Research paper Expanding the chemical space of polyketides through structure- guided mutagenesis of Vitis vinifera stilbene synthase Namita Bhan a, 1 , Brady F. Cress a , Robert J. Linhardt a, b, c, d , Mattheos Koffas a, c, * a Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Center for Biotechnology and Interdisciplinary Studies, Troy, NY, USA b Department of Chemistry and Chemical Biology, Rensselaer Polytechnic Institute, Center for Biotechnology and Interdisciplinary Studies, Troy, NY, USA c Department of Biological Sciences, Rensselaer Polytechnic Institute, Center for Biotechnology and Interdisciplinary Studies, Troy, NY, USA d Department of Biomedical Engineering, Rensselaer Polytechnic Institute, Center for Biotechnology and Interdisciplinary Studies, Troy, NY, USA article info Article history: Received 9 April 2015 Accepted 22 May 2015 Available online 3 June 2015 Keywords: Stilbene synthase Aromatic polyketides Type III polyketide synthases Flavonoids Resveratrol abstract Several natural polyketides (PKs) have been associated with important pharmaceutical properties. Type III polyketide synthases (PKS) that generate aromatic PK polyketides have been studied extensively for their substrate promiscuity and product diversity. Stilbene synthase-like (STS) enzymes are unique in the type III PKS class as they possess a hydrogen bonding network, furnishing them with thioesterase-like properties, resulting in aldol condensation of the polyketide intermediates formed. Chalcone syn- thases (CHS) in contrast, lack this hydrogen-bonding network, resulting primarily in the Claisen condensation of the polyketide intermediates formed. We have attempted to expand the chemical space of this interesting class of compounds generated by creating structure-guided mutants of Vitis vinifera STS. Further, we have utilized a previously established workflow to quickly compare the wild-type reaction products to those generated by the mutants and identify novel PKs formed by using XCMS analysis of LC-MS and LC-MS/MS data. Based on this approach, we were able to generate 15 previously unreported PK molecules by exploring the substrate promiscuity of the wild-type enzyme and all mu- tants using unnatural substrates. These structures were specific to STSs and cannot be formed by their closely related CHS-like counterparts. © 2015 Elsevier B.V. and Soci et e Française de Biochimie et Biologie Mol eculaire (SFBBM). All rights reserved. 1. Introduction Polyketides (PKs) are a chemically important class of com- pounds with several beneficial pharmaceutical properties [1,2]. Natural PKs generated by type III polyketide synthases (PKSs) have been associated with the slowing of the aging process in model organisms [3,4], anti-inflammatory and anti-cancer prop- erties [5e10] and have shown potential to ameliorate diabetes and nervous system disorders related complications [11,12]. Type III PKSs are found in several plants, bacteria and fungi [13e15]. They are homodimeric enzymes that catalyze iterative conden- sation of repeating units to a CoA-tethered starter substrate through a conserved Cys-His-Asn catalytic triad. Functionally diverse type III PKSs arise due to variable substitutions in non- catalytic residues present in the three catalytically important cavities: the substrate binding pocket, composed of the important residues S133, Q192, T194, T197, S338; the CoA binding tunnel, composed of L55, R58, L62; and the cyclization pocket, composed of T132, M137, F215, I254, G256, F265, P375 (residues numbered according to the Vitis vinifera stilbene synthase (VvSTS)). Altering these residues results in diversity of preference for starter sub- strates, number of extender substrates incorporated through iterative condensations and mechanism of cyclization of the poly- b-keto intermediate formed through Claisen condensation, aldol condensation or lactonization. Moreover, type III PKSs possess unusually broad substrate promiscuity and can accept several non-natural substrates to form novel non-natural PKs [2,16]. Apart from the naturally occurring type III PKSs, several intuitive and structure-guided mutations have also been carried out to alter their enzymatic activity, so as to expand the chemical diversity of PKs [17,18]. Some of the novel non-natural PKs formed through these processes has also been demonstrated to possess important biological activities [16]. Abbreviations: PKS, polyketide synthase; PK, polyketide; Vv, Vitis vinifera; STS, stilbene synthase; CHS, chalcone synthase; Wt, wild-type; HBN, hydrogen-bonding network; BNY, bisnoryangonin; CATL, p-coumaroyltriacetic acid lactone. * Corresponding author. E-mail address: koffam@rpi.edu (M. Koffas). 1 Present address: Northwestern University, Evanston, Illinois, USA. Contents lists available at ScienceDirect Biochimie journal homepage: www.elsevier.com/locate/biochi http://dx.doi.org/10.1016/j.biochi.2015.05.019 0300-9084/© 2015 Elsevier B.V. and Soci et e Française de Biochimie et Biologie Mol eculaire (SFBBM). All rights reserved. Biochimie 115 (2015) 136e143