Natural Products DOI: 10.1002/ange.200906114 Closthioamide: An Unprecedented Polythioamide Antibiotic from the Strictly Anaerobic Bacterium Clostridium cellulolyticum** Thorger Lincke, Swantje Behnken, Keishi Ishida, Martin Roth, and Christian Hertweck* Bacteria belonging to the genus Clostridium are among the most prominent microorganisms that lead an obligate anae- robic lifestyle. Clostridia occur in gastrointestinal tracts and are ubiquitously distributed in soil and sediments, rapidly decaying organic matter. [1] Owing to their potent catabolic properties, interest in these organisms has grown rapidly over the past few years. Not only are clostridia routinely employed to degrade anthropogenic cellulosic waste products, they have also been increasingly exploited to meet the need for renewable chemicals and biofuels. [2] Although various patho- genic species produce the most powerful neurotoxins known to mankind, the tetanus and botulinum toxins, respectively, [3] no secondary metabolites have yet been isolated from these or any other strictly anaerobic bacteria. However, bioinfor- matics analysis (“mining”) of the recently sequenced genomes of Clostridium spp., for example, Clostridium kluyveri [4] and Clostridium cellulolyticum, indicated that these bacteria harbor genes for the biosynthesis of secondary metabolites. Since the encoded cryptic natural products have been over- looked so far, it appears the biosynthesis genes remain dormant under standard laboratory conditions and are only triggered in the presence of particular stimuli. Herein we report the discovery of the first secondary metabolite, a hitherto fully unprecedented type of polythioamide, from a strictly anaerobic bacterium, Clostridium cellulolyticum. C. cellulolyticum is an anaerobic nonruminal Gram-pos- itive bacterium that was isolated from decayed grass compost and is an important industrial strain as a result of its ability to degrade crystalline cellulose. [5] Yet, no secondary metabolite has been reported from this well-known model organism. To investigate secondary-metabolite production, we cultivated a strain of C. cellulolyticum, DSM 5812 (= ATCC 35319), in 1 L fermenters with pH control by using complex media. How- ever, under these standard growth conditions as reported for this strain, no secondary-metabolite formation was observed. Therefore, we sought to induce natural product biosynthesis by applying external triggers. Several supplements, such as nutrients and various chemicals, were administered, and stress conditions were used (see the Supporting Information). All cultures were extracted and analyzed by reversed-phase (RP) HPLC. Unfortunately, no secondary metabolites were detected in any of the extracts. We then sought to exploit yet unknown environmental cues that occur in the natural habitat. Thus, as the bacterium had been isolated from decayed grass compost, we added an aqueous soil extract to the fermenter prior to inoculation. Most surprisingly, under these conditions, the RP-HPLC profile showed new peaks with a maximum absorbance at 270 nm (Figure 1). This effect could be reproduced with various soil samples (fen, compost) from different sources, geographical locations, and layers. Careful analysis of the soil extracts demonstrated unequivocally that the new compounds were not components of the supplement (see the Supporting Information). The major metabolite, compound 1, was isolated from 5 L of culture broth and purified by sequential open-column chromatography and RP-HPLC, which yielded 1.07 mg of a pale-yellow compound. According to HRMS measurements, 1 had the molecular formula C 29 H 38 N 6 O 2 S 6 and thus appeared to be remarkably rich in sulfur atoms. Furthermore, NMR spectroscopic data showed that the molecule was symmet- rical: the 13 C NMR and DEPT spectra indicated the presence of six methylene, two methine, and five quaternary carbon atoms. In the 1 H NMR spectrum, signals corresponding to two olefinic and six methylene hydrogen atoms were observed. The HMBC correlation between 2-H (d = 3.62 ppm) and C2’ (d = 44.1 ppm) confirmed the symmetrical structure of 1. Extensive 1 H- 1 H COSY, single-bond 1 H- 13 C HSQC, and 1 H- 13 C long-range HMBC analyses revealed one p-hydroxyben- zoyl, one diaminopropane, and two b-alanine units. However, several lines of evidence excluded the possibility of a canon- ical amide linkage of these building blocks. First, an amide carbonyl carbon shift would be expected at around 170 ppm. Instead, the signals of the quaternary carbon atoms C9 (d = 199.1 ppm), C6 (d = 203.3 ppm), and C3 (d = 202.9 ppm) [*] Dipl.-Chem. T. Lincke, [+] Dipl.-Biochem. S. Behnken, [+] Dr. K. Ishida, Dr. M. Roth, Prof. Dr. C. Hertweck Leibniz-Institut für Naturstoff-Forschung und Infektionsbiologie HKI, Biomolekulare Chemie und Biotechnikum Beutenbergstrasse 11a, 07745 Jena (Germany) Fax: (+ 49) 3641-532-0804 E-mail: christian.hertweck@hki-jena.de Prof. Dr. C. Hertweck Friedrich-Schiller-Universität, Jena (Germany) [ + ] These authors contributed equally to this study. [**] We thank A. Perner and F. Rhein for MS and NMR measurements, C. Weigel and Dr. H.-M. Dahse for performing biological assays, M. Cyrulies and U. Knüpfer for fermentations, Dr. S. Nietzsche (Electron Microscopy Center of the University Hospital, Jena) for providing scanning electron micrographs, and A. Thuille (MPI for Biogeochemistry) for soil samples. This research was supported financially by the “Pakt für Forschung und Innovation” of the Free State of Thuringia, the Federal Ministry of Science and Technology (BMBF, Germany), the DFG-funded graduate school of excellence Jena School for Microbial Communication (JSMC), and the International Leibniz Research School for Microbial and Biomolec- ular Interactions (ILRS). Supporting information for this article, including experimental details, is available on the WWW under http://dx.doi.org/10.1002/ anie.200906114. Angewandte Chemie 2055 Angew. Chem. 2010, 122, 2055 –2057  2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim