Characterization of the cystargolide biosynthetic gene cluster and functional analysis of the methyltransferase CysG.

Cystargolides are natural products originally isolated from Kitasatospora cystarginea NRRL B16505 as inhibitors of the proteasome. They are composed of a dipeptide backbone linked to a ß-lactone warhead. Recently, we identified the cystargolide biosynthetic gene cluster, but systematic genetic analyses had not been carried out because of the lack of a heterologous expression system. Here, we report the discovery of a homologous cystargolide biosynthetic pathway in Streptomyces durhamensis NRRL-B3309 by genome mining. The gene cluster was cloned via transformation-associated recombination and heterologously expressed in Streptomyces coelicolor M512. We demonstrate that it contains all genes necessary for the production of cystargolide A and B. Single gene deletion experiments reveal that only five of the eight genes from the initially proposed gene cluster are essential for cystargolide synthesis. Additional insights into the cystargolide pathway could be obtained from in vitro assays with CysG and chemical complementation of the respective gene knockout. This could be further supported by the in vitro investigation of the CysG homolog BelI from the belactosin biosynthetic gene cluster. Thereby, we confirm that CysG and BelI catalyze a cryptic SAM-dependent transfer of a methyl group that is critical for the construction of the cystargolide and belactosin ß-lactone warheads.

Structure of Staphylococcus aureus ClpP Bound to the Covalent Active-Site Inhibitor Cystargolide A.

The caseinolytic protease is a highly conserved serine protease, crucial to prokaryotic and eukaryotic protein homeostasis, and a promising antibacterial and anticancer drug target. Herein, we describe the potent cystargolides as the first natural ß-lactone inhibitors of the proteolytic core ClpP. Based on the discovery of two clpP genes next to the cystargolide biosynthetic gene cluster in Kitasatospora cystarginea, we explored ClpP as a potential cystargolide target. We show the inhibition of Staphylococcus aureus ClpP by cystargolide A and B by different biochemical methods in vitro. Synthesis of semisynthetic derivatives and probes with improved cell penetration allowed us to confirm ClpP as a specific target in S. aureus cells and to demonstrate the anti-virulence activity of this natural product class. Crystal structures show cystargolide A covalently bound to all 14 active sites of ClpP from S. aureus, Aquifex aeolicus, and Photorhabdus laumondii, and reveal the molecular mechanism of ClpP inhibition by ß-lactones, the predominant class of ClpP inhibitors.

Metabolic engineering of the shikimate pathway in Amycolatopsis strains for optimized glycopeptide antibiotic production.

Glycopeptide antibiotics (GPA) consist of a glycosylated heptapeptide backbone enriched in aromatic residues originating from the shikimate pathway. Since the enzymatic reactions within the shikimate pathway are highly feedback-regulated, this raises the question as to how GPA producers control the delivery of precursors for GPA assembly. We chose Amycolatopsis balhimycina, the producer of balhimycin, as a model strain for analyzing the key enzymes of the shikimate pathway. A. balhimycina contains two copies each of the key enzymes of the shikimate pathway, deoxy-d-arabino-heptulosonate-7-phosphate synthase (Dahp) and prephenate dehydrogenase (Pdh), with one pair (Dahpsec and Pdhsec) encoded within the balhimycin biosynthetic gene cluster and one pair (Dahpprim and Pdhprim) in the core genome. While overexpression of the dahpsec gene resulted in a significant (>4-fold) increase in balhimycin yield, no positive effects were observed after overexpression of the pdhprim or pdhsec genes. Investigation of allosteric enzyme inhibition revealed that cross-regulation between the tyrosine and phenylalanine pathways plays an important role. Tyrosine, a key precursor of GPAs, was found to be a putative activator of prephenate dehydratase (Pdt), which catalyzes the first step reaction from prephenate to phenylalanine in the shikimate pathway. Surprisingly, overexpression of pdt in A. balhimycina led to an increase in antibiotic production in this modified strain. In order to demonstrate that this metabolic engineering approach is generally applicable to GPA producers, we subsequently applied this strategy to Amycolatopsis japonicum and improved the production of ristomycin A, which is used in diagnosis of genetic disorders. Comparison of "cluster-specific" enzymes with the isoenzymes from the primary metabolism's pathway provided insights into the adaptive mechanisms used by producers to ensure adequate precursor supply and GPA yields. These insights further demonstrate the importance of a holistic approach in bioengineering efforts that takes into account not only peptide assembly but also adequate precursor supply.

