Production of Epoxyketone Peptide-Based Proteasome Inhibitors by Streptomyces sp. BRA-346: Regulation and Biosynthesis

Abstract

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.

NP3 MS Workflow: An Open-Source Software System to Empower Natural Product-Based Drug Discovery Using Untargeted Metabolomics

“…Among the suggestions, the compound with the lowest m/z error was TMC-86A (MQscore 0.27, m/z error 1.87 ppm), a bioactive epoxy-β-aminoketone known as a specialized metabolite produced by Streptomyces, 47 including BRA-346. 17,36 The clean consensus spectrum of m/z ion 343.187 exhibited fragment ions of m/z 325.176, 251.103, 186.113, 168.102, 150.091, and 130.050 (Figures S18A and S19), which match the experimental spectra of isolated TMC-86A 48,49 (Figure S18C), supporting the initial structure annotated by Tremolo using the UNPD-ISDB database. Although TMC-86A was not ranked with the highest score, its lowest m/z error was a determining factor among Tremolo suggestions.…”

“…The suggested compounds were ranked according to the highest Tremolo score and the lowest m / z error (Table S3). Among the suggestions, the compound with the lowest m / z error was TMC-86A (MQscore 0.27, m / z error 1.87 ppm), a bioactive epoxy-β-aminoketone known as a specialized metabolite produced by Streptomyces, including BRA-346. , …”

“…BRA-346, isolated from a Brazilian endemic tunicate, 17 was reported to produce the epoxy-β-aminoketones TMC-86A, dihydroeponemycin, and eponemycin and analogues that exhibited high cytotoxicity against glioma cell lines through proteasome inhibition. 17,36 Streptomyces sp. BRA-346 extract was fractionated, resulting in 96 fractions.…”

“…Other TMC-86A analogues were also detected in the BRA-346 extract (Figure 3A, in black), which also represent epoxy-β-aminoketone proteasome inhibitors produced by the same BGC. 36,49 These TMC-86A analogues, detected in the BRA-346 extract, are present in the second peak of activity (fractions 83−93), which were not used in this NP 3 MS Workflow analysis (more information in Supporting Information, Note S13).…”

“…BRA-346. 36 In addition to TMC-86 being a known proteasome inhibitor with IC 50 value in the low micromolar range, 47 a duplicated copy of the bacterial proteasome gene was also found within the TMC-86A biosynthetic gene cluster in the Streptomyces sp. BRA-346 genome.…”