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Belactosins and hormaomycins are peptide natural products containing 3-(2-aminocyclopropyl)alanine and 3-(2-nitrocyclopropyl)alanine residues, respectively, with opposite stereoconfigurations of the cyclopropane ring. Herein we demonstrate that the heme oxygenase-like enzymes BelK and HrmI catalyze the N-oxygenation of l-lysine to generate 6-nitronorleucine. The nonheme iron enzymes BelL and HrmJ then cyclize the nitroalkane moiety to the nitrocyclopropane ring with the desired stereochemistry found in the corresponding natural products. We also show that both cyclopropanases remove the 4-proS-H of 6-nitronorleucine during the cyclization, establishing the inversion and retention of the configuration at C4 during the BelL and HrmJ reactions, respectively. This study reveals the unique strategy for stereocontrolled cyclopropane synthesis in nature.

Nocardia spp. are filamentous Actinobacteria of the order Corynebacteriales and mostly known for their ability to cause localized and systemic infections in humans. However, the onset and progression of nocardiosis is only poorly understood, in particular the mechanisms of strain-specific presentations. Recent genome sequencing has revealed an extraordinary capacity for the production of specialized small molecules. Such secondary metabolites are often crucial for the producing microbe to survive the challenges of different environmental conditions. An interesting question thus concerns the role of these natural products in Nocardia-associated pathogenicity and immune evasion in a human host. In this review, a summary and discussion of Nocardia metabolites is presented, which may play a part in nocardiosis because of their cytotoxic, immunosuppressive and metal-chelating properties or otherwise vitally important functions. This review also contains so far unpublished data concerning the biosynthesis of these molecules that were obtained by detailed bioinformatic analyses.

We report a genomics‐guided exploration of the metabolic potential of the brasilicardin producer strain Nocardia terpenica IFM 0406. Bioinformatics analysis of the whole genome sequence revealed the presence of a biosynthetic gene cluster presumably responsible for the generation of formerly unknown nocobactin derivatives. Mass spectrometry‐assisted isolation led to the identification of three new siderophores, terpenibactins A (1), B (2) and C (3), which belong to the class of nocobactins. Their structures were elucidated by employing spectroscopic techniques. Compounds 1–3 demonstrated inhibitory activity towards the muscarinic M3 receptor, while exhibiting only a low cytotoxicity., Nature's combinatorial biosynthesis produces new bioactive nocobactin derivatives. Genome mining led to the discovery and characterization of terpenibactins A−C, three new nocobactins from the bacterium Nocardia terpenica IFM 0406 that exhibit unprecedented combinations of asteroidic acid, lipid and modified lysine residues. They demonstrate low cytotoxicity but potent inhibitory activity towards the muscarinic M3 receptor.

Pyrones comprise a structurally diverse class of compounds. Although they are widespread in nature, their specific physiological functions remain unknown in most cases. We recently described that triketide pyrones mediate the sulfotransfer in caprazamycin biosynthesis. Herein, we report the identification of conexipyrones A-C, three previously unrecognized tetra-substituted α-pyrones, from the soil actinobacterium Conexibacter woesei. Insights into their biosynthesis via a type III polyketide synthase were obtained by feeding studies using isotope-enriched precursors. In vitro assays employing the genetically associated 3'-phosphoadenosine-5'-phosphosulfate (PAPS)-dependent sulfotransferase CwoeST revealed conexipyrones as the enzymes' genuine sulfate acceptor substrates. Furthermore, conexipyrones were determined to function as sulfate shuttles in a two-enzyme assay, because their sulfated derivatives were accepted as donor molecules by the PAPS-independent arylsulfate sulfotransferase (ASST) Cpz4 to yield sulfated caprazamycin intermediates.

International audience; Genomics and metabolomics are widely used to explore specialized metabolite diversity. The Paired Omics Data Platform is a community initiative to systematically document links between metabolome and (meta)genome data, aiding identification of natural product biosynthetic origins and metabolite structures.