Origin of the 3-methylglutaryl moiety in caprazamycin biosynthesis.

BACKGROUND: Caprazamycins are liponucleoside antibiotics showing bioactivity against Gram-positive bacteria including clinically relevant Mycobacterium tuberculosis by targeting the bacterial MraY-translocase. Their chemical structure contains a unique 3-methylglutaryl moiety which they only share with the closely related liposidomycins. Although the biosynthesis of caprazamycin is understood to some extent, the origin of 3-methylglutaryl-CoA for caprazamycin biosynthesis remains elusive. RESULTS: In this work, we demonstrate two pathways of the heterologous producer Streptomyces coelicolor M1154 capable of supplying 3-methylglutaryl-CoA: One is encoded by the caprazamycin gene cluster itself including the 3-hydroxy-3-methylglutaryl-CoA synthase Cpz5. The second pathway is part of primary metabolism of the host cell and encodes for the leucine/isovalerate utilization pathway (Liu-pathway). We could identify the liu cluster in S. coelicolor M1154 and gene deletions showed that the intermediate 3-methylglutaconyl-CoA is used for 3-methylglutaryl-CoA biosynthesis. This is the first report of this intermediate being hijacked for secondary metabolite biosynthesis. Furthermore, Cpz20 and Cpz25 from the caprazamycin gene cluster were found to be part of a common route after both individual pathways are merged together. CONCLUSIONS: The unique 3-methylglutaryl moiety in caprazamycin originates both from the caprazamycin gene cluster and the leucine/isovalerate utilization pathway of the heterologous host. Our study enhanced the knowledge on the caprazamycin biosynthesis and points out the importance of primary metabolism of the host cell for biosynthesis of natural products.

Antitumor astins originate from the fungal endophyte Cyanodermella asteris living within the medicinal plant Aster tataricus.

Medicinal plants are a prolific source of natural products with remarkable chemical and biological properties, many of which have considerable remedial benefits. Numerous medicinal plants are suffering from wildcrafting, and thus biotechnological production processes of their natural products are urgently needed. The plant Aster tataricus is widely used in traditional Chinese medicine and contains unique active ingredients named astins. These are macrocyclic peptides showing promising antitumor activities and usually containing the highly unusual moiety 3,4-dichloroproline. The biosynthetic origins of astins are unknown despite being studied for decades. Here we show that astins are produced by the recently discovered fungal endophyte Cyanodermella asteris. We were able to produce astins in reasonable and reproducible amounts using axenic cultures of the endophyte. We identified the biosynthetic gene cluster responsible for astin biosynthesis in the genome of C. asteris and propose a production pathway that is based on a nonribosomal peptide synthetase. Striking differences in the production profiles of endophyte and host plant imply a symbiotic cross-species biosynthesis pathway for astin C derivatives, in which plant enzymes or plant signals are required to trigger the synthesis of plant-exclusive variants such as astin A. Our findings lay the foundation for the sustainable biotechnological production of astins independent from aster plants.

A Second Gamma-Glutamylpolyamine Synthetase, GlnA2, Is Involved in Polyamine Catabolism in Streptomyces coelicolor.

