Nonlinear Biosynthetic Assembly of Alpiniamide by a Hybrid <i>cis</i>/<i>trans</i>-AT PKS-NRPS

Sigrist, Renata; Luhavaya, Hanna; McKinnie, Shaun M. K.; Silva, Amanda Ferreira da; Jurberg, Igor D.; Moore, Bradley S.; Oliveira, Luciana G. de

doi:10.1021/acschembio.0c00081

Cited by 16 publications

(14 citation statements)

References 86 publications

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“…We then cloned the epn/tmc BGC of BRA-346 using the transformation-associated recombination (TAR) approach ( Zhang et al, 2019 ; Sigrist et al, 2020 ), inserting the target BGC under the control of a strong promoter into a S. coelicolor M1146 “antibiotic null” host organism ( Gomez-Escribano and Bibb, 2011 ). The heterologous organism S. coelicolor M1446- epn/tmc was cultured in A1 medium and its metabolites were analyzed in parallel with the wild-type BRA-346 and the host organism Streptomyces coelicolor M1446 ( Figure 5A ; Supplementary Figure S12 ).…”

Section: Resultsmentioning

confidence: 99%

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

et al. 2022

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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.

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Section: Resultsmentioning

confidence: 99%

“…The epn/tmc BGC was cloned by PCR-based transformation-associated recombination (TAR) method, as described previously ( Zhang et al, 2019 ; Sigrist et al, 2020 ), with minor modifications as detailed in the Supplementary Material.…”

Section: Methodsmentioning

confidence: 99%

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

et al. 2022

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“…This antibiotic has an unexpected nonlinear synthesis that involves a hybrid polyketide synthase-nonribosomal peptide synthetase. It is assembled in two halves and is then ligated into the mature molecule [ 37 , 38 ]. Another compound produced by one of the isolated strains is the polyketide alteramide A.…”

Section: Discussionmentioning

confidence: 99%

Characterization of Actinomycetes Strains Isolated from the Intestinal Tract and Feces of the Larvae of the Longhorn Beetle Cerambyx welensii

et al. 2020

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Actinomycetes constitute a large group of Gram-positive bacteria present in different habitats. One of these habitats involves the association of these bacteria with insects. In this work, we have studied twenty-four actinomycetes strains isolated from the intestinal tract and feces from larvae of the xylophagous coleopteran Cerambyx welensii and have shown that seventeen strains present hydrolytic activity of some of the following substrates: cellulose, hemicellulose, starch and proteins. Fourteen of the isolates produce antimicrobial molecules against the Gram-positive bacteria Micrococcus luteus. Analysis of seven strains led us to identify the production of a wide number of compounds including streptanoate, alpiniamide A, alteramides A and B, coproporphyrin III, deferoxamine, demethylenenocardamine, dihydropicromycin, nocardamine, picromycin, surugamides A, B, C, D and E, tirandamycins A and B, and valinomycin. A significant number of other compounds, whose molecular formulae are not included in the Dictionary of Natural Products (DNP), were also present in the extracts analyzed, which opens up the possibility of identifying new active antibiotics. Molecular identification of ten of the isolated bacteria determined that six of them belong to the genus Streptomyces, two of them are included in the genus Amycolatopsis and two in the genus Nocardiopsis.

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“…Our recent genome-mining studies revealed Streptomyces sp. CBMAI 2042, an endophytic strain isolated from Citrus sinensis branches, to be a promising source of secondary metabolites ( Sigrist, Paulo et al, 2020;Sigrist, Luhavaya et al, 2020;Paulo, Sigrist, Angolini, Eberlin et al, 2019). Additionally, biotransformation experiments using whole cells revealed the capability of the strain to promote epoxide hydrolysis (unpublished results).…”

Section: Introductionmentioning

confidence: 99%

An epoxide hydrolase from endophyticStreptomycesshows unique structural features and wide biocatalytic activity

Tormet‐González

Wilson

Oliveira

et al. 2020

Acta Cryst Sect D Struct Biol

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The genus Streptomyces is characterized by the production of a wide variety of secondary metabolites with remarkable biological activities and broad antibiotic capabilities. The presence of an unprecedented number of genes encoding hydrolytic enzymes with industrial appeal such as epoxide hydrolases (EHs) reveals its resourceful microscopic machinery. The whole-genome sequence of Streptomyces sp. CBMAI 2042, an endophytic actinobacterium isolated from Citrus sinensis branches, was explored by genome mining, and a putative α/β-epoxide hydrolase named B1EPH2 and encoded by 344 amino acids was selected for functional and structural studies. The crystal structure of B1EPH2 was obtained at a resolution of 2.2 Å and it was found to have a similar fold to other EHs, despite its hexameric quaternary structure, which contrasts with previously solved dimeric and monomeric EH structures. While B1EPH2 has a high sequence similarity to EHB from Mycobacterium tuberculosis, its cavity is similar to that of human EH. A group of 12 aromatic and aliphatic racemic epoxides were assayed to determine the activity of B1EPH2; remarkably, this enzyme was able to hydrolyse all the epoxides to the respective 1,2-diols, indicating a wide-range substrate scope acceptance. Moreover, the (R)- and (S)-enantiomers of styrene oxide, epichlorohydrin and 1,2-epoxybutane were used to monitor enantiopreference. Taken together, the functional and structural analyses indicate that this enzyme is an attractive biocatalyst for future biotechnological applications.

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Nonlinear Biosynthetic Assembly of Alpiniamide by a Hybrid cis/trans-AT PKS-NRPS

Cited by 16 publications

References 86 publications

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

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

Characterization of Actinomycetes Strains Isolated from the Intestinal Tract and Feces of the Larvae of the Longhorn Beetle Cerambyx welensii

An epoxide hydrolase from endophyticStreptomycesshows unique structural features and wide biocatalytic activity

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