Our work emphasizes that Nocardia represent a prolific source for natural products rivaling better-characterized genera such as Streptomyces or Amycolatopsis. Furthermore, we showed that large-scale analysis of biosynthetic gene clusters using similarity networks with high stringency allows the distinction and prediction of natural product structural variations. This will facilitate future genomics-driven drug discovery campaigns., Using automated genome analysis tools, it is often unclear to what degree genetic variability in homologous biosynthetic pathways relates to structural variation. This hampers strain prioritization and compound identification and can lead to overinterpretation of chemical diversity. Here, we assessed the metabolic potential of Nocardia, an underinvestigated actinobacterial genus that is known to comprise opportunistic human pathogens. Our analysis revealed a plethora of putative biosynthetic gene clusters of various classes, including polyketide, nonribosomal peptide, and terpenoid pathways. Furthermore, we used the highly conserved biosynthetic pathway for nocobactin-like siderophores to investigate how gene cluster differences correlate to structural differences in the produced compounds. Sequence similarity networks generated by BiG-SCAPE (Biosynthetic Gene Similarity Clustering and Prospecting Engine) showed the presence of several distinct gene cluster families. Metabolic profiling of selected Nocardia strains using liquid chromatography-mass spectrometry (LC-MS) metabolomics data, nuclear magnetic resonance (NMR) spectroscopy, and GNPS (Global Natural Product Social molecular networking) revealed that nocobactin-like biosynthetic gene cluster (BGC) families above a BiG-SCAPE threshold of 70% can be assigned to distinct structural types of nocobactin-like siderophores. IMPORTANCE Our work emphasizes that Nocardia represent a prolific source for natural products rivaling better-characterized genera such as Streptomyces or Amycolatopsis. Furthermore, we showed that large-scale analysis of biosynthetic gene clusters using similarity networks with high stringency allows the distinction and prediction of natural product structural variations. This will facilitate future genomics-driven drug discovery campaigns.

Metalloproteinase inhibitors often feature hydroxamate moieties to facilitate the chelation of metal ions in the catalytic center of target enzymes. Actinonin and matlystatins are potent metalloproteinase inhibitors that comprise rare N-hydroxy-2-pentyl-succinamic acid warheads. Here we report the identification and characterization of their biosynthetic pathways. By gene cluster comparison and a combination of precursor feeding studies, heterologous pathway expression and gene deletion experiments we are able to show that the N-hydroxy-alkyl-succinamic acid warhead is generated by an unprecedented variation of the ethylmalonyl-CoA pathway. Moreover, we present evidence that the remarkable structural diversity of matlystatin congeners originates from the activity of a decarboxylase-dehydrogenase enzyme with high similarity to enzymes that form epoxyketones. We further exploit this mechanism to direct the biosynthesis of non-natural matlystatin derivatives. Our work paves the way for follow-up studies on these fascinating pathways and allows the identification of new protease inhibitors by genome mining., Metalloproteinase inhibitors are leads for drug development, but their biosynthetic pathways are often unknown. Here the authors show that the acyl branched warhead of actinonin and matlystatins derives from an ethylmalonyl-CoA-like pathway and the structural diversity of matlystatins is due to the activity of a decarboxylase-dehydrogenase enzyme.

Belactosins and cystargolides are natural product proteasome inhibitors from Actinobacteria. Both feature dipeptidic backbones and a unique beta-lactone building block. Herein, we present a detailed investigation of their biosynthesis. Identification and analysis of the corresponding gene clusters indicated that both compounds are assembled by rare single-enzyme amino acid ligases. Feeding experiments with isotope-labeled precursors and in vitro biochemistry showed that the formation of the beta-lactone warhead is unprecedented and reminiscent of leucine biosynthesis, and that it involves the action of isopropylmalate synthase homologues.

