@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{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={Site 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.Site occupancy methods were developed to study acyltransferase activity. To further evaluate these methods, several standard peptides containing one cysteine residue were modified with alkylation reagents of increasing hydrophobicity to study the MS signal as a function of NPSA.A consistent trend in MS response was observed which is dependent on the NPSA of the analyte. An optimal NPSA zone was observed for the peptides studied.Nonpolar surface area can be used as metric to determine relative LC/MS response for peptides and evaluate site occupancy measurements.}, 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{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} }
 @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} }
 @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{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. Detailed facts of importance to specialist readers are published as ”Supporting Information”. Such documents are peer-reviewed, but not copy-edited or typeset. They are made available as submitted by the authors. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.}, number={15}, journal={ChemBioChem}, publisher={Wiley}, author={Koryakina, Irina and Williams, Gavin J.}, year={2011}, month={Aug}, pages={2289–2293} }