structural Biology}, publisher={Elsevier BV}, author={Williams, Gavin J}, year={2013}, month={Aug}, pages={603–612} }
 @article{koryakina_mcarthur_draelos_williams_2013, title={Promiscuity of a modular polyketide synthase towards natural and non-natural extender units}, volume={11}, ISSN={1477-0520 1477-0539}, url={http://dx.doi.org/10.1039/c3ob40633d}, DOI={10.1039/c3ob40633d}, abstractNote={Combinatorial biosynthesis approaches that involve modular type I polyketide synthases (PKSs) are proven strategies for the synthesis of polyketides. In general however, such strategies are usually limited in scope and utility due to the restricted substrate specificity of polyketide biosynthetic machinery. Herein, a panel of chemo-enzymatically synthesized acyl-CoA's was used to probe the promiscuity of a polyketide synthase. Promiscuity determinants were dissected, revealing that the KS is remarkably tolerant to a diverse array of extender units, while the AT likely discriminates between extender units that are native to the producing organism. Our data provides a clear blueprint for future enzyme engineering efforts, and sets the stage for harnessing extender unit promiscuity by employing various in vivo polyketide diversification strategies.}, number={27}, journal={Organic & Biomolecular Chemistry}, publisher={Royal Society of Chemistry (RSC)}, author={Koryakina, Irina and McArthur, John B. and Draelos, Matthew M. and Williams, Gavin J.}, year={2013}, pages={4449} }
 @inbook{williams_koryakina_mcarthur_draelos_randal_muddimanl_2013, series={ACS Symposium Series}, title={Reprogramming the Biosynthesis of Natural Products by Directed Evolution}, ISBN={0841229104 0841229112}, ISSN={0097-6156 1947-5918}, url={http://dx.doi.org/10.1021/bk-2013-1125.ch009}, DOI={10.1021/bk-2013-1125.ch009}, abstractNote={Directed evolution is a powerful biochemical tool used to alter the function and properties of enzymes. Modification of the activity of key enzymes responsible for the biosynthesis of complex, biologically active natural products may provide routes to increase the diversity of natural products or generate novel and more potent analogues. Here we describe our combined efforts aimed to utilize and improve upon combinatorial biosynthesis techniques to reprogram the biosynthesis of polyketide natural products. We describe a synthetic biology approach that uses directed enzyme evolution to broaden the substrate specificities of enzymes used in natural product biosynthesis. These engineered mutant enzymes will be used to construct artificial biosynthetic pathways for the synthesis and installation of a broad array of natural and non-natural building blocks into polyketides.}, booktitle={Developments in Biotechnology and Bioprocessing}, publisher={American Chemical Society}, author={Williams, Gavin and Koryakina, Irina and McArthur, John and Draelos, Matthew and Randal, Shan and Muddimanl, David}, editor={Kantardjieff, Anne and Asuri, Prashanth and Coffman, Jonathan L. and Jayapal, KarthikEditors}, year={2013}, month={Jan}, pages={147–163}, collection={ACS Symposium Series} }
 @inbook{mcarthur_williams_2012, place={Weinheim}, title={Engineering Glycosyltransferases}, volume={3}, ISBN={9783527666997}, booktitle={The Protein Engineering Handbook}, publisher={Wiley-HCH}, author={McArthur, J. and Williams, G.J.}, editor={Lutz, S and Bornscheuer, U.T.Editors}, year={2012}, pages={303–326} }
 @article{koryakina_mcarthur_randall_draelos_musiol_muddiman_weber_williams_2012, title={Poly Specific trans-Acyltransferase Machinery Revealed via Engineered Acyl-CoA Synthetases}, volume={8}, ISSN={1554-8929 1554-8937}, url={http://dx.doi.org/10.1021/cb3003489}, DOI={10.1021/cb3003489}, abstractNote={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.}, number={1}, journal={ACS Chemical Biology}, publisher={American Chemical Society (ACS)}, author={Koryakina, Irina and McArthur, John and Randall, Shan and Draelos, Matthew M. and Musiol, Ewa M. and Muddiman, David C. and Weber, Tilmann and Williams, Gavin J.}, year={2012}, month={Oct}, pages={200–208} }
 @inproceedings{koryakina_ye_mcarthur_williams_2012, place={Washington, DC}, title={Reprogramming the biosynthesis of natural products by directed evolution.}, booktitle={Abstracts of Papers of the American Chemical Society}, publisher={American Chemical Society}, author={Koryakina, I. and Ye, Z. and McArthur, J. and Williams, G.J.}, year={2012}, month={Mar} }
