No description available.

Strain MS14, exhibiting antifungal activity, was classified to belong to Burkholderia contaminans. Occidiofungin produced by strain MS14 is an octapeptide dedicated to a broad range of antifungal activities of the bacterium. The 58.2-kb genomic fragment containing 18 open reading frames (ORFs), named occidiofungin (occ) gene cluster, is required for occidiofungin production. Putative proteins encoded by five nonribosomal peptide synthetase genes (occA – occE) of the gene cluster were predicted to contain the catalytic modules responsible for the biosynthesis of occidiofungin. Transcription of all the ORFs identified in the region except ORF1 and ORF16 was regulated by both ambR1 and ambR2, the LuxR-type regulatory genes located at the left border of the cluster. The functional ambR1 gene was essential for transcription of ambR2, and constitutive expression of ambR2 did not restore the phenotype of the mutant MS14GG44(ambR1::nptII). Sequence analysis revealed that the occ gene cluster shared high similarity (99% nucleotide coverage and 91% identity) to an uncharacterized DNA region of B. ambifaria strain AMMD. The gene cluster was not found in other Burkholderia strains available in GenBank (nucleotide coverage < 24%). Analysis of G+C composition and prediction using “IslandPick” indicate that the occ gene cluster has possibly been horizontally transferred between bacteria. In addition, the absence of the gene cluster in clinical strains of Burkholderia indicates that occidiofungin is not required for potential human pathogenesis. The findings have provided insights into the development of antifungal medicines and agricultural fungicides based on occidiofungin.

Heterocycles play an important role in many biological processes and are widespread among natural products. Oxazole-containing natural products possess a broad range of bioactivities and are of great interest in the pharmaceutical and agrochemical industries. Herein, the biosynthetic routes to the oxazole-containing phthoxazolins and the bis(benzoxaozle) AJI9561, were investigated. Phthoxazolins A-D are a group of oxazole trienes produced by a polyketide synthase-nonribosomal peptide synthetase (PKS-NRPS) pathway in Streptomyces sp. KO-7888 and Streptomyces sp. OM-5714. The phthoxazolin pathway was used as a model to study 5-oxazole and primary amide formation in PKS-NRPS pathways. An unusually large gene cluster for phthoxazolin biosynthesis was identified from the complete genome sequence of the producer strains and various gene deletions were performed to define the minimal gene cluster. PhoxP was proposed to encode an ATP-dependent cyclodehydratase for 5-oxazole formation on an enzyme-bound N-formylglycylacyl-intermediate, and its deletion abolished phthoxazolin production. In vitro reconstitution of the early steps of phthoxazolin biosynthesis was attempted to validate the role of PhoxP, but was unsuccessful. Furthermore, Orf3515, a putative flavin-dependent monooxygenase coded by a remote gene, was proposed to hydroxylate glycine-extended polyketide-peptide chain(s) at the α-position to yield phthoxazolins with the primary amide moiety. On the other hand, an in vitro approach was employed to establish the enzymatic logic of the biosynthesis of AJI9561, a bis(benzoxazole) antibiotic isolated from Streptomyces sp. AJ9561. The AJI9561 pathway was reconstituted using the precursors 3-hydroxyanthranilic acid and 6-methylsalicylic acid and five purified enzymes previously identified from the pathway as key enzymes for benzoxazole formation, including two adenylation enzymes for precursor activation, an acyl carrier protein (ACP), a 3-oxoacyl-ACP synthase and an amidohydrolase-like cyclase. Intermediates and shunt products isolated from enzymatic reactions containing different enzyme and precursor combinations were assessed for their competence for various steps of AJI9561 biosynthesis. Further bioinformatic analysis and in silico modelling of the amidohydrolase-like cyclase shed light on the oxazole cyclisation that represents a novel catalytic function of the amidohydrolase superfamily.