Streptomyces coelicolor is a soil bacterium living in a habitat with very changeable nutrient availability. This organism possesses a complex nitrogen metabolism and is able to utilize the polyamines putrescine, cadaverine, spermidine, and spermine and the monoamine ethanolamine. We demonstrated that GlnA2 (SCO2241) facilitates S. coelicolor to survive under high toxic polyamine concentrations. GlnA2 is a gamma-glutamylpolyamine synthetase, an enzyme catalyzing the first step in polyamine catabolism. The role of GlnA2 was confirmed in phenotypical studies with a glnA2 deletion mutant as well as in transcriptional and biochemical analyses. Among all GS-like enzymes in S. coelicolor, GlnA2 possesses the highest specificity towards short-chain polyamines (putrescine and cadaverine), while its functional homolog GlnA3 (SCO6962) prefers long-chain polyamines (spermidine and spermine) and GlnA4 (SCO1613) accepts only monoamines. The genome-wide RNAseq analysis in the presence of the polyamines putrescine, cadaverine, spermidine, or spermine revealed indication of the occurrence of different routes for polyamine catabolism in S. coelicolor involving GlnA2 and GlnA3. Furthermore, GlnA2 and GlnA3 are differently regulated. From our results, we can propose a complemented model of polyamine catabolism in S. coelicolor, which involves the gamma-glutamylation pathway as well as other alternative utilization pathways.

Stereodivergent Nitrocyclopropane Formation during Biosynthesis of Belactosins and Hormaomycins.

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.

Mining Indonesian Microbial Biodiversity for Novel Natural Compounds by a Combined Genome Mining and Molecular Networking Approach.

Indonesia is one of the most biodiverse countries in the world and a promising resource for novel natural compound producers. Actinomycetes produce about two thirds of all clinically used antibiotics. Thus, exploiting Indonesia's microbial diversity for actinomycetes may lead to the discovery of novel antibiotics. A total of 422 actinomycete strains were isolated from three different unique areas in Indonesia and tested for their antimicrobial activity. Nine potent bioactive strains were prioritized for further drug screening approaches. The nine strains were cultivated in different solid and liquid media, and a combination of genome mining analysis and mass spectrometry (MS)-based molecular networking was employed to identify potential novel compounds. By correlating secondary metabolite gene cluster data with MS-based molecular networking results, we identified several gene cluster-encoded biosynthetic products from the nine strains, including naphthyridinomycin, amicetin, echinomycin, tirandamycin, antimycin, and desferrioxamine B. Moreover, 16 putative ion clusters and numerous gene clusters were detected that could not be associated with any known compound, indicating that the strains can produce novel secondary metabolites. Our results demonstrate that sampling of actinomycetes from unique and biodiversity-rich habitats, such as Indonesia, along with a combination of gene cluster networking and molecular networking approaches, accelerates natural product identification.

A Regulator Based "Semi-Targeted" Approach to Activate Silent Biosynthetic Gene Clusters.

By culturing microorganisms under standard laboratory conditions, most biosynthetic gene clusters (BGCs) are not expressed, and thus, the products are not produced. To explore this biosynthetic potential, we developed a novel "semi-targeted" approach focusing on activating "silent" BGCs by concurrently introducing a group of regulator genes into streptomycetes of the Tübingen strain collection. We constructed integrative plasmids containing two classes of regulatory genes under the control of the constitutive promoter ermE*p (cluster situated regulators (CSR) and Streptomyces antibiotic regulatory proteins (SARPs)). These plasmids were introduced into Streptomyces sp. TÜ17, Streptomyces sp. TÜ10 and Streptomyces sp. TÜ102. Introduction of the CSRs-plasmid into strain S. sp. TÜ17 activated the production of mayamycin A. By using the individual regulator genes, we proved that Aur1P, was responsible for the activation. In strain S. sp. TÜ102, the introduction of the SARP-plasmid triggered the production of a chartreusin-like compound. Insertion of the CSRs-plasmid into strain S. sp. TÜ10 resulted in activating the warkmycin-BGC. In both recombinants, activation of the BGCs was only possible through the simultaneous expression of aur1PR3 and griR in S. sp. TÜ102 and aur1P and pntR in of S. sp. TÜ10.

Engineering of Streptoalloteichus tenebrarius 2444 for Sustainable Production of Tobramycin.