 @article{koryakina_neville_nonaka_van lanen_williams_2011, title={A High-Throughput Screen for Directed Evolution of the Natural Product Sulfotransferase LipB}, volume={16}, ISSN={1087-0571 1552-454X}, url={http://dx.doi.org/10.1177/1087057111413273}, DOI={10.1177/1087057111413273}, abstractNote={In this article, the authors describe a colorimetric, high-throughput assay suitable for optimizing the activity of the recently discovered sulfotransferase LipB, by directed evolution. Crucially, LipB uses para-nitrophenol sulfate as donor in the sulfation of the nucleoside antibiotic liposidomycin B-I and other acceptor surrogates. Thus, using a robotic liquid-handling device, crude cell extracts were prepared from an Escherichia coli strain that overproduced LipB in wells of a microplate, and production of para-nitrophenol at 405 nm was monitored spectrophotometrically. Enzyme activity could be detected only in the presence of both LipB substrates and overexpressed LipB. The screen displays a suitable standard deviation for directed evolution and importantly is not limited to the natural desulfo-liposidomycin acceptor. The authors plan to use the screen to identify LipB variants with altered acceptor specificity and promiscuity for use in sulfation of natural products and other small-molecule therapeutics.}, number={8}, journal={Journal of Biomolecular Screening}, publisher={SAGE Publications}, author={Koryakina, Irina and Neville, Jessica and Nonaka, Koichi and Van Lanen, Steven G. and Williams, Gavin J.}, year={2011}, month={Jul}, pages={845–851} }
 @article{parajuli_williams_2011, title={A high-throughput screen for directed evolution of aminocoumarin amide synthetases}, volume={419}, ISSN={0003-2697}, url={http://dx.doi.org/10.1016/j.ab.2011.07.037}, DOI={10.1016/j.ab.2011.07.037}, abstractNote={The biosynthesis of aminocoumarin antibiotics involves the action of amide synthetases which construct amide bonds between aminocoumarins and various acyl moieties. Libraries of aminocoumarin analogues have been generated by in vivo fermentation, via feeding known amide synthetase substrates into producing microbial strains. Critically, such feeding studies rely on the inherent or engineered substrate promiscuity of each amide synthetase. We have initiated a program of directed evolution in order to create mutant amide synthetases for the synthesis of new nonnatural amino coumarin analogues. We used the clorobiocin enzyme CloL as a model amide synthetase to design and validate a fluorimetric high-throughput screen, which can be used to report the activity of mutant amide synthetases toward a broad range of coumarin and acyl donor substrates. Our assay monitors the decrease in fluorescence of aminocoumarins on acylation. The utility of the assay was illustrated by screening a library of amide synthetase mutants created by error-prone PCR. The substrate specificity of an amide synthetase was also rapidly probed using this assay, affording several newly identified substrates. It is anticipated that this high-throughput screen will accelerate the creation of amide synthetase mutants with new specificities by directed evolution.}, number={1}, journal={Analytical Biochemistry}, publisher={Elsevier BV}, author={Parajuli, Niranjan and Williams, Gavin J.}, year={2011}, month={Dec}, pages={61–66} }
 @article{ye_bair_desai_williams_2011, title={A photocrosslinking assay for reporting protein interactions in polyketide and fatty acid synthases}, volume={7}, ISSN={1742-206X 1742-2051}, url={http://dx.doi.org/10.1039/c1mb05270e}, DOI={10.1039/c1mb05270e}, abstractNote={Understanding protein-protein interactions that occur between ACP and KS domains of polyketide synthases and fatty acid synthases is critical to improving the scope and efficiency of combinatorial biosynthesis efforts aimed at producing non-natural polyketides. Here, we report a facile strategy for rapidly reporting such ACP-KS interactions based on the incorporation of an amino acid with photocrosslinking functionality. Crucially, this photocrosslinking strategy can be applied to any polyketide or fatty acid synthase regardless of substrate specificity, and can be adapted to a high-throughput format for directed evolution studies.}, number={11}, journal={Molecular BioSystems}, publisher={Royal Society of Chemistry (RSC)}, author={Ye, Zhixia and Bair, Morgan and Desai, Hemant and Williams, Gavin J.}, year={2011}, pages={3152} }
