Unusual Sesquiterpenes from Streptomyces olindensis DAUFPE 5622.

In nature, the vast majority of sesquiterpenes are produced by type I mechanisms, and glycosylated sesquiterpenes are rare in actinobacteria. Streptomyces olindensis DAUFPE 5622 produces the sesquiterpenes olindenones A-G, a new class of rearranged drimane sesquiterpenes. Olindenones B-D are oxygenated derivatives of olindenone A, while olindenones E-G are analogs glycosylated with dideoxysugars. 13C-isotope labeling studies demonstrated olindenone A biosynthesis occurs via the methylerythritol phosphate (MEP) pathway and suggested the rearrangement is only partially concerted. Based on the structures, one potential mechanism of olindenone A formation proceeds by cyclization of the linear terpenoid precursor, likely occurring via a terpene cyclase-mediated type II mechanism whereby the terminal alkene of the precursor is protonated, triggering carbocation-driven cyclization followed by rearrangement. Diphosphate hydrolysis may occur either before or after cyclization. Although a biosynthetic route is proposed, the terpene cyclase gene responsible for producing olindenones currently remains unidentified.

Genome mining of Streptomyces sp. BRB081 reveals the production of the antitumor pyrrolobenzodiazepine sibiromycin.

Employing a genome mining approach, this work aimed to further explore the secondary metabolism associated genes of Streptomyces sp. BRB081, a marine isolate. The genomic DNA of BRB081 was sequenced and assembled in a synteny-based pipeline for biosynthetic gene clusters (BGCs) annotation. A total of 27 BGCs were annotated, including a sibiromycin complete cluster, a bioactive compound with potent antitumor activity. The production of sibiromycin, a pyrrolobenzodiazepine, was confirmed by the analysis of obtained BRB081 extract by HPLC-MS/MS, which showed the presence of the sibiromycin ions themselves, as well as its imine and methoxylated forms. To verify the presence of this cluster in other genomes available in public databases, a genome neighborhood network (GNN) was constructed with the non-ribosomal peptide synthetase (NRPS) gene from Streptomyces sp. BRB081. Although the literature does not report the occurrence of the sibiromycin BGC in any other microorganism than Streptosporangium sibiricum, we have located this BGC in 10 other genomes besides the BRB081 isolate, all of them belonging to the Actinomycetia class. These findings strengthen the importance of uninterrupted research for new producer strains of secondary metabolites with uncommon biological activities. These results reinforced the accuracy and robustness of genomics in the screening of natural products. Furthermore, the unprecedented nature of this discovery confirms the unknown metabolic potential of the Actinobacteria phylum and the importance of continuing screening studies in this taxon. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-022-03305-0.

Mycothiol Peroxidase Activity as a Part of the Self-Resistance Mechanisms against the Antitumor Antibiotic Cosmomycin D.

Antibiotic-producing microorganisms usually require one or more self-resistance determinants to survive antibiotic production. The effectors of these mechanisms are proteins that inactivate the antibiotic, facilitate its transport, or modify the target to render it insensitive to the molecule. Streptomyces bacteria biosynthesize various bioactive natural products and possess resistance systems for most metabolites, which are coregulated with antibiotic biosynthesis genes. Streptomyces olindensis strain DAUFPE 5622 produces the antitumor antibiotic cosmomycin D (COSD), a member of the anthracycline family. In this study, we propose three self-resistance mechanisms, anchored or based in the COSD biosynthetic gene cluster. These include cosIJ (an ABC transporter), cosU (a UvrA class IIa protein), and a new self-resistance mechanism encoded by cosP, which shows response against peroxides by the enzyme mycothiol peroxidase (MPx). Activity-based investigations of MPx and its mutant enzyme confirmed peroxidation during the production of COSD. Overexpression of the ABC transporter, the UvrA class IIa protein, and the MPx led to an effective response against toxic anthracyclines, such as cosmomycins. Our findings help to understand how thiol peroxidases play an antioxidant role in the anthracycline producer S. olindensis DAUFPE 5622, a mechanism which has been reported for neoplastic cells that are resistant to doxorubicin (DOX). IMPORTANCE Anthracycline compounds are DNA intercalating agents widely used in cancer chemotherapeutic protocols. This work focused on the self-resistance mechanisms developed by the cosmomycin-producing bacterium Streptomyces olindensis. Our findings showed that cysteine peroxidases, such as mycothiol peroxidase, encoded by the gene cosP, protected S. olindensis against peroxidation during cosmomycin production. This observation can contribute to much better understanding of resistance both in the producers, eventually enhancing production, and in some tumoral cell lines.