Tobramycin is a broad-spectrum aminoglycoside antibiotic agent. The compound is obtained from the base-catalyzed hydrolysis of carbamoyltobramycin (CTB), which is naturally produced by the actinomycete Streptoalloteichus tenebrarius. However, the strain uses the same precursors to synthesize several structurally related aminoglycosides. Consequently, the production yields of tobramycin are low, and the compound's purification is very challenging, costly, and time-consuming. In this study, the production of the main undesired product, apramycin, in the industrial isolate Streptoalloteichus tenebrarius 2444 was decreased by applying the fermentation media M10 and M11, which contained high concentrations of starch and dextrin. Furthermore, the strain was genetically engineered by the inactivation of the aprK gene (∆aprK), resulting in the abolishment of apramycin biosynthesis. In the next step of strain development, an additional copy of the tobramycin biosynthetic gene cluster (BGC) was introduced into the ∆aprK mutant. Fermentation by the engineered strain (∆aprK_1-17L) in M11 medium resulted in a 3- to 4-fold higher production than fermentation by the precursor strain (∆aprK). The phenotypic stability of the mutant without selection pressure was validated. The use of the engineered S. tenebrarius 2444 facilitates a step-saving, efficient, and, thus, more sustainable production of the valuable compound tobramycin on an industrial scale.

Genetic engineering approaches for the fermentative production of phenylglycines.

L-phenylglycine (L-Phg) is a rare non-proteinogenic amino acid, which only occurs in some natural compounds, such as the streptogramin antibiotics pristinamycin I and virginiamycin S or the bicyclic peptide antibiotic dityromycin. Industrially, more interesting than L-Phg is the enantiomeric D-Phg as it plays an important role in the fine chemical industry, where it is used as a precursor for the production of semisynthetic ß-lactam antibiotics. Based on the natural L-Phg operon from Streptomyces pristinaespiralis and the stereo-inverting aminotransferase gene hpgAT from Pseudomonas putida, an artificial D-Phg operon was constructed. The natural L-Phg operon, as well as the artificial D-Phg operon, was heterologously expressed in different actinomycetal host strains, which led to the successful production of Phg. By rational genetic engineering of the optimal producer strains S. pristinaespiralis and Streptomyces lividans, Phg production could be improved significantly. Here, we report on the development of a synthetic biology-derived D-Phg pathway and the optimization of fermentative Phg production in actinomycetes by genetic engineering approaches. Our data illustrate a promising alternative for the production of Phgs.

Characterization of the Actinonin Biosynthetic Gene Cluster.

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.

Xanthocidin Derivatives from the Endophytic Streptomyces sp. AcE210 Provide Insight into Xanthocidin Biosynthesis.

Xanthocidin and six new derivatives were isolated from the endophytic Streptomyces sp. AcE210. Their planar structures were elucidated by 1D and 2D NMR spectroscopy as well as by HRMS. The absolute configuration of one compound was determined by using vibrational circular dichroism spectroscopy (VCD). The structural similarities of xanthocidin and some of the isolated xanthocidin congeners to the methylenomycins A, B, and C suggested that the biosynthesis of these compounds might follow a similar route. Feeding studies with isotopically labelled [13 C5 ]-l-valine showed that instead of utilizing acetyl-CoA as starter unit, which has been proposed for the methylenomycin biosynthesis, Streptomyces sp. AcE210 employs an isobutyryl-CoA starter unit, resulting in a branched side chain in xanthocidin. Further evidence for a comparable biosynthesis was given by the analysis of the genome sequence of Streptomyces sp. AcE210 that revealed a cluster of homologues to the mmy genes involved in methylenomycin biosynthesis.

High Frequency and Diversity of Antimicrobial Activities Produced by Nasal Staphylococcus Strains against Bacterial Competitors.