Belactosine und Cystargolide sind von Actinobakterien produzierte Naturstoffe mit proteasominhibitorischen Eigenschaften. Beide weisen ein Peptidruckgrat aus zwei Aminosauren sowie einen einzigartigen β-Lacton-Baustein auf. Hier wird eine detaillierte Untersuchung der Biosynthese der beiden Naturstoffe beschrieben. Die Identifizierung und Analyse der entsprechenden Gencluster weist darauf hin, dass beide Stoffe uber seltene Aminosaure-Ligasen gebildet werden. Futterungsversuche mit isotopenmarkierten Vorstufen und In-vitro-Biochemie zeigen einen bislang unbeschriebenen Mechanismus zur β-Lacton-Biosynthese, der sich aus der Leucin-Bildung ableitet und die Beteiligung eines Isopropylmalat-Synthase-ahnlichen Enzyms vorsieht.

Bacteria are a rich source for small-molecule inhibitors of proteases and protease-like enzymes with various therapeutic applications. These molecules often comprise distinct structural moieties, so called warheads which mediate specific interactions with the target enzymes. Knowledge on the biosynthesis of the protease inhibitors warheads can be used to search for orphan pathways with similar capacities in bacterial genomes. The identified biosynthetic gene clusters likely encode the production of new protease inhibitors with the same mode of action as the template compound.

Covering: up to 2019 Inhibitors of proteases and related enzymes have versatile applications in medicine and other areas. They are used in the clinic e.g. for the treatment of cancer, hypertension, thrombosis, diabetes as well as viral and bacterial infections. Most of these drugs are produced synthetically but a substantial part of them has been developed from or are inspired by natural products. Protease inhibitors typically mimic the peptide substrates of the target enzymes and feature specialized moieties that specifically interact with catalytic residues in the active centre. Such moieties may consist of electrophilic warheads e.g. β-lactones, Michael systems, epoxyketones or β-lactams that are attacked by active site nucleophiles and generate covalent adducts. Metalloproteinase inhibitors often contain functional groups which facilitate the chelation of active site metal ions e.g. hydroxamates, carboxylates or phosphoramidates. Other reversible protease inhibitors feature γ-amino acids or ketomethylene pseudopeptides to form stable substrate or transition state analogs. The discovery of such functional groups in natural products has been extensively exploited by medicinal chemistry to generate synthetic protease inhibitors. However, the biosynthetic principles for many of these moieties have remained obscure until recently. This review summarizes the current knowledge on the biosynthesis of important and/or structurally interesting inhibitors of proteases and related enzymes. Understanding the genetic basis that directs the formation of the specialized, activity-conferring moieties in protease inhibitors will allow targeted genome mining for the discovery of new derivatives.

Phosphoramidon is a potent metalloprotease inhibitor and a widespread tool in cell biology research. It contains a dipeptide backbone that is uniquely linked to a 6-deoxysugar via a phosphoramidate bridge. Herein, we report the identification of a gene cluster for the formation of phosphoramidon and its detailed characterization. In vitro reconstitution of the biosynthesis established TalE as a phosphoramidate-forming kinase and TalC as the glycosyltransferase which installs the L-rhamnose moiety by phosphoester linkage.

The α',β'-epoxyketone moiety of proteasome inhibitors confers high binding specificity to the N-terminal threonine in catalytic proteasome β-subunits. We recently identified the epoxomicin and eponemycin biosynthetic gene clusters and have now conducted isotope-enriched precursor feeding studies and comprehensive gene deletion experiments to shed further light on their biosynthetic pathways. Leucine and two methyl groups from S-adenosylmethionine were readily incorporated into the epoxyketone warhead, suggesting decarboxylation of the thioester intermediate. Formation of the α',β'-epoxyketone is likely mediated by conserved acyl-CoA dehydrogenase-like enzymes, as indicated by complete loss of epoxomicin and eponemycin production in the respective knockout mutants. Our results clarify crucial questions in the formation of epoxyketone compounds and lay the foundation for in vitro biochemical studies on the biosynthesis of this pharmaceutically important class of proteasome inhibitors.