Os microrganismos atraem considerável atenção por serem uma fonte de compostos biotecnológicos e farmacêuticos. Diversos produtos naturais peptídicos produzidos por fungos e bactérias são sintetizados por grandes enzimas, conhecidas como peptídeo sintetase não ribossômica (NRPS) e policetídeo sintase (PKS). A bioprospecção dos microrganismos isolados do solo de Terra Preta Antropogênica (TPA) da Amazônia Ocidental é de grande importância para o conhecimento deste bioma tropical. Este estudo correlacionou a presença de sideróforos e de compostos antimicrobianos produzidos pelos microrganismos isolados de TPA e dos solos adjacentes com a presença dos genes que codificam para NRPS e PKS. Linhagens bacterianas foram isoladas das amostras do solo coletadas de 10, 20 e 40 cm de profundidade. Os isolados foram cultivados em meio líquido específico por 2 dias a 28oC. Um total de 143 isolados foi testado para a atividade de sideróforo e para isso, as linhagens foram inoculadas em um meio com baixa concentração de ferro (MM9) contendo o complexo cromoazurol S-Fe3. Do total, 72 isolados apresentaram reação positiva para a produção de sideróforo. O DNA genômico dos isolados foi extraído e a amplificação por PCR foi realizada usando iniciadores específicos para NRPS e PKS. Os resultados mostraram que quinze isolados apresentaram o gene que codifica para NRPS, vinte isolados para PKS e somente dez isolados apresentaram ambos os genes. A presença de genes de NRPS e PKS em 31% dos isolados testados sugere que a produção dos sideróforos possa ocorrer pela via não ribossomal. Dois isolados foram selecionados para estudos de identificação e caracterização dos compostos. O isolado TP11 foi identificado como Pseudomonas putida através de seqüenciamento do 16S rRNA e apresentou resultado negativo para hidroxamato e catecol, sugerindo que o tipo de sideróforo não possui nenhum destes grupos funcionais. O isolado TP16 foi identificado como Pseudomonas putida e apresentou produção de sideróforo do tipo catecol e hidroxamato, sugerindo a produção de mais de um sideróforo. Além disso, esta linhagem produziu um composto antimicrobiano, com atividade de sideróforo identificado por espectrometria de massas como pseudomonina com massa molar de 330 Da. / Microorganisms have attracted considerable attention as a source for biotechnological and pharmaceutical agents. Several peptidic natural products synthesized by fungi and bacteria are assembled by large enzymes, referred as nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS). Bioprospection of microorganisms isolated from Anthropological Dark Earth soil of Brazilian Occidental Amazon is of great importance to the knowledge of this tropical biome. This study aimed to correlate the presence of siderophores and antimicrobial compounds produced by microorganisms isolated from Dark Earth and adjacent soils of Brazilian Amazon with the presence of genes encoding NRPS and PKS. Bacterial strains were isolated from soil samples collected at 10, 20 and 40 cm depth. The isolates were grown in specific liquid medium for 2 d at 28oC. A total of 143 isolates were screened for siderophore activity and for this, bacterial strains were inoculated on plates containing an iron-limited medium (MM9) amended with a chromeazurol S-Fe3 complex. From the total, seventy-two isolates showed positive reaction for siderophore production. Genomic DNA of the isolates was extracted and PCR amplification was carried out using specific primers for NRPS and PKS. The results showed that fifteen isolates presented NRPS, twenty isolates presented PKS and only ten isolates showed both genes. The presence of NRPS and PKS genes in 31% of the isolates tested suggests that production of siderophores may occur by a nonribosomal pathway. Two isolates were selected for further studies. Isolate TP11 was identified as Pseudomonas putida by 16S rDNA sequencing analysis and was negative for hydroxamate and catechol, suggesting that the siderophore type has no hydroxamate- or catechol-type functional groups. The isolate TP16 was identified as Pseudomonas putida and showed the production of catechol and hydroxamate siderophore-type, suggesting the production of more than one siderophore. In addition, this strain produced an antimicrobial compound, with siderophore activity identified through mass spectrometry as pseudomonine with a molar mass of 330 Da.