The human nasal microbiota is highly variable and dynamic often enclosing major pathogens such as Staphylococcus aureus. The potential roles of bacteriocins or other mechanisms allowing certain bacterial clones to prevail in this nutrient-poor habitat have hardly been studied. Of 89 nasal Staphylococcus isolates, unexpectedly, the vast majority (84%) was found to produce antimicrobial substances in particular under habitat-specific stress conditions, such as iron limitation or exposure to hydrogen peroxide. Activity spectra were generally narrow but highly variable with activities against certain nasal members of the Actinobacteria, Proteobacteria, Firmicutes, or several groups of bacteria. Staphylococcus species and many other Firmicutes were insusceptible to most of the compounds. A representative bacteriocin was identified as a nukacin-related peptide whose inactivation reduced the capacity of the producer Staphylococcus epidermidis IVK45 to limit growth of other nasal bacteria. Of note, the bacteriocin genes were found on mobile genetic elements exhibiting signs of extensive horizontal gene transfer and rearrangements. Thus, continuously evolving bacteriocins appear to govern bacterial competition in the human nose and specific bacteriocins may become important agents for eradication of notorious opportunistic pathogens from human microbiota.

Streptomyces AcH 505 triggers production of a salicylic acid analogue in the fungal pathogen Heterobasidion abietinum that enhances infection of Norway spruce seedlings.

The necrotrophic fungus Heterobasidion spp. is the causal agent of 'annosum root rot' of Norway spruce. In the presence of the rhizosphere bacterium Streptomyces AcH 505, enhanced colonization of Norway spruce roots with Heterobasidion abietinum 331 has previously been observed. By analyzing dual cultures of H. abietinum 331 and Streptomyces AcH 505 with HPLC, a fungal metabolite was identified that was increased in the presence of Streptomyces AcH 505. Likewise, challenge of H. abietum 331 with common antifungals produced by soil streptomycetes rendered the same effect. The structure of the compound, 5-formylsalicylic acid (5-FSA), was elucidated by HPLC-HR-ESI-Orbitrap-mass spectrometry and NMR spectroscopy. Based on in vivo measurements of maximum photosystem II efficiency of Norway spruce seedlings, 5-FSA did not influence plant vitality. However, when challenged with H. abietinum 331, ergosterol amounts in infected roots increased significantly for 5-FSA pre-treated seedlings. The severity of the infection was comparable to that observed in the presence of Streptomyces AcH 505. 5-FSA is a structural analogue of salicylic acid, an important signalling molecule active in plant defence. Thus, the expression of two defence-response related marker genes (PR1, Hel) was analysed in 5-FSA treated Arabidopsis thaliana seedlings by Northern blot analysis. The transcription of both marker genes was altered, indicating that 5-FSA is perceived by Arabidopsis in a similar manner to salicylic acid and is able to interfere with Arabidopsis defence signalling. The role of 5-FSA as a potential virulence factor of H. abietinum 331 in the presence of Streptomyces AcH 505 is discussed.

Pharmacological Potential of Phylogenetically Diverse Actinobacteria Isolated from Deep-Sea Coral Ecosystems of the Submarine Avilés Canyon in the Cantabrian Sea.

Marine Actinobacteria are emerging as an unexplored source for natural product discovery. Eighty-seven deep-sea coral reef invertebrates were collected during an oceanographic expedition at the submarine Avilés Canyon (Asturias, Spain) in a range of 1500 to 4700 m depth. From these, 18 cultivable bioactive Actinobacteria were isolated, mainly from corals, phylum Cnidaria, and some specimens of phyla Echinodermata, Porifera, Annelida, Arthropoda, Mollusca and Sipuncula. As determined by 16S rRNA sequencing and phylogenetic analyses, all isolates belong to the phylum Actinobacteria, mainly to the Streptomyces genus and also to Micromonospora, Pseudonocardia and Myceligenerans. Production of bioactive compounds of pharmacological interest was investigated by high-performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GC-MS) techniques and subsequent database comparison. Results reveal that deep-sea isolated Actinobacteria display a wide repertoire of secondary metabolite production with a high chemical diversity. Most identified products (both diffusible and volatiles) are known by their contrasted antibiotic or antitumor activities. Bioassays with ethyl acetate extracts from isolates displayed strong antibiotic activities against a panel of important resistant clinical pathogens, including Gram-positive and Gram-negative bacteria, as well as fungi, all of them isolated at two main hospitals (HUCA and Cabueñes) from the same geographical region. The identity of the active extracts components of these producing Actinobacteria is currently being investigated, given its potential for the discovery of pharmaceuticals and other products of biotechnological interest.