The hydroxamate moiety of the natural product actinonin mediates inhibition of metalloproteinases because of its chelating properties towards divalent cations in the active site of those enzymes. Owing to its antimicrobial activity, actinonin has served as a lead compound for the development of new antibiotic drug candidates. Recently, we identified a putative gene cluster for the biosynthesis of actinonin. Here, we confirm and characterize this cluster by heterologous pathway expression and gene-deletion experiments. We assigned the biosynthetic gene cluster to actinonin production and determine the cluster boundaries. Furthermore, we establish that ActI, an AurF-like oxygenase, is responsible for the N-hydroxylation reaction that forms the hydroxamate warhead. Our findings provide the basis for more detailed investigations of actinonin biosynthesis.

M.H.M. was supported by a Rubicon fellowship of the Netherlands Organization for Scientific Research (NWO;Rubicon 825.13.001). The work of R.K. was supported by the European Union’s Seventh Framework Programme(Joint Call OCEAN.2011–2: Marine microbial diversity—new insights into marine ecosystems functioning and its biotechnological potential) under the grant agreement no.287589 (Micro B3). M.C. was supported by a Biotechnology and Biological Sciences Research Council (BBSRC)studentship (BB/J014478/1). The GSC is supported by funding from the Natural Environment Research Council(UK), the National Institute for Energy Ethics and Society(NIEeS; UK), the Gordon and Betty Moore Foundation,the National Science Foundation (NSF; US) and the US Department of Energy. The Manchester Synthetic Biology Research Centre, SYNBIOCHEM, is supported by BBSRC/Engineering and Physical Sciences Research Council(EPSRC) grant BB/M017702/1, Medema, M.H., Kottmann, R., Yilmaz, P., Cummings, M., Biggins, J.B., Blin, K., De Bruijn, I., Chooi, Y.H., Claesen, J., Coates, R.C., Cruz-Morales, P., Duddela, S., Düsterhus, S., Edwards, D.J., Fewer, D.P., Garg, N., Geiger, C., Gomez-Escribano, J.P., Greule, A., Hadjithomas, M., Haines, A.S., Helfrich, E.J.N., Hillwig, M.L., Ishida, K., Jones, A.C., Jones, C.S., Jungmann, K., Kegler, C., Kim, H.U., Kötter, P., Krug, D., Masschelein, J., Melnik, A.V., Mantovani, S.M., Monroe, E.A., Moore, M., Moss, N., Nützmann, H.-W., Pan, G., Pati, A., Petras, D., Reen, F.J., Rosconi, F., Rui, Z., Tian, Z., Tobias, N.J., Tsunematsu, Y., Wiemann, P., Wyckoff, E., Yan, X., Yim, G., Yu, F., Xie, Y., Aigle, B., Apel, A.K., Balibar, C.J., Balskus, E.P., Barona-Gómez, F., Bechthold, A., Bode, H.B., Borriss, R., Brady, S.F., Brakhage, A.A., Caffrey, P., Cheng, Y.Q., Clardy, J., Cox, R.J., De Mot, R., Donadio, S., Donia, M.S., Van Der Donk, W.A., Dorrestein, P.C., Doyle, S., Driessen, A.J.M., Ehling-Schulz, M., Entian, K.-D., Fischbach, M.A., Gerwick, L., Gerwick, W.H., Gross, H., Gust, B., Hertweck, C., Höfte, M., Jensen, S.E., Ju, J., Katz, L., Kaysser, L., Klassen, J.L., Keller, N.P., Kormanec, J., Kuipers, O.P., Kuzuyama, T., Kyrpides, N.C., Kwon, H.-J., Lautru, S., Lavigne, R., Lee, C.Y., Linquan, B., Liu, X., Liu, W., Luzhetskyy, A., Mahmud, T., Mast, Y., Méndez, C., Metsä-Ketelä, M., Micklefield, J., Mitchell, D.A., Moore, B.S., Moreira, L.M., Müller, R., Neilan, B.A., Nett, M., Nielsen, J., O'Gara, F., Oikawa, H., Osbourn, A., Osburne, M.S., Ostash, B., Payne, S.M., Pernodet, J.-L., Petricek, M., Piel, J., Ploux, O., Raaijmakers, J.M., Salas, J.A., Schmitt, E.K., Scott, B., Seipke, R.F., Shen, B., Sherman, D.H., Sivonen, K., Smanski, M.J., Sosio, M., Stegmann, E., Süssmuth, R.D., Tahlan, K., Thomas, C.M., Tang, Y., Truman, A.W., Viaud, M., Walton, J.D., Walsh, C.T., Weber, T., Van Wezel, G.P., Wilkinson, B., Willey, J.M., Wohlleben, W., Wright, G.D., Ziemert, N., Zhang, C., Zotchev, S.B., Breitling, R., Takano, E., Glöckner, F.O.