 @article{koryakina_williams_2011, title={Inside Cover: Mutant Malonyl-CoA Synthetases with Altered Specificity for Polyketide Synthase Extender Unit Generation (ChemBioChem 15/2011)}, volume={12}, ISSN={1439-4227}, url={http://dx.doi.org/10.1002/cbic.201190070}, DOI={10.1002/cbic.201190070}, abstractNote={The inside cover picture shows the shift in substrate specificity of malonyl-CoA synthetase towards non-native malonate analogues afforded by active-site saturation mutagenesis followed by colorimetric screening for improved activity. Mutant synthetases could provide extender units for probing the activity of polyketide synthases. For further details, see the paper by G. J. Williams and I. Koryakina on p. 2289 ff.}, number={15}, journal={ChemBioChem}, publisher={Wiley}, author={Koryakina, Irina and Williams, Gavin J.}, year={2011}, month={Oct}, pages={2230–2230} }
 @article{koryakina_williams_2011, title={Mutant Malonyl-CoA Synthetases with Altered Specificity for Polyketide Synthase Extender Unit Generation}, volume={12}, ISSN={1439-4227}, url={http://dx.doi.org/10.1002/cbic.201100383}, DOI={10.1002/cbic.201100383}, abstractNote={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.}, number={15}, journal={ChemBioChem}, publisher={Wiley}, author={Koryakina, Irina and Williams, Gavin J.}, year={2011}, month={Aug}, pages={2289–2293} }
 @article{williams_yang_zhang_thorson_2011, title={Recombinant E. coli Prototype Strains for in Vivo Glycorandomization}, volume={6}, ISSN={1554-8929 1554-8937}, url={http://dx.doi.org/10.1021/cb100267k}, DOI={10.1021/cb100267k}, abstractNote={In vitro glycorandomization is a powerful strategy to alter the glycosylation patterns of natural products and small molecule therapeutics. Yet, such in vitro methods are often difficult to scale and can be costly given the requirement to provide various nucleotides and cofactors. Here, we report the construction of several recombinant E. coli prototype strains that allow the facile production of a range of small molecule glycosides. This strategy relies on the engineered promiscuity of three key enzymes, an anomeric kinase, a sugar-1-phosphate nucleotidyltransferase, and a glycosyltransferase, as well as the ability of diverse small molecules to freely enter E. coli. Subsequently, this work is the first demonstration of "in vivo glycorandomization" and offers vast combinatorial potential by simple fermentation.}, number={1}, journal={ACS Chemical Biology}, publisher={American Chemical Society (ACS)}, author={Williams, Gavin J. and Yang, Jie and Zhang, Changsheng and Thorson, Jon S.}, year={2011}, month={Jan}, pages={95–100} }
 @inbook{williams_thorson_2009, title={Natural Product Glycosyltransferases: Properties and Applications}, ISBN={9780470392881 9780471235842}, ISSN={1934-4694}, url={http://dx.doi.org/10.1002/9780470392881.ch2}, DOI={10.1002/9780470392881.ch2}, abstractNote={This chapter contains sections titled: Introduction Glycosyltransferase Sequence, Structure, and Mechanism In Vitro Characterization of Natural Product Glycosyltransferases Engineering Glycosyltransferases Conclusions References}, booktitle={Advances in Enzymology - and Related Areas of Molecular Biology}, publisher={John Wiley & Sons, Inc.}, author={Williams, Gavin J. and Thorson, Jon S.}, year={2009}, month={May}, pages={55–119} }
 @article{williams_thorson_2008, title={A high-throughput fluorescence-based glycosyltransferase screen and its application in directed evolution}, volume={3}, ISSN={1754-2189 1750-2799}, url={http://dx.doi.org/10.1038/nprot.2007.538}, DOI={10.1038/nprot.2007.538}, abstractNote={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.}, number={3}, journal={Nature Protocols}, publisher={Springer Science and Business Media LLC}, author={Williams, Gavin J and Thorson, Jon S}, year={2008}, month={Feb}, pages={357–362} }
 @article{williams_goff_zhang_thorson_2008, title={Optimizing Glycosyltransferase Specificity via “Hot Spot” Saturation Mutagenesis Presents a Catalyst for Novobiocin Glycorandomization}, volume={15}, ISSN={1074-5521}, url={http://dx.doi.org/10.1016/j.chembiol.2008.02.017}, DOI={10.1016/j.chembiol.2008.02.017}, abstractNote={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.}, number={4}, journal={Chemistry & Biology}, publisher={Elsevier BV}, author={Williams, Gavin J. and Goff, Randal D. and Zhang, Changsheng and Thorson, Jon S.}, year={2008}, month={Apr}, pages={393–401} }