Biosynthesis of the ß-Lactone Proteasome Inhibitors Belactosin and Cystargolide.

Belactosins and cystargolides are natural product proteasome inhibitors from Actinobacteria. Both feature dipeptidic backbones and a unique ß-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 ß-lactone warhead is unprecedented and reminiscent of leucine biosynthesis, and that it involves the action of isopropylmalate synthase homologues.

Distinct mechanisms contribute to immunity in the lantibiotic NAI-107 producer strain Microbispora ATCC PTA-5024.

The investigation of self-resistance in antibiotic producers is important to understand the emergence of antibiotic resistance in pathogens and to improve antibiotic production. Lantibiotics are ribosomally synthesized antibiotics that mostly target peptidoglycan biosynthesis. The actinomycete Microbispora ATCC PTA-5024 produces the lantibiotic NAI-107, which interferes with peptidoglycan biosynthesis by binding bactoprenol-pyrophosphate-coupled peptidoglycan precursors. In order to understand how Microbispora counteracts the action of its own antibiotic, its peptidoglycan composition was analysed in detail. Microbispora peptidoglycan consists of muropeptides with D-Ala and Gly in similar proportion at the fourth position of the peptide stems and alternative 3-3 cross-links besides the classical 4-3 cross-links. In addition, the NAI-107 biosynthetic gene cluster (mlb) was analysed for the expression of immunity proteins. We show that distinct immunity determinants are encoded in the mlb cluster: the ABC transporter MlbYZ acting cooperatively with the transmembrane protein MlbJ and the lipoprotein MlbQ. NMR structural analysis of MlbQ revealed a hydrophobic surface patch, which is proposed to bind the cognate lantibiotic. This study demonstrates that immunity in Microbispora is not only based on one determinant but on the action of the distinct immunity proteins MlbQ, MlbYZ and MlbJ.

The cyclochlorotine mycotoxin is produced by the nonribosomal peptide synthetase CctN in Talaromyces islandicus ('Penicillium islandicum').

Talaromyces islandicus ('Penicillium islandicum') is a widespread foodborne mold that produces numerous secondary metabolites, among them potent mycotoxins belonging to different chemical classes. A notable metabolite is the hepatotoxic and carcinogenic pentapeptide cyclochlorotine that contains the unusual amino acids ß-phenylalanine, 2-aminobutyrate and 3,4-dichloroproline. Although the chemical structure has been known for over five decades, nothing is known about the biosynthetic pathway of cyclochlorotine. Bioinformatic analysis of the recently sequenced genome of T. islandicus identified a wealth of gene clusters potentially coding for the synthesis of secondary metabolites. Here, we show by RNA interference-mediated gene silencing that a nonribosomal peptide synthetase, CctN, is responsible for the synthesis of cyclochlorotine. Moreover, we identified novel cyclochlorotine chemical variants, whose production also depended on cctN expression. Surprisingly, the halogenase required for cyclochlorotine biosynthesis is not encoded in the cct cluster. Nonetheless, our findings enabled us to propose a detailed model for cyclochlorotine biosynthesis. In addition, comparative genomics revealed that cct-like clusters are present in all of the sequenced Talaromyces strains indicating a high prevalence of cyclochlorotine production ability.

Epoxomicin and Eponemycin Biosynthesis Involves gem-Dimethylation and an Acyl-CoA Dehydrogenase-Like Enzyme.

The α',ß'-epoxyketone moiety of proteasome inhibitors confers high binding specificity to the N-terminal threonine in catalytic proteasome ß-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',ß'-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.