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1.
Nat Prod Res ; 35(7): 1090-1096, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31303055

RESUMO

The culture broth of endophytic Streptomyces sp. AB100, isolated from the shoots of medicinal plant Atropa belladonna (L.) was investigated for the presence of antibacterial compounds. After initial testing followed by bioactivity-guided fractionation, six new piperazic acid (PA)-containing congeners of two known peptides, JBIR-39 and JBIR-40, were identified by HR-MS/MS and NMR analyses. Only the dehydroxylated hexapeptidic derivatives with unusual incorporation of four PA moieties exhibited weak antibacterial activity against Gram-positive test organism Bacillus subtilis. A 16S rDNA-based phylogenetic tree of known Streptomyces spp. producing PA-containing hexapeptides isolated from different habitats and endophyte Streptomyces AB100 showed considerable diversity, suggesting that these metabolites may play an important environmental role beyond their antibacterial activity.


Assuntos
Atropa belladonna/microbiologia , Endófitos/química , Peptídeos/farmacologia , Plantas Medicinais/química , Piridazinas/farmacologia , Streptomyces/química , Streptomyces/isolamento & purificação , Antibacterianos/farmacologia , Bacillus subtilis/efeitos dos fármacos , DNA Ribossômico/genética , Espectroscopia de Ressonância Magnética , Testes de Sensibilidade Microbiana , Filogenia , Brotos de Planta/microbiologia , Espectrometria de Massas em Tandem
2.
J Nat Prod ; 83(8): 2381-2389, 2020 08 28.
Artigo em Inglês | MEDLINE | ID: mdl-32786880

RESUMO

Extracts from Streptomyces sp. S4.7 isolated from the rhizosphere of edelweiss, an alpine medicinal plant, exhibited activity against Gram-positive bacteria. LC-HRMS analyses of the extracts resulted in the detection of two unknown, structurally related lipopeptides that were assumed to be responsible for the antibiotic activity. LC-MS guided isolation and structure elucidation of viennamycins A and B (1 and 2) by HR-MS/MS, 1D and 2D NMR, and Marfey's analyses revealed them to be novel compounds, with viennamycin A containing cysteic acid, a unique feature for lipopeptides. Tests for antibacterial, antifungal, and cytotoxic activities of purified viennamycins, both with and without divalent cations, did not reveal any bioactivity, suggesting that their biological function, which could not be determined in the tests used, is atypical for lipopeptides. The genome of Streptomyces sp. S4.7 was sequenced and analyzed, revealing the viennamycin biosynthetic gene cluster. Detailed bioinformatics-based analysis of the viennamycin gene cluster allowed elucidation of the biosynthetic pathway for these lipopeptides.


Assuntos
Lipopeptídeos/biossíntese , Streptomyces/metabolismo , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Lipopeptídeos/farmacologia , Testes de Sensibilidade Microbiana , Análise Espectral/métodos
3.
Nat Commun ; 8(1): 1965, 2017 12 06.
Artigo em Inglês | MEDLINE | ID: mdl-29213087

RESUMO

Metalloproteinase inhibitors often feature hydroxamate moieties to facilitate the chelation of metal ions in the catalytic center of target enzymes. Actinonin and matlystatins are  potent metalloproteinase inhibitors that comprise rare N-hydroxy-2-pentyl-succinamic acid warheads. Here we report the identification and characterization of their biosynthetic pathways. By gene cluster comparison and a combination of precursor feeding studies, heterologous pathway expression and gene deletion experiments we are able to show that the N-hydroxy-alkyl-succinamic acid warhead is generated by an unprecedented variation of the ethylmalonyl-CoA pathway. Moreover, we present evidence that the remarkable structural diversity of matlystatin congeners originates from the activity of a decarboxylase-dehydrogenase enzyme with high similarity to enzymes that form epoxyketones. We further exploit this mechanism to direct the biosynthesis of non-natural matlystatin derivatives. Our work paves the way for follow-up studies on these fascinating pathways and allows the identification of new protease inhibitors by genome mining.


Assuntos
Inibidores de Metaloproteinases de Matriz/química , Inibidores de Metaloproteinases de Matriz/metabolismo , Metaloproteases/efeitos dos fármacos , Inibidores de Proteases/química , Inibidores de Proteases/metabolismo , Acetilcisteína/análogos & derivados , Acetilcisteína/antagonistas & inibidores , Acetilcisteína/química , Actinobacteria/genética , Actinobacteria/metabolismo , Acil Coenzima A , Vias Biossintéticas/genética , Carboxiliases , Deleção de Genes , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos/genética , Ácidos Hidroxâmicos/antagonistas & inibidores , Ácidos Hidroxâmicos/química , Ácidos Hidroxâmicos/metabolismo , Inibidores de Metaloproteinases de Matriz/farmacologia , Família Multigênica , Ornitina/metabolismo , Oxirredutases , Propionatos/metabolismo , Inibidores de Proteases/farmacologia , Piridazinas/antagonistas & inibidores , Piridazinas/química , Piridazinas/metabolismo , Deleção de Sequência , Streptomyces/genética , Streptomyces/metabolismo
4.
Arch Pharm (Weinheim) ; 349(8): 594-601, 2016 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-27258165

