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Genetic basis for the biosynthesis of the pharmaceutically important class of epoxyketone proteasome inhibitors.

ABSTRACT: The epoxyketone proteasome inhibitors are an established class of therapeutic agents for the treatment of cancer. Their unique ?',?'-epoxyketone pharmacophore allows binding to the catalytic ?-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 anticancer 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 nonribosomal 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.

SUBMITTER: Schorn M

PROVIDER: S-EPMC4041076 | biostudies-literature | 2014 Jan

REPOSITORIES: biostudies-literature

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Genetic basis for the biosynthesis of the pharmaceutically important class of epoxyketone proteasome inhibitors.

Schorn Michelle M Zettler Judith J Noel Joseph P JP Dorrestein Pieter C PC Moore Bradley S BS Kaysser Leonard L

ACS chemical biology 20131108 1

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 anticancer drug Kyprolis, and for eponemycin were identified in the actino ...[more]

PMID: 24168704

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Project description:Streptomyces sp. BRA-346 is an Actinobacteria isolated from the Brazilian endemic tunicate Euherdmania sp. We have reported that this strain produces epoxyketone peptides, as dihydroeponemycin (DHE) and structurally related analogs. This cocktail of epoxyketone peptides inhibits the proteasome chymotrypsin-like activity and shows high cytotoxicity to glioma cells. However, low yields and poor reproducibility of epoxyketone peptides production by BRA-346 under laboratory cultivation have limited the isolation of epoxyketone peptides for additional studies. Here, we evaluated several cultivation methods using different culture media and chemical elicitors to increase the repertoire of peptide epoxyketone production by this bacterium. Furthermore, BRA-346 genome was sequenced, revealing its broad genetic potential, which is mostly hidden under laboratory conditions. By using specific growth conditions, we were able to evidence different classes of secondary metabolites produced by BRA-346. In addition, by combining genome mining with untargeted metabolomics, we could link the metabolites produced by BRA-346 to its genetic capacity and potential regulators. A single biosynthetic gene cluster (BGC) was related to the production of the target epoxyketone peptides by BRA-346. The candidate BGC displays conserved biosynthetic enzymes with the reported eponemycin (EPN) and TMC-86A (TMC) BGCs. The core of the putative epoxyketone peptide BGC (ORFs A-L), in which ORF A is a LuxR-like transcription factor, was cloned into a heterologous host. The recombinant organism was capable to produce TMC and EPN natural products, along with the biosynthetic intermediates DH-TMC and DHE, and additional congeners. A phylogenetic analysis of the epn/tmc BGC revealed related BGCs in public databases. Most of them carry a proteasome beta-subunit, however, lacking an assigned specialized metabolite. The retrieved BGCs also display a diversity of regulatory genes and TTA codons, indicating tight regulation of this BGC at the transcription and translational levels. These results demonstrate the plasticity of the epn/tmc BGC of BRA-346 in producing epoxyketone peptides and the feasibility of their production in a heterologous host. This work also highlights the capacity of BRA-346 to tightly regulate its secondary metabolism and shed light on how to awake silent gene clusters of Streptomyces sp. BRA-346 to allow the production of pharmacologically important biosynthetic products.

Project description:Ergot alkaloids are specialized fungal metabolites that are important as the bases of several pharmaceuticals. Many ergot alkaloids are derivatives of lysergic acid (LA) and have vasoconstrictive activity, whereas several dihydrolysergic acid (DHLA) derivatives are vasorelaxant. The pathway to LA is established, with the P450 monooxygenase CloA playing a key role in oxidizing its substrate agroclavine to LA. We analyzed the activities of products of cloA alleles from different fungi relative to DHLA biosynthesis by expressing them in a mutant of the fungus Neosartorya fumigata that accumulates festuclavine, the precursor to DHLA. Transformants expressing CloA from Epichloë typhina × Epichloë festucae, which oxidizes agroclavine to LA, failed to oxidize festuclavine to DHLA. In substrate feeding experiments, these same transformants oxidized exogenously supplied agroclavine to LA, indicating that a functional CloA was produced. A genomic clone of cloA from Claviceps africana, a sorghum ergot fungus that produces a DHLA derivative, was cloned and expressed in the festuclavine-accumulating mutant of N. fumigata, but several introns in this genomic clone were not processed properly. Expression of a synthetic intron-free version of C. africanacloA resulted in the accumulation of DHLA as assessed by fluorescence high-pressure liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS). In substrate feeding experiments, the C. africana CloA also accepted agroclavine as the substrate, oxidizing it to LA. The data indicate that a specialized allele of cloA is required for DHLA biosynthesis and that the pharmaceutically important compound DHLA can be produced in engineered N. fumigataIMPORTANCE Ergot alkaloids are fungal metabolites that have impacted humankind historically as poisons and more recently as pharmaceuticals used to treat dementia, migraines, and other disorders. Much is known about the biosynthesis of ergot alkaloids that are derived from lysergic acid (LA), but important questions remain about a parallel pathway to ergot alkaloids derived from dihydrolysergic acid (DHLA). DHLA-derived alkaloids have minor structural differences compared to LA-derived alkaloids but can have very different activities. To understand how DHLA is made, we analyzed activities of a key enzyme in the DHLA pathway and found that it differed from its counterpart in the LA pathway. Our data indicate a critical difference between the two pathways and provide a strategy for producing DHLA by modifying a model fungus. The ability to produce DHLA in a model fungus may facilitate synthesis of DHLA-derived pharmaceuticals.

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Genetic basis for the biosynthesis of the pharmaceutically important class of epoxyketone proteasome inhibitors.

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Genetic basis for the biosynthesis of the pharmaceutically important class of epoxyketone proteasome inhibitors.

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