Metabolomic study of marine Streptomyces sp.: Secondary metabolites and the production of potential anticancer compounds.

Resorting to a One Strain Many Compounds (OSMAC) approach, the marine Streptomyces sp. BRB081 strain was grown in six different media settings over 1, 2, 3 or 7 days. Extractions of mycelium and broth were conducted separately for each media and cultivation period by sonication using methanol/acetone 1:1 and agitation with ethyl acetate, respectively. All methanol/acetone and ethyl acetate crude extracts were analysed by HPLC-MS/MS and data treatment was performed through GNPS platform using MZmine 2 software. In parallel, the genome was sequenced, assembled and mined to search for biosynthetic gene clusters (BGC) of secondary metabolites using the AntiSMASH 5.0 software. Spectral library search tool allowed the annotation of desferrioxamines, fatty acid amides, diketopiperazines, xanthurenic acid and, remarkably, the cyclic octapeptides surugamides. Molecular network analysis allowed the observation of the surugamides cluster, where surugamide A and the protonated molecule corresponding to the B-E isomers, as well as two potentially new analogues, were detected. Data treatment through MZmine 2 software allowed to distinguish that the largest amount of surugamides was obtained by cultivating BRB081 in SCB medium during 7 days and extraction of culture broth. Using the same data treatment, a chemical barcode was created for easy visualization and comparison of the metabolites produced overtime in all media. By genome mining of BRB081 four regions of biosynthetic gene clusters of secondary metabolites were detected supporting the metabolic data. Cytotoxic evaluation of all crude extracts using MTT assay revealed the highest bioactivity was also observed for extracts obtained in the optimal conditions as those for surugamides production, suggesting these to be the main active compounds herein. This method allowed the identification of compounds in the crude extracts and guided the selection of best conditions for production of bioactive compounds.

Phenotypic traits of Burkholderia spp. associated with ecological adaptation and plant-host interaction.

Burkholderia species have different lifestyles establishing mutualist or pathogenic associations with plants and animals. Changes in the ecological behavior of these bacteria may depend on genetic variations in response to niche adaptation. Here, we studied 15 Burkholderia strains isolated from different environments with respect to genetic and phenotypic traits. By Multilocus Sequence Analysis (MLSA) these isolates fell into 6 distinct groups. MLSA clusters did not correlate with strain antibiotic sensitivity, but with the bacterial ability to produce antimicrobial compounds and control orchid necrosis. Further, the B. seminalis strain TC3.4.2R3, a mutualistic bacterium, was inoculated into orchid plants and the interaction with the host was evaluated by analyzing the plant response and the bacterial oxidative stress response in planta. TC3.4.2R3 responded to plant colonization by increasing its own growth rate and by differential gene regulation upon oxidative stress caused by the plant, while reducing the plant's membrane lipid peroxidation. The bacterial responses to oxidative stress were recapitulated by bacterial exposure to the herbicide paraquat. We suggest that the ability of Burkholderia species to successfully establish in the rhizosphere correlates with genetic variation, whereas traits associated with antibiotic resistance are more likely to be categorized as strain specific.

Correction for Ichiwaki et al., "Genome Sequence of Micromonospora sp. NBS 11-29, an Antibiotic and Hydrolytic Enzyme Producer, Isolated from River Sediment in Brazil".

[This corrects the article DOI: 10.1128/genomeA.00552-17.].

Genome Sequence of Micromonospora sp. NBS 11-29, an Antibiotic and Hydrolytic Enzyme Producer, Isolated from River Sediment in Brazil.

The genus Micromonospora comprises actinomycetes with high biotechnological potential, due to their ability to produce secondary metabolites and enzymes. In this study, we report the draft genome sequence of Micromonospora sp. NBS 11-29, which showed antibacterial, cellulolytic, and xylanolytic activities under in vitro conditions.