 @article{gantt_goff_williams_thorson_2008, title={Probing the Aglycon Promiscuity of an Engineered Glycosyltransferase}, volume={47}, ISSN={1433-7851 1521-3773}, url={http://dx.doi.org/10.1002/anie.200803508}, DOI={10.1002/anie.200803508}, abstractNote={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.}, number={46}, journal={Angewandte Chemie International Edition}, publisher={Wiley}, author={Gantt, Richard W. and Goff, Randal D. and Williams, Gavin J. and Thorson, Jon S.}, year={2008}, month={Nov}, pages={8889–8892} }
 @article{williams_gantt_thorson_2008, title={The impact of enzyme engineering upon natural product glycodiversification}, volume={12}, ISSN={1367-5931}, url={http://dx.doi.org/10.1016/j.cbpa.2008.07.013}, DOI={10.1016/j.cbpa.2008.07.013}, abstractNote={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.}, number={5}, journal={Current Opinion in Chemical Biology}, publisher={Elsevier BV}, author={Williams, Gavin J and Gantt, Richard W and Thorson, Jon S}, year={2008}, month={Oct}, pages={556–564} }
 @article{nelson_williams_woodhall_farnsworth_berry_2007, title={Stereochemically Complementary Biocatalysts Created by Directed Evolution}, volume={2007}, ISSN={1861-1958 1861-194X}, url={http://dx.doi.org/10.1055/s-2006-955791}, DOI={10.1055/s-2006-955791}, abstractNote={A pair of stereochemically complementary biocatalysts for the synthesis of sialic acid mimetics has been created. Directed evolution using both error-prone PCR as well as saturation mutagenesis starting from the E192N variant of wild-type enzyme N-acetylneuramic acid lyase (NAL) has provided S-selective E192N/T167G and R-selective E192N/T167V/S208V, respectively. These mutants mediate the aldol reaction of aldehyde 1 and pyruvate 2 to give 4S- and 4R-configured dipropylamides 3 and 4 in moderate yields and high diastereoselectivities.}, number={2}, journal={Synfacts}, publisher={Georg Thieme Verlag KG}, author={Nelson, A. and Williams, G. and Woodhall, T. and Farnsworth, L. and Berry, A.}, year={2007}, month={Feb}, pages={0208–0208} }
 @article{williams_woodhall_farnsworth_nelson_berry_2006, title={Creation of a Pair of Stereochemically Complementary Biocatalysts}, volume={128}, ISSN={0002-7863 1520-5126}, url={http://dx.doi.org/10.1021/ja065233q}, DOI={10.1021/ja065233q}, abstractNote={N-Acetylneuraminic acid lyase (NAL) exhibits poor facial selectivity during carbon-carbon formation, and as such, its utility as a catalyst for use in synthetic chemistry is limited. For example, the NAL-catalyzed condensation between pyruvate and (2R,3S)-2,3-dihydroxy-4-oxo-N,N-dipropylbutyramide yields ca. 3:1 mixtures of diastereomeric products under either kinetic or thermodynamic control. Engineering the stereochemical course of NAL-catalyzed reactions could remove this limitation. We used directed evolution to create a pair of stereochemically complementary variant NALs for the synthesis of sialic acid mimetics. The E192N variant, a highly efficient catalyst for aldol reactions of (2R,3S)-2,3-dihydroxy-4-oxo-N,N-dialkylbutyramides, was chosen as a starting point. Initially, error-prone PCR identified residues in the active site of NAL that contributed to the stereochemical control of an aldolase-catalyzed reaction. Subsequently, an intense structure-guided program of saturation and site-directed mutagenesis was used to identify a complementary pair of variants, E192N/T167G and E192N/T167V/S208V, which were approximately 50-fold selective toward the cleavage of the alternative 4S- and 4R-configured condensation products, respectively. It was shown that wild-type NAL could not be used for the highly stereoselective synthesis of a 6-dipropylamide sialic acid mimetic because the 4S-configured product was only approximately 3-fold kinetically favored and only approximately 3-fold thermodynamically favored over the alternative 4R-configured product. However, the complementary 4R- and 4S-selective variants allowed the highly (>98:<2) diastereoselective synthesis of both 4S- and 4R-configured products under kinetic control from the same starting materials. Conversion of an essentially nonselective aldolase into a pair of complementary biocatalysts will be of enormous interest to synthetic chemists. Furthermore, since residues identified as critical for stereoselectivity are conserved among members of the NAL superfamily, the approach might be extended to the evolution of other useful biocatalysts for the stereoselective synthesis of biologically active molecules.