The vanadium-dependent chloroperoxidase Mcl24 was discovered to mediate a complex series of unprecedented transformations in the biosynthesis of the merochlorin meroterpenoid antibiotics. In particular, a site-selective naphthol chlorination is followed by a sequence of oxidative dearomatization/terpene cyclization reactions to build up the stereochemically complex carbon framework of the merochlorins in one step. Inspired by the enzyme reactivity, we developed a chemical chlorination protocol paralleling the biocatalytic process. These chemical studies led to the identification of previously overlooked merochlorin natural products.

The polycycles merochlorin A and B are complex halogenated meroterpenoid natural products with significant antibacterial activities and are produced by the marine bacterium Streptomyces sp. strain CNH-189. Heterologously produced enzymes and chemical synthesis are employed herein to fully reconstitute the merochlorin biosynthesis in vitro. The interplay of a dedicated type III polyketide synthase, a prenyl diphosphate synthase, and an aromatic prenyltransferase allow formation of a highly unusual aromatic polyketide-terpene hybrid intermediate which features an unprecedented branched sesquiterpene moiety from isosesquilavandulyl diphosphate. As supported by in vivo experiments, this precursor is furthermore chlorinated and cyclized to merochlorin A and isomeric merochlorin B by a single vanadium-dependent haloperoxidase, thus completing the remarkably efficient pathway.

Caprazamycins (CPZs) belong to a group of liponucleoside antibiotics inhibiting the bacterial MraY translocase, an essential enzyme involved in peptidoglycan biosynthesis. We have recently identified analogs that are decorated with a sulfate group at the 2″-hydroxy of the aminoribosyl moiety, and we now report an unprecedented two-step sulfation mechanism during the biosynthesis of CPZs. A type III polyketide synthase (PKS) known as Cpz6 is used in the biosynthesis of a group of new triketide pyrones that are subsequently sulfated by an unusual 3'-phosphoadenosine-5'-phosphosulfate (PAPS)-dependent sulfotransferase (Cpz8) to yield phenolic sulfate esters, which serve as sulfate donors for a PAPS-independent arylsulfate sulfotransferase (Cpz4) to generate sulfated CPZs. This finding is to our knowledge the first demonstration of genuine sulfate donors for an arylsulfate sulfotransferase and the first report of a type III PKS to generate a chemical reagent in bacterial sulfate metabolism.

