Natural Products Hot Paper DOI: 10.1002/anie.201402632 Discovery of Clostrubin, an Exceptional Polyphenolic Polyketide Antibiotic from a Strictly Anaerobic Bacterium** Sacha Pidot, Keishi Ishida, Michael Cyrulies, and Christian Hertweck* Abstract: Genome mining of the strictly anaerobic bacterium Clostridium beijerinckii, an industrial producer of solvents, revealed the presence of several cryptic gene clusters for secondary metabolite biosynthesis. To unearth its metabolic potential, a C. beijerinckii strain was cultured under various conditions, which led to the discovery of a deep purple pigment. This novel metabolite, named clostrubin (1), was isolated and its structure was fully elucidated. The pentacyclic polyphenol features a benzo[a]tetraphene ring topology that is unprece- dented for natural products. Stable-isotope labeling experi- ments showed that 1 is an aromatic polyketide that folds in a noncanonical manner to form the unusual perifused ring system. In addition to being the first reported polyketide from an anaerobic bacterium, 1 is a potent antibiotic with pro- nounced activity against various pathogenic bacteria, such as MRSA, VRE, and mycobacteria, with minimum inhibitory concentrations (MIC) of 0.12–0.97 mm. Bacterial infectious diseases are a persistent global concern and causing millions of deaths annually. According to the WHO, the rapid emergence of resistant pathogens and the lack of new therapeutics portend a return to the pre-antibiotic era. [1] Increased infections caused by the hospital-acquired pathogens methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterocci (VRE), com- bined with the emergence of multidrug-resistant tuberculosis (MDR-TB), demand the urgent development of new anti- biotic agents. [2, 3] Natural products, in particular microbial secondary metabolites, are the most important source of pharmacologically active compounds. [4] Traditionally, actino- mycetes have been regarded as the mainstay of natural- product discovery in bacteria. [5] However, a problem inherent in screening these well-investigated organisms is the high rediscovery rate of known compounds and scaffolds. [6] In addition to myxobacteria [7] , neglected bacterial species, the secondary metabolic potential of which has been poorly studied, have thus been considered as alternative sources of new bioactive compounds. [8] Indeed, the analyses of sequenced bacterial genomes for gene clusters for secondary metabolite biosynthesis have shown that unidentified leads may be unearthed from obscure bacterial lineages. [9] Even bacteria that were once completely discounted as a source of natural products, such as the anaerobes, contain the genetic material for the production of a wide array of secondary metabolites. [10] This holds true for the clostridia, a heteroge- neous group of Gram-positive spore-forming obligate anaer- obes that includes well-known pathogens that produce severe neurotoxins and numerous non-pathogenic strains that are used on an industrial scale for cellulose degradation and solvent production. [11] Through the isolation of the rare polythioamide antibiotic closthioamide from Clostridium cellulolyticum, we have shown that these organisms can indeed produce unusual biologically active compounds with novel structural features. [12] Herein, we report the discovery of an unprecedented type of antibiotic from Clostridium beijer- inckii and demonstrate its high potency against nosocomial pathogens such as MRSA, and VRE, and mycobacteria. We also report this new compound as the first polyketide from an anaerobic organism and demonstrate that the unusual poly- phenolic ring system derives from a noncanonical chain- folding pattern. In the course of investigating different Clostridium species for their potential to produce secondary metabolites, we noted that the genome of C. beijerinckii (NCIMB 8052, JGI project ID 3634512) harbors orphan secondary metabolite biosynthesis genes. [10a] Surprisingly, C. beijerinckii has been extensively used for the biotechnological production of the solvents acetone, butanol, and ethanol, [13] yet no secondary metabolite has been reported from this organism. Given the presence of secondary metabolite biosynthesis genes in the type strain C. beijerinckii (NCIMB 8052), we reasoned that other C. beijerinckii strains may also possess these genes, and because the industrially used strain was not readily available, we investigated a related C. beijerinckii strain from the HKI strain collection. Initial fermentation of C. beijerinckii (HKI0724) in liquid media showed the cultures to be deep red to purple in color, most likely owing to the formation of an unknown pigment. Interestingly, similarly colored C. beijer- inckii strains have not been reported in the literature, and in general, pigments are not a common feature of Clostridium species. As such, we sought to identify the type and composition of this pigment. [*] Dr. S. Pidot, [+] Dr. K. Ishida, [+] M. Cyrulies, Prof. Dr. C. Hertweck Department of Biomolecular Chemistry and BioPilot Plant Leibniz Institute for Natural Product Research and Infection Biology, HKI Beutenbergstrasse 11a, 07745 Jena (Germany) E-mail: christian.hertweck@hki-jena.de Prof. Dr. C. Hertweck Chair of Natural Product Chemistry, Friedrich Schiller University, Jena (Germany) [ + ] These authors contributed equally to this work. [**] We thank Andrea Perner, Heike Heinecke, and Christiane Weigel for mass spectrometry, NMR spectroscopy, and antibacterial assays, respectively. This project was supported by the Pakt für Forschung und Innovation. S.P. is the recipient of an Alexander von Humboldt Postdoctoral Fellowship. Supporting information (including experimental details) for this article is available on the WWW under http://dx.doi.org/10.1002/ anie.201402632. . Angewandte Communications 7856  2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Angew. Chem. Int. Ed. 2014, 53, 7856 –7859