Identification by Genome Mining of a Type I Polyketide Gene Cluster From

fmicb-08-00194 February 8, 2017 Time: 14:51 # 1 View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Repositorio Institucional de la Universidad de Oviedo ORIGINAL RESEARCH published: 10 February 2017 doi: 10.3389/fmicb.2017.00194 Identification by Genome Mining of a Type I Polyketide Gene Cluster from Streptomyces argillaceus Involved in the Biosynthesis of Pyridine and Piperidine Alkaloids Argimycins P Suhui Ye1, Brian Molloy1, Alfredo F. Braña1, Daniel Zabala1, Carlos Olano1, Jesús Cortés2, Francisco Morís2, José A. Salas1 and Carmen Méndez1* 1 Departamento de Biología Funcional e Instituto Universitario de Oncología del Principado de Asturias, Universidad de Oviedo, Oviedo, Spain, 2 EntreChem, S.L, Oviedo, Spain Edited by: Mattijs Julsing, Genome mining of the mithramycin producer Streptomyces argillaceus ATCC 12956 Technical University of Dortmund, Germany revealed 31 gene clusters for the biosynthesis of secondary metabolites, and allowed Reviewed by: to predict the encoded products for 11 of these clusters. Cluster 18 (renamed Shawn Chen, cluster arp) corresponded to a type I polyketide gene cluster related to the previously Revive Genomics Inc., USA described coelimycin P1 and streptazone gene clusters. The arp cluster consists Markus Nett, Technical University of Dortmund, of fourteen genes, including genes coding for putative regulatory proteins (a SARP- Germany like transcriptional activator and a TetR-like transcriptional repressor), genes coding *Correspondence: for structural proteins (three PKSs, one aminotransferase, two dehydrogenases, two Carmen Méndez [email protected]. cyclases, one imine reductase, a type II thioesterase, and a flavin reductase), and one gene coding for a hypothetical protein. Identification of encoded compounds by Specialty section: this cluster was achieved by combining several strategies: (i) inactivation of the type I This article was submitted to Microbiotechnology, Ecotoxicology PKS gene arpPIII; (ii) inactivation of the putative TetR-transcriptional repressor arpRII; and Bioremediation, (iii) cultivation of strains in different production media; and (iv) using engineered strains a section of the journal with higher intracellular concentration of malonyl-CoA. This has allowed identifying six Frontiers in Microbiology new alkaloid compounds named argimycins P, which were purified and structurally Received: 06 October 2016 Accepted: 26 January 2017 characterized by mass spectrometry and nuclear magnetic resonance spectroscopy. Published: 10 February 2017 Some argimycins P showed a piperidine ring with a polyene side chain (argimycin Citation: PIX); others contain also a fused five-membered ring (argimycins PIV-PVI). Argimycins Ye S, Molloy B, Braña AF, Zabala D, Olano C, Cortés J, Morís F, Salas JA PI-PII showed a pyridine ring instead, and an additional N-acetylcysteinyl moiety. and Méndez C (2017) Identification These compounds seem to play a negative role in growth and colony differentiation by Genome Mining of a Type I in S. argillaceus, and some of them show weak antibiotic activity. A pathway for the Polyketide Gene Cluster from Streptomyces argillaceus Involved biosynthesis of argimycins P is proposed, based on the analysis of proposed enzyme in the Biosynthesis of Pyridine functions and on the structure of compounds encoded by the arp cluster. and Piperidine Alkaloids Argimycins P. Front. Microbiol. 8:194. Keywords: cryptic, type I polyketide synthase, pyridine, piperidine, thioester reductase, growth, alkaloid, doi: 10.3389/fmicb.2017.00194 Streptomyces Frontiers in Microbiology| www.frontiersin.org 1 February 2017| Volume 8| Article 194 fmicb-08-00194 February 8, 2017 Time: 14:51 # 2 Ye et al. New Alkaloid Compounds by Genome Mining INTRODUCTION that make suitable as potent drugs, but steep technological challenges associated with screening and manufacturing of these Natural products have been a productive source of bioactive molecules has stifled the discovery and development of natural compounds and drugs. By the mid-1990s, >50% of all new drugs products. The recent development of genomics, metagenomics were either natural products or analogs (Cragg et al., 1997). and high-throughput screening has increased exponentially the This frequency has decreased since then, due to the increased volume of useful genetic sequence information that can be efforts in synthetic small molecules, while the isolation and employed for natural products discovery. Additionally, a new characterization of new natural products from crude extracts manufacturing paradigm employing metabolic engineering and remains perceived as time-consuming and labor-intensive. synthetic biology as its engine has greatly accelerated the path of However, natural products and their