RESUMO

The fast growing genome databases provide us with a large number of so far unknown secondary metabolite biosynthetic gene clusters. A key method to study these gene clusters is their heterologous expression in an engineered host strain. Gene clusters derived from actinomycetes are usually expressed in a Streptomyces host strain to identify and investigate the corresponding compounds. However, heterologous expression is often accompanied with some challenges affecting the production rates of secondary metabolites. The first step is therefore the selection of a suitable expression vector and host strain. Once production has been established, there are several possibilities to improve compound yields either by media screens, by overexpression of regulatory or transport genes or by introduction of constitutive or inducible promoters. A surely important, but hitherto little studied factor is also the regulation of a heterologously expressed gene cluster by its host strain. This review gives a short overview on the chances and challenges provided by heterologous production of secondary metabolites in Streptomyces.


Assuntos
Antibacterianos/biossíntese , Streptomyces/genética , Streptomyces/metabolismo , Regulação Bacteriana da Expressão Gênica , Vetores Genéticos , Família Multigênica , Metabolismo Secundário/genética
5.
PLoS One ; 11(5): e0153249, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27145180

RESUMO

ROK-family proteins have been described to act either as sugar kinases or as transcriptional regulators. Few ROK-family regulators have been characterized so far and most of them are involved in carbon catabolite repression. RokB (Sco6115) has originally been identified in a DNA-affinity capturing approach as a possible regulator of the heterologously expressed novobiocin biosynthetic gene cluster in Streptomyces coelicolor M512. Interestingly, both, the rokB deletion mutants as well as its overexpressing mutants showed significantly reduced novobiocin production in the host strain S.coelicolor M512. We identified the DNA-binding site for RokB in the promoter region of the novobiocin biosynthetic genes novH-novW. It overlaps with the novH start codon which may explain the reduction of novobiocin production caused by overexpression of rokB. Bioinformatic screening coupled with surface plasmon resonance based interaction studies resulted in the discovery of five RokB binding sites within the genome of S. coelicolor. Using the genomic binding sites, a consensus motif for RokB was calculated, which differs slightly from previously determined binding motifs for ROK-family regulators. The annotations of the possible members of the so defined RokB regulon gave hints that RokB might be involved in amino acid metabolism and transport. This hypothesis was supported by feeding experiments with casamino acids and L-tyrosine, which could also explain the reduced novobiocin production in the deletion mutants.


Assuntos
Proteínas de Bactérias/metabolismo , DNA/metabolismo , Regulon , Streptomyces coelicolor/metabolismo , Aminoácidos/metabolismo , Sítios de Ligação , Genes Bacterianos , Regiões Promotoras Genéticas , Streptomyces coelicolor/genética
6.
Appl Microbiol Biotechnol ; 100(10): 4495-509, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-26795961

RESUMO

Understanding the regulation of a heterologously expressed gene cluster in a host organism is crucial for activation of silent gene clusters or overproduction of the corresponding natural product. In this study, Streptomyces coelicolor M512(nov-BG1) containing the novobiocin biosynthetic gene cluster from Streptomyces niveus NCIMB 11891 was chosen as a model. An improved DNA affinity capturing assay (DACA), combined with semi-quantitative mass spectrometry, was used to identify proteins binding to the promoter regions of the novobiocin gene cluster. Altogether, 2475 proteins were identified in DACA studies with the promoter regions of the pathway-specific regulators novE (PnovE) and novG (PnovG), of the biosynthetic genes novH-W (PnovH) and of the vegetative σ-factor hrdB (PhrdB) as a negative control. A restrictive classification for specific binding reduced this number to 17 proteins. Twelve of them were captured by PnovH, among them, NovG, two were captured by PnovE, and three by PnovG. Unexpectedly some well-known regulatory proteins, such as the global regulators NdgR, AdpA, SlbR, and WhiA were captured in similar intensities by all four tested promoter regions. Of the 17 promoter-specific proteins, three were studied in more detail by deletion mutagenesis and by overexpression. Two of them, BxlRSc and BxlR2Sc, could be identified as positive regulators of novobiocin production in S. coelicolor M512. Deletion of a third gene, sco0460, resulted in reduced novobiocin production, while overexpression had no effect. Furthermore, binding of BxlRSc to PnovH and to its own promoter region was confirmed via surface plasmon resonance spectroscopy.


Assuntos
Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Família Multigênica , Novobiocina/biossíntese , Streptomyces coelicolor/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Meios de Cultura , DNA Bacteriano/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Deleção de Genes , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Plasmídeos/genética , Regiões Promotoras Genéticas , Fator sigma/genética , Fator sigma/metabolismo , Streptomyces coelicolor/metabolismo
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