Genome Sequence of Streptomyces olindensis DAUFPE 5622, Producer of the Antitumoral Anthracycline Cosmomycin D.

Streptomyces olindensis DAUFPE 5622, which was isolated from a Brazilian soil sample, produces the antitumor anthracycline cosmomycin D. The genome sequence is 9.4 Mb in length, with a G+C content of 71%. Thirty-four putative secondary metabolite biosynthetic gene clusters were identified, including the cosmomycin D cluster.

DNA damage induced by the anthracycline cosmomycin D in DNA repair-deficient cells.

PURPOSE: Anthracyclines have been widely used as antitumor agents, playing a crucial role in the successful treatment of many types of cancer, despite some side effects related to cardiotoxicity. New anthracyclines have been designed and tested, but the first ones discovered, doxorubicin and daunorubicin, continue to be the drugs of choice. Despite their extensive use in chemotherapy, little is known about the DNA repair mechanisms involved in the removal of lesions caused by anthracyclines. The anthracycline cosmomycin D is the main product isolated from Streptomyces olindensis, characterized by a peculiar pattern of glycosylation with two trisaccharide rings attached to the A ring of the tetrahydrotetracene. METHODS: We assessed the induction of apoptosis (Sub-G1) by cosmomycin D in nucleotide excision repair-deficient fibroblasts (XP-A and XP-C) as well as the levels of DNA damage (alkaline comet assay). RESULTS: Treatment of XP-A and XP-C cells with cosmomycin D resulted in apoptosis in a time-dependent manner, with highest apoptosis levels observed 96 h after treatment. The effects of cosmomycin D were equivalent to those obtained with doxorubicin. The broad caspase inhibitor Z-VAD-FMK strongly inhibited apoptosis in these cells, and DNA damage induced by cosmomycin D was confirmed by alkaline comet assay. CONCLUSIONS: Cosmomycin D induced time-dependent apoptosis in nucleotide excision repair-deficient fibroblasts. Despite similar apoptosis levels, cosmomycin D caused considerably lower levels of DNA damage compared to doxorubicin. This may be related to differences in structure between cosmomycin D and doxorubicin.

Insights in the glycosylation steps during biosynthesis of the antitumor anthracycline cosmomycin: characterization of two glycosyltransferase genes.

Glycosylation pattern in cosmomycins is a distinctive feature among anthracyclines. These antitumor compounds possess two trisaccharide chains attached at C-7 and C-10, each of them with structural variability, mainly at the distal deoxysugar moieties. We have characterized a 14-kb chromosomal region from Streptomyces olindensis containing 13 genes involved in cosmomycin biosynthesis. Two of the genes, cosG and cosK, coding for glycosyltransferase were inactivated with the generation of five new derivatives. Structural elucidation of these compounds showed altered glycosylation patterns indicating the capability of both glycosyltransferases of transferring deoxysugars to both sides of the aglycone and the flexibility of CosK with respect to the deoxysugar donor. A model is proposed for the glycosylation steps during cosmomycins biosynthesis.

Effect of pH on the production of the antitumor antibiotic retamycin by Streptomyces olindensis.

The effect of pH on cell growth and retamycin production in batch bioreactor cultures of Streptomyces olindensis ICB20 was investigated. In fermentations pH-controlled over the range 6.0-8.0, the highest retamycin production was achieved at pH 7.0, and the maximum concentration of retamycin, about 1.36 A (absorbance) units, was about 43, 58 and 232% higher than the values obtained at pH 7.5, 6.0 and 8.0 respectively.

DNA-binding properties of cosmomycin D, an anthracycline with two trisaccharide chains.

Cosmomycin D (CosD) is the major constituent fraction isolated from a culture of Streptomyces olindensis ICB20. The ability of this compound to intercalate with double-stranded DNA was studied by gel mobility shift assays and electrospray ionization mass spectrometry (ESI-MS). ESI-MS experiments showed that the complex of CosD with 16-mer double-stranded DNA was at least as stable as a complex of daunorubicin with the same DNA sequence. This is the first study showing DNA binding properties of an anthracycline containing a beta-rhodomycinone aglycone chromophore O-linked to two trisaccharide chains.