Meroterpenoids are mixed polyketide-terpenoid natural products with a broad range of biological activities. Herein, we present the structures of four new meroterpenoid antibiotics, merochlorins A-D, produced by the marine bacterium Streptomyces sp. strain CNH-189, which possess novel chemical skeletons unrelated to known bacterial agents. Draft genome sequencing, mutagenesis, and heterologous biosynthesis in the genome-minimized model actinomycete Streptomyces coelicolor provided the 57.6 kb merochlorin gene cluster that contains two genes encoding rare bacterial vanadium-dependent haloperoxidase (VHPO) genes. Pathway expression of two different fosmid clones that differ largely by the presence or absence of the VHPO gene mcl40 resulted in the differential biosynthesis of merochlorin C, suggesting that Mcl40 catalyzes an unprecedented 15-membered chloronium-induced macrocyclization reaction converting merochlorin D to merochlorin C.

Terpenoids are arguably the largest and most diverse family of natural products, featuring prominently in e.g. signalling, self-defence, UV-protection and electron transfer. Prenyltransferases are essential players in terpenoid and hybrid isoprenoid biosynthesis that install isoprene units on target molecules and thereby often modulate their bioactivity. In our search for new prenyltransferase biocatalysts we focused on the marine-derived Streptomyces sp. CNQ-509, a particularly rich source of meroterpenoid chemistry. Sequencing and analysis of the genome of Streptomyces sp. CNQ-509 revealed seven putative phenol/phenazine-specific ABBA prenyltransferases, and one putative indole-specific ABBA prenyltransferase. To elucidate the substrate specificity of the ABBA prenyltransferases and to learn about their role in secondary metabolism, CnqP1 –CnqP8 were produced in Escherichia coli and incubated with various aromatic and isoprenoid substrates. Five of the eight prenyltransferases displayed enzymatic activity. The efficient conversion of dihydroxynaphthalene derivatives by CnqP3 (encoded by AA958_24325) and the co-location of AA958_24325 with genes characteristic for the biosynthesis of THN (tetrahydroxynaphthalene)-derived natural products indicates that the enzyme is involved in the formation of debromomarinone or other naphthoquinone-derived meroterpenoids. Moreover, CnqP3 showed high flexibility towards a range of aromatic and isoprenoid substrates and thus represents an interesting new tool for biocatalytic applications.

The development of reliable analytic methods, capable of separating mixtures of secondary metabolites as well as providing structural information, is essential for the investigation of secondary metabolites, e.g. from Streptomyces. Here we report a liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) method using a triple quadrupole mass analyzer for the structural elucidation of caprazamycins and liposidomycins from culture extracts of the wild-type producer strains. Comparison of the fragmentation patterns in positive as well as in negative ionization mode revealed several characteristic product ions used for identification of six new caprazamycins. Furthermore, a chromatographic method for the purification of nucleosides from cell cultures using a boronic acid gel was adapted for the partial purification of the culture extracts. Copyright © 2011 John Wiley & Sons, Ltd.

Napsamycins are potent inhibitors of bacterial translocase I, an essential enzyme in peptidoglycan biosynthesis, and are classified as uridylpeptide antibiotics. They comprise an N-methyl diaminobutyric acid, an ureido group, a methionine and two non-proteinogenic aromatic amino acid residues in a peptide backbone that is linked to a 5'-amino-3'-deoxyuridine by an unusual enamide bond. The napsamycin gene cluster was identified in Streptomyces sp. DSM5940 by using PCR probes from a putative uridylpeptide biosynthetic cluster found in S. roseosporus NRRL15998 by genome mining. Annotation revealed 29 hypothetical genes encoding for resistance, regulation and biosynthesis of the napsamycins. Analysis of the gene cluster indicated that the peptide core structure is assembled by a nonlinear non-ribosomal peptide synthetase (NRPS)-like mechanism that involves several discrete single or didomain proteins. Some genes could be assigned, for example, to the synthesis of the N-methyl diaminobutyric acid, to the generation of m-tyrosine and to the reduction of the uracil moiety. The heterologous expression of the gene cluster in Streptomyces coelicolor M1154 resulted in the production of napsamycins and mureidomycins as demonstrated by LC-ESI-MS and MS/MS analysis. The napsamycin gene cluster provides a molecular basis for the detailed study of the biosynthesis of this class of structurally unusual compounds.