}, number={50}, journal={Journal of the American Chemical Society}, publisher={American Chemical Society (ACS)}, author={Williams, Gavin J. and Woodhall, Thomas and Farnsworth, Lorna M. and Nelson, Adam and Berry, Alan}, year={2006}, month={Dec}, pages={16238–16247} }
 @article{williams_woodhall_nelson_berry_2005, title={Structure-guided saturation mutagenesis of N-acetylneuraminic acid lyase for the synthesis of sialic acid mimetics}, volume={18}, ISSN={1741-0134 1741-0126}, url={http://dx.doi.org/10.1093/protein/gzi027}, DOI={10.1093/protein/gzi027}, abstractNote={Analogues of N-acetylneuraminic acid (sialic acid, NANA, Neu5Ac), including 6-dipropylcarboxamides, have been found to be selective and potent inhibitors of influenza sialidases. Sialic acid analogues are, however, difficult to synthesize by traditional chemical methods and the enzyme N-acetylneuraminic acid lyase (NAL) has previously been used for the synthesis of a number of analogues. The activity of this enzyme towards 6-dipropylcarboxamides is, however, low. Here, we used structure-guided saturation mutagenesis to produce variants of NAL with improved activity and specificity towards 6-dipropylcarboxamides. Three residues were targeted for mutagenesis, Asp191, Glu192 and Ser208. Only substitution at position 192 produced significant improvements in activity towards the dipropylamide. One variant, E192N, showed a 49-fold improvement in catalytic efficiency towards the target analogue and a 690-fold shift in specificity from sialic acid towards the analogue. These engineering efforts provide a scaffold for the further tailoring of NAL for the synthesis of sialic acid mimetics.}, number={5}, journal={Protein Engineering, Design and Selection}, publisher={Oxford University Press (OUP)}, author={Williams, G.J. and Woodhall, T. and Nelson, A. and Berry, A.}, year={2005}, month={May}, pages={239–246} }
 @article{williams_berry_2003, title={Directed evolution: Creating new enzymes}, volume={25}, DOI={10.1042/BIO02504013}, abstractNote={One of the goals for protein engineers has been the acquisition of the knowledge to design and build proteins for any given function. This has usually been achieved by modifying an existing protein with a similar function. However, the protein engineer has been faced with an intensive task of gathering and understanding a whole realm of information about the enzyme's structure and mechanism and the role that each amino acid plays in the catalytic process. Although rational enzyme redesign has resulted in some notable successes, it has highlighted more often our relatively poor understanding of the intricacies of enzyme recognition and catalysis. The search was therefore on to develop an alternative to rational enzyme redesign. The techniques of molecular biology coupled with more automated methods for handling repetitive tasks have provided an answer: directed evolution.}, number={4}, journal={The Biochemist}, author={Williams, G.J. and Berry, A.}, year={2003}, month={Aug}, pages={13–15} }
 @article{williams_domann_nelson_berry_2003, title={Modifying the stereochemistry of an enzyme-catalyzed reaction by directed evolution}, volume={100}, ISSN={0027-8424 1091-6490}, url={http://dx.doi.org/10.1073/pnas.0635924100}, DOI={10.1073/pnas.0635924100}, abstractNote={
            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
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            toward the non-natural substrate fructose 1,6-bisphosphate, resulting in a 100-fold change in stereospecificity.
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            P 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.
          }, number={6}, journal={Proceedings of the National Academy of Sciences}, publisher={Proceedings of the National Academy of Sciences}, author={Williams, G. J. and Domann, S. and Nelson, A. and Berry, A.}, year={2003}, month={Mar}, pages={3143–3148} }