Poly Specific trans-Acyltransferase Machinery Revealed via Engineered Acyl-CoA Synthetases Irina Koryakina, † John McArthur, † Shan Randall, ‡ Matthew M. Draelos, † Ewa M. Musiol, § David C. Muddiman, ‡ Tilmann Weber, § and Gavin J. Williams* ,† † Department of Chemistry and ‡ W. M. Keck FT-ICR Mass Spectrometry Laboratory, North Carolina State University, Raleigh, North Carolina, United States § Eberhard-Karls-Universitä t Tü bingen, Interfakultä res Institut fü r Mikrobiologie und Infektionsmedizin, Mikrobiologie/Biotechnologie, Tü bingen, Germany * S Supporting Information ABSTRACT: 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. P olyketides are a large class of pharmaceutically relevant natural products with wide-ranging biological activities that include antimicrobial, anticancer, and immunosuppressant. 1 Moreover, polyketides constitute a significant fraction of approved and top-selling drugs, annual sales of which total $20 billion. 2,3 The broad and potent biological activities of polyketides are determined by their chemical structures, the carbon backbones of which are biosynthesized via iterative Claisen condensations between activated malonate extender units and acyl thioesters, catalyzed by polyketide synthases (PKSs). 4 A modest variety of malonate extender units substituted at the C2 position are cumulatively available to polyketide biosynthetic pathways, 5,6 although producing organisms typically only possess biosynthetic routes to a small number of extender units, depending on the structures of the corresponding polyketides. The most common PKS extender units include malonyl-Coenzyme A (CoA) (1), methylmalonyl-CoA (2), and ethylmalonyl-CoA (3) (Figure 1). A smaller number of PKSs use specialized extender units that are functionalized directly on standalone acyl carrier proteins (ACPs) and include methoxymalonyl-ACP 7 (4), hydroxymalonyl-ACP 8 (5), aminomalonyl-ACP 8 (6), and allylmalonyl-ACP 9 (7) (Figure 1). Additionally, it has recently emerged that several extender units, including chloroethyl, propyl, hexyl, and isobutylmalonyl-CoA (8-11, respectively) (Figure 1) are available to a subset of PKSs via the reductive carboxylation of α,β-unsaturated acyl-CoA precursors by crotonyl-CoA carboxylase/reductase (CCR) homologues. 10-12 The selection and incorporation of extender units by PKSs contributes to significant portions of polyketide structure, in addition to the vast structural diversity across the polyketide family, as illustrated by the structures of concanamycin A aglycone (12), zwittermicin A (13), salinosporamides (14a- 14d) and the FK506 series (e.g., 15a and 15b) (Figure 1). Moreover, variation of extender unit selection and incorpo- ration at specific positions in polyketide backbones has been shown to directly modulate biological activities of polyketides produced by natural PKSs. 9,13 Even though Nature provides a modest selection of potential PKS extender units, most polyketide-producing organisms provide biosynthetic routes to only a relatively small number of suitable acyl-CoAs. Subsequently, there is a growing body of evidence that PKSs can tolerate non-natural extender units that are not normally provided by the producing host. 14,15 However, Received: July 11, 2012 Accepted: October 19, 2012 Articles pubs.acs.org/acschemicalbiology © XXXX American Chemical Society A dx.doi.org/10.1021/cb3003489 | ACS Chem. Biol. XXXX, XXX, XXX-XXX