Os microrganismos atraem considerável atenção por serem uma fonte de compostos biotecnológicos e farmacêuticos. Diversos produtos naturais peptídicos produzidos por fungos e bactérias são sintetizados por grandes enzimas, conhecidas como peptídeo sintetase não ribossômica (NRPS) e policetídeo sintase (PKS). A bioprospecção dos microrganismos isolados do solo de Terra Preta Antropogênica (TPA) da Amazônia Ocidental é de grande importância para o conhecimento deste bioma tropical. Este estudo correlacionou a presença de sideróforos e de compostos antimicrobianos produzidos pelos microrganismos isolados de TPA e dos solos adjacentes com a presença dos genes que codificam para NRPS e PKS. Linhagens bacterianas foram isoladas das amostras do solo coletadas de 10, 20 e 40 cm de profundidade. Os isolados foram cultivados em meio líquido específico por 2 dias a 28oC. Um total de 143 isolados foi testado para a atividade de sideróforo e para isso, as linhagens foram inoculadas em um meio com baixa concentração de ferro (MM9) contendo o complexo cromoazurol S-Fe3. Do total, 72 isolados apresentaram reação positiva para a produção de sideróforo. O DNA genômico dos isolados foi extraído e a amplificação por PCR foi realizada usando iniciadores específicos para NRPS e PKS. Os resultados mostraram que quinze isolados apresentaram o gene que codifica para NRPS, vinte isolados para PKS e somente dez isolados apresentaram ambos os genes. A presença de genes de NRPS e PKS em 31% dos isolados testados sugere que a produção dos sideróforos possa ocorrer pela via não ribossomal. Dois isolados foram selecionados para estudos de identificação e caracterização dos compostos. O isolado TP11 foi identificado como Pseudomonas putida através de seqüenciamento do 16S rRNA e apresentou resultado negativo para hidroxamato e catecol, sugerindo que o tipo de sideróforo não possui nenhum destes grupos funcionais. O isolado TP16 foi identificado como Pseudomonas putida e apresentou produção de sideróforo do tipo catecol e hidroxamato, sugerindo a produção de mais de um sideróforo. Além disso, esta linhagem produziu um composto antimicrobiano, com atividade de sideróforo identificado por espectrometria de massas como pseudomonina com massa molar de 330 Da. / Microorganisms have attracted considerable attention as a source for biotechnological and pharmaceutical agents. Several peptidic natural products synthesized by fungi and bacteria are assembled by large enzymes, referred as nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS). Bioprospection of microorganisms isolated from Anthropological Dark Earth soil of Brazilian Occidental Amazon is of great importance to the knowledge of this tropical biome. This study aimed to correlate the presence of siderophores and antimicrobial compounds produced by microorganisms isolated from Dark Earth and adjacent soils of Brazilian Amazon with the presence of genes encoding NRPS and PKS. Bacterial strains were isolated from soil samples collected at 10, 20 and 40 cm depth. The isolates were grown in specific liquid medium for 2 d at 28oC. A total of 143 isolates were screened for siderophore activity and for this, bacterial strains were inoculated on plates containing an iron-limited medium (MM9) amended with a chromeazurol S-Fe3 complex. From the total, seventy-two isolates showed positive reaction for siderophore production. Genomic DNA of the isolates was extracted and PCR amplification was carried out using specific primers for NRPS and PKS. The results showed that fifteen isolates presented NRPS, twenty isolates presented PKS and only ten isolates showed both genes. The presence of NRPS and PKS genes in 31% of the isolates tested suggests that production of siderophores may occur by a nonribosomal pathway. Two isolates were selected for further studies. Isolate TP11 was identified as Pseudomonas putida by 16S rDNA sequencing analysis and was negative for hydroxamate and catechol, suggesting that the siderophore type has no hydroxamate- or catechol-type functional groups. The isolate TP16 was identified as Pseudomonas putida and showed the production of catechol and hydroxamate siderophore-type, suggesting the production of more than one siderophore. In addition, this strain produced an antimicrobial compound, with siderophore activity identified through mass spectrometry as pseudomonine with a molar mass of 330 Da.

Leiodolide A is a unique natural product isolated from Pacific marine sponges which has provided an interesting target for total synthesis due to its complex structure and undefined stereochemistry. Although synthetic work towards the synthesis of sister compound leiodolide B has been published, the total synthesis of leiodolide A is yet to be achieved but remains an important target due to high potency against leukaemia, non-small lung and ovarian cancers. The convergent strategy towards the synthesis of leiodolide A involved the synthesis of three subunits; a synthetic route to the C21-C25 vinyl stannane is described, and efforts towards the synthesis of the bidirectional C11-C20 subunit are detailed. Asymmetric vinylogous aldol methodology was developed for the installation of the 1,2-syn propionate motif found in the C1-C10 subunit and in other polypropionate natural products, and was shown to be applicable to a range of substrates in moderate diastereoselectivity and excellent enantioselectivity.