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.

Keywords: Streptomyces sp. BRA-346; biosynthesis of natural products; epoxyketone peptides; genome mining; mass spectrometry; molecular networking; proteasome inhibitors; transformation-associated recombination cloning.

Figure 1

Chemical structures of the epoxyketone peptides discussed in this work.

Figure 2

The biosynthesis of BRA-346 secondary metabolites is tightly regulated by the culture media used for bacteria growth. (A) Spectra similarity molecular network (SSMN) of the different BRA-346 cultures media A1 (cultures C3a–c and C5, in red tones), TSB (cyan), TSBY (light blue), or ISP2 (dark blue). MS/MS spectra detected in the crude extracts are shown as the network nodes. For clarity, only the MS/MS spectra pointed as the protonated ion ([M + H]⁺) and nodes with at least one connection (self-loops and single nodes were removed) are shown. The MS/MS spectra were annotated by spectra matches with UNPD-ISDB and GNPS databases, visually inspected and compared to literature data. Chemical classes were annotated, and the MS/MS clusters grouped accordantly. The annotated chemical classes could also be linked to the Streptomyces sp. BRA-346 draft genome. DHE (1) and DH-TMC (3) are pointed by arrows. The MS/MS annotations for 1 and 3 were based on the described by Furtado et al. (2021) and Zabala et al. (2016), respectively. The parameter used for node size was intensity value ([minimum value; 100,000]: linear interpolation, [100,000; maximum value]: constant size). (B) Heat map constructed from all nodes detected in the SSMN shown in (A), considering the seven BRA-346 cultures analysed. The MS1 peak area was normalized by the maximum area of a given m/z and used for a cosine similarity analysis of the detection of each node across the different cultures. (C) Extracted ion chromatogram (EIC) of m/z 401.26 ± 0.01 in BRA-346 cultures with the different media used. The region 5–10 min (“x” axis) was selected for clarity. (D) Detail of the epoxyketone peptide MS/MS cluster extracted from the SSMN shown in (A). (E) EIC of m/z 401.26 ± 0.01 in the four BRA-346 cultures in the A1 medium.

Figure 3

Chemical elicitors, especially ampicillin, induce the production of epoxyketone peptides by BRA-346. (A) SSMN of BRA-346 cultures using the A1 medium and different chemical elicitors [no elicitor, as a control, (i); ampicillin 100 μg/ml (ii); sodium butyrate 50 μM (iii); procaine 100 μM (iv)]. Only the [M + H]⁺ spectra and nodes with at least one connection are shown for clarity. In each node, the external ring chart represents the crude extracts, and the internal pie chart represents the F50 (50% methanol) fractions. (B) Detail of the epoxyketone MS/MS clusters containing compounds 1, 3 and 4. (C) and (D) represent the EIC of m/z 343.19 ± 0.01 (TMC, 4) and m/z 401.26 ± 0.01 (DHE, 1), respectively. Chromatograms of the crude extract and F50 fractions samples are represented in purple and cyan, respectively.

Figure 4

Biosynthetic gene cluster of epoxyketone peptides in BRA-346. The epn/tmc BGC in BRA-346 was identified using antiSMASH. The related BGC of eponemycin (epn, Streptomyces hygroscopicus ATCC53708; Schorn et al., 2014) and TMC-86A (tmc, Streptomyces chromofuscus ATCC49982; Zabala et al., 2016) already reported are shown for comparison.

Figure 5

Heterologous expression of the epn/tmc BGC of BRA-346. (A) SSMN evidencing the production of epoxyketone peptides by the heterologous (purple) and the wild type (pink) organisms. (B) Zoom at the SSMN clusters containing compounds 1–4. (C) Extracted ion chromatogram of the dihydrointermediates 1 (m/z 401.26) and 3 (m/z 345.20). (D) Extracted ion chromatogram of the final biosynthetic products 2 (m/z 399.25) and 4 (m/z 343.19).

Figure 6

Phylogenetic analysis of BGCs related to BRA-346 epn/tmc. (A) Maximum likelihood phylogenetic tree based on the alignment of the 16 retrieved BGCs related to BRA-346 epn/tmc BGC. (B) Schematic representation of the 17 BGCs and (C) their related biosynthetic products. BGCs with experimentally validated biosynthetic products are highlighted. ORFs predicted as regulatory proteins are numbered and listed.