Caprazamycins are potent anti-mycobacterial liponucleoside antibiotics isolated from Streptomyces sp. MK730-62F2 and belong to the translocase I inhibitor family. Their complex structure is derived from 5′-(β-O-aminoribosyl)-glycyluridine and comprises a unique N-methyldiazepanone ring. The biosynthetic gene cluster has been identified, cloned, and sequenced, representing the first gene cluster of a translocase I inhibitor. Sequence analysis revealed the presence of 23 open reading frames putatively involved in export, resistance, regulation, and biosynthesis of the caprazamycins. Heterologous expression of the gene cluster in Streptomyces coelicolor M512 led to the production of non-glycosylated bioactive caprazamycin derivatives. A set of gene deletions validated the boundaries of the cluster and inactivation of cpz21 resulted in the accumulation of novel simplified liponucleoside antibiotics that lack the 3-methylglutaryl moiety. Therefore, Cpz21 is assigned to act as an acyltransferase in caprazamycin biosynthesis. In vivo and in silico analysis of the caprazamycin biosynthetic gene cluster allows a first proposal of the biosynthetic pathway and provides insights into the biosynthesis of related uridyl-antibiotics.

These are the first examples of total enzymatic syntheses of meroterpenoid natural products.

Streptomyces sp. CNQ-509 is a marine actinomycete belonging to the MAR4 streptomycete lineage. MAR4 strains have been linked to the production of diverse and otherwise rare meroterpenoid compounds. The genome sequence of Streptomyces sp. CNQ-509 was found to contain 29 putative gene clusters for the biosynthesis of secondary metabolites, some of them potentially involved in the formation of meroterpenoid molecules.

The epoxyketone proteasome inhibitors are an established class of therapeutic agents for the treatment of cancer. Their unique α′,β′-epoxyketone pharmacophore allows binding to the catalytic β-subunits of the proteasome with extraordinary specificity. Here we report the characterization of the first gene clusters for the biosynthesis of natural peptidyl-epoxyketones. The clusters for epoxomicin, the lead compound for the anti-cancer drug Kyprolis™, and for eponemycin were identified in the actinobacterial producer strains ATCC 53904 and Streptomyces hygroscopicus ATCC 53709, respectively, using a modified protocol for Ion Torrent PGM genome sequencing. Both gene clusters code for a hybrid non-ribosomal peptide synthetase/polyketide synthase multifunctional enzyme complex and homologous redox enzymes. Epoxomicin and eponemycin were heterologously produced in Streptomyces albus J1046 via whole pathway expression. Moreover, we employed mass spectral molecular networking for a new comparative metabolomics approach in a heterologous system and discovered a number of putative epoxyketone derivatives. With this study we have definitively linked epoxyketone proteasome inhibitors and their biosynthesis genes for the first time in any organism, which will now allow for their detailed biochemical investigation.

The development of reliable analytic methods, capable of separating mixtures of secondary metabolites as well as providing structural information, is essential for the investigation of secondary metabolites, e.g. from Streptomyces. Here we report a liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) method using a triple quadrupole mass analyzer for the structural elucidation of caprazamycins and liposidomycins from culture extracts of the wild-type producer strains. Comparison of the fragmentation patterns in positive as well as in negative ionization mode revealed several characteristic product ions used for identification of six new caprazamycins. Furthermore, a chromatographic method for the purification of nucleosides from cell cultures using a boronic acid gel was adapted for the partial purification of the culture extracts.