analogs still account for development for microbial natural product drugs (Rutledge and >40% of small molecule drugs under development (Newman Challis, 2015; Katz and Baltz, 2016; Pawar et al., 2016). Genome and Cragg, 2016). The importance of drug development based mining consists of searching a genome for genes that encode on natural products and their analogs is, in part, associated with enzymes involved in a particular process. In recent years, genome their structural diversity, which facilitates opening up unexplored mining has been applied to streptomycetes and has become a chemical spaces (Bauer et al., 2010). The growing appreciation of new and quick approach to identify previously unknown gene functional assays and phenotypic screens may further contribute clusters (Olano et al., 2014; Rutledge and Challis, 2015; Ochi, to a revival of interest in natural products for drug discovery 2016). In most cases these clusters are silent or lowly expressed (Harvey et al., 2015). under laboratory conditions. Therefore, several strategies have Streptomycetes are Gram-positive bacteria with high GC been developed to awake and/or increase their expression, and DNA content that are mainly found in terrestrial and aquatic to identify the compounds encoded by these gene clusters, which environments, and show a complex development cycle, involving involve both the use of genetic engineering approaches and the formation of substrate and aerial mycelium, and spores (Flärdh use of different media and culture conditions (Olano et al., 2014; and Buttner, 2009; Barka et al., 2016). Moreover, they are Rutledge and Challis, 2015; Ochi, 2016). one of the most important sources of microbial bioactive In this paper we report the genome mining of Streptomyces compounds. These compounds are secondary metabolites argillaceus ATCC 12956, a producer of the known antitumoral that show, among others, antibiotic, antifungal, antiparasitic, drug mithramycin, and the use of this approach to uncover immunosuppressive or herbicide activities. In addition, some cryptic pathways, lowly or not expressed by the wild type strain. of them also play a role in colony development in these One of these pathways lowly expressed under standard laboratory microorganisms (Barka et al., 2016). Most bioactive compounds conditions, has been overexpressed and the products isolated and belong to the polyketide, peptide, hybrid polyketide-peptide characterized chemically and biologically, namely the argimycin and terpene groups. Polyketides (PK) are synthesized by P family of compounds. macroenzyme complexes called polyketide synthases (PKS) through the condensation of small acyl-CoA units. Three different types of PKS have been described so far. Type I or MATERIALS AND METHODS modular PKS are typically organized into modules, each one responsible for a single elongation cycle. Each module contains Bacterial Strains, Culture Conditions, a b-ketoacyl synthase (KS), an acyltransferase (AT) and an acyl Plasmids and DNA Manipulations carrier protein (ACP) domain. AT domains select the appropriate Streptomyces argillaceus ATCC 12956, and S. argillaceus GIH, acyl-CoA unit that will be used in the corresponding extension S. argillaceus AFTA and S. argillaceus AFTA-GIH (Zabala cycle; KS domains are responsible for the decarboxylative et al., 2013) were used as source of DNA and for gene Claisen condensations of acyl-CoA units; ACPs are non-catalytic replacement and expression experiments, and/or production domains that tether the growing PK chain and building block of argimycins P. For sporulation the strains were grown on its phosphopantetheine arm. After every condensation step, for 7 days at 30◦C on agar plates containing medium A the resulting b-keto group of the biosynthesis intermediate (Fernández et al., 1998). Streptomyces sp. NRRL S-1022 was could be modified before the next elongation step by functional used to test argimycins P production. SM10 and SM17 media domains optionally present in the corresponding module: were used for argimycins P production by S. argillaceus and ketoreductase (KR), dehydratase (DH), and enoyl reductase Streptomyces S-1022, respectively. When required, antibiotics (ER). KRs stereospecifically reduce the carbonyl group of the were added to media at the following final concentrations: b-ketoacyl-ACP intermediates leading to the formation of a ampicillin (100 mg/mL), kanamycin (50 mg/mL), nalidixic b-hydroxyl group; DH domains catalyze reversible dehydration acid (25 mg/mL), apramycin (25 mg/mL), and thiostrepton events to yield double bond formation between the a- and (50 mg/mL). A pKC505-based cosmid library of S. argillaceus b-carbons in the polyketide chain; ER domains catalyze the genome DNA was used to identify cosmids containing

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