Caprazamycins are potent antimycobacterials isolated from Streptomyces sp. MK730F-62F2 (23). In a pulmonary tuberculosis mouse model, they showed a therapeutic effect but no significant toxicity (24). The caprazamycins are assigned to the translocase I inhibitors (27) due to their structural similarity to the liposidomycins (34, 43), which have been studied in more detail. Translocase I catalyzes the first step in the membrane-linked reaction cycle of bacterial cell wall formation (52): the transfer of phospho-N-acetylmuramic acid-l-Ala-γ-d-Glu-m-diaminopimelic acid-d-Ala-d-Ala from UMP to the lipid carrier undecaprenyl phosphate. Structurally unique in nature, the caprazamycins and liposidomycins share a 5′-β-O-aminoribosyl-glycyluridine and a rare N-methylated diazepanone as their characteristic feature (Fig. ​(Fig.1)1) (25, 53). Attached at the 3″ position are β-hydroxylated fatty acid groups of different chain lengths, carrying a 3-methylglutarate. While the liposidomycins are sulfated, the caprazamycins lack this group. Instead, they are glycosylated with a 2,3,4-O-methyl-l-rhamnose and therefore belong to the large number of bioactive compounds containing 6-deoxyhexoses. Usually, these moieties contribute significantly to the compounds' properties, influencing, e.g., molecule-target interactions, cell import and export, pharmacokinetics, and solubility (56). The biosynthesis of deoxysugars has been studied in detail and generally starts from NDP-activated hexoses via 4-keto-6-deoxy intermediates (36). The formation of l-rhamnose involves four enzymes, a dTDP-d-glucose synthase, a dTDP-d-glucose 4,6-dehydratase, a dTDP-3,5-deoxyglucose epimerase, and a 4-ketoreductase. Recent advances in combinatorial biosynthesis have led to a variety of novel natural products with an engineered glycosylation pattern and altered bioactivity (37, 44). The key step in this approach is the attachment of different deoxysugars to the aglycones, which demands substrate-flexible glycosyltransferases. FIG. 1. Structure of caprazamycins and organization of the caprazamycin gene cluster (cpz9 to cpz31) lacking the genes for deoxysugar formation. Assignments of genes to different steps in the biosynthesis are indicated. We recently reported cloning and heterologous expression of cosmid cpzLK09 containing the first identified gene cluster of a translocase I inhibitor, the caprazamycins. However, genes for the formation of the dTDP-l-rhamnose could not be identified on this cosmid (Fig. ​(Fig.1)1) and therefore only caprazamycin aglycones were produced by the heterologous host, Streptomyces coelicolor M512/cpzLK09 (31). The absence of genes for the formation of the l-rhamnose moiety within the corresponding biosynthetic gene cluster has previously been reported for aranciamycin (38), steffimycin (16), spinosyn (55), and elloramycin (9). Since potential genes for O methylation (cpz28 to cpz30) and a glycosyltransferase (cpz31) are encoded in the caprazamycin gene cluster, we speculated that only four genes are missing for successful heterologous production of intact caprazamycins. Here we report the identification of the genes required for the biosynthesis of the caprazamycin deoxysugar moiety elsewhere on the genome of Streptomyces sp. MK730-62F2. A new strategy was developed, based on Red/ET-mediated recombination, to assemble the identified subcluster into cosmid cpzLK09. Expression of the assembled cluster readily resulted in the production of intact caprazamycins in the heterologous producer strain. Moreover, in vitro studies demonstrated that Cpz31 is the glycosyltransferase in caprazamycin biosynthesis.

Several strains that grow on medium-chain-length alkanes and catalyze interesting hydroxylation and epoxidation reactions do not possess integral membrane nonheme iron alkane hydroxylases. Using PCR, we show that most of these strains possess enzymes related to CYP153A1 and CYP153A6, cytochrome P450 enzymes that were characterized as alkane hydroxylases. A vector for the polycistronic coexpression of individual CYP153 genes with a ferredoxin gene and a ferredoxin reductase gene was constructed. Seven of the 11 CYP153 genes tested allowed Pseudomonas putida GPo12 recombinants to grow well on alkanes, providing evidence that the newly cloned P450s are indeed alkane hydroxylases.