Avanços recentes em biossíntese combinatória de policetídeos: perspectivas e desafios Recent advances in combinatorial biosynthesis of polyketides: perspectives and challenges

Natural products continue to inspire scientists on the search for biological active structures. Recent advances in synthetic biology allow modifying genes to extend structural diversity present on natural products by changing important functional groups guided through genetic information. In this context, polyketides synthases (PKS) represent a class of enzymes from where the first applications and well succeed modifications were achieved. During the last two decades PKSs have been extensively studied, sequenced and deposited in data banks available to scientific community around the world. This newly wide information associated to different methodologies accessible to modify the PKSs genetic circuits including genetic insertion, gene deletion and gene replacement leads to discovery of new natural products. In this review, the recent advances in combinatorial biosynthesis, as well as modern techniques in synthetic biology to promote structural diversity on natural products will be discussed

Gene deletion leads to improved valinomycin production by Streptomyces sp. CBMAI 2042

The genus Streptomyces represents one of the largest producers of molecules with antibiotic activity. The whole genome sequencing of the endophytic microorganism Streptomyces sp. CBMAI 2042 revealed 35 gene clusters encoding for secondary metabolism including 3 non-ribosomal peptide synthetases (NRPS) and 7 NRPS-hybrids. Combining genome mining and cultivation profile analysis, the depsipeptide ionophore valinomycin was identified as one of the main metabolites produced by this strain. To better understand the metabolic machinery codified in CBMAI 2042 genome an adenylation domain from a hybrid NRPS cluster was deleted through a double crossing knockout experiment. Though the deletion was not plentiful to elucidate the encoded NRP metabolite related to the adenilation domain, an astounding increase of 10.5-fold in valinomycin production was observed in the mutant. These results suggest a metabolic flux redistribution of common substrates as an outcome of a gene target deletion

Whole-genome sequence of the endophytic Streptomyces sp. strain CBMAI 2042, isolated from Citrus sinensis

The whole-genome sequence of Streptomyces sp. strain CBMAI 2042, an endophytic actinobacterium isolated from Citrus sinensis branches, is described. The strain has the ability to inhibit the growth of Xylella fastidiosa and other human pathogens. In silico analysis highlighted the presence of nonribosomal peptide and polyketide synthases, revealing promising antibiotic assembly lines

Genome mining of endophytic streptomyces wadayamensis reveals high antibiotic production capability

The actinobacteria Streptomyces wadayamensis A23, an endophitic strain, was recently sequenced and previous work showed qualitatively that the strain inhibits the growth of some pathogens. Herein we report the genome analysis of S. wadayamensis which reveals several antibiotic biosynthetic pathways. Using mass spectrometry, we were able to identify desferoxamines, several antimycins and candicidin, as predicted. Additionally, it was possible to confirm that the biosynthetic machinery of the strain when compared to identified known metabolites is far underestimated. As suggested by biochemical qualitative tests, genome encoded information reveals that the strain A23 has high capability to produce antibiotics.The actinobacteria Streptomyces wadayamensis A23, an endophitic strain, was recently sequenced and previous work showed qualitatively that the strain inhibits the growth of some pathogens. Herein we report the genome analysis of S. wadayamensis which reve27814651475FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCNPQ - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO2014/12727-5; 2010/51677-2; 2013/12598-8; 2015/01013-4162191/2015-4; 130933/2015-5We gratefully acknowledge FAPESP (project grant 2014/12727-5 to L. G. O. and 2010/51677-2 to M. N. E.), PETROBRAS (grant 4712-0), and the University of Campinas. C. F. F. A. and B. S. P. acknowledges CNPq (studentships 162191/2015-4 and 130933/2015-5). A

"Genome mining" and cloning strategies for characterization of PKS and NRPS biosynthetic pathways from "Streptomyces" sp. CBMAI 2042

Orientador: Luciana Gonzaga de OliveiraTese (doutorado) - Universidade Estadual de Campinas, Instituto de QuímicaResumo: Actinobacteria é um filo de bactérias Gram-positivas que habitam ambientes terrestres ou aquáticos e são de grande relevância para a pesquisa acadêmica e a indústria farmacêutica devido ao grande potencial de biossintetizar diferentes classes de produtos naturais. Os policetídeos e os peptídeos não ribossomais destacam-se por combinar alta complexidade estrutural a uma ampla gama de atividades terapêuticas, tais como antibiótica, antitumoral e imonussupressora. Os genes biossintéticos que codificam para enzimas produtoras desses compostos podem ser mapeados pelo sequenciamento e genome mining de diferentes linhagens de Streptomyces, de modo a auxiliar e agilizar a busca por novos e importantes alvos terapêuticos. O microrganismo endofítico Streptomyces sp. CBMAI 2042, isolado de Citrus sinensis, foi sequenciado e a análise in silico do genoma permitiu identificar um cluster de genes órfão composto por uma PKS do tipo 1, trans-AT PKS e NRPS. O estudo aqui apresentado visou a clonagem desse cluster de genes biossintético através de bibliotecas genômicas em vetores do tipo BAC e ESAC (empresa Bio S&T) e de técnicas de recombinação de DNA, como l- RED, Gibson Assembly® e TAR-cloning, para posterior expressão em organismo heterólogo S. coelicolor. O cluster órfão obtido via TAR-cloning e expresso em S. coelicolor M1146 foi associado a produção de alpiniamida. A deleção do domínio de adenilação confirmou a correlação da via de biossíntese com a molécula isolada e caracterizada. Experimentos com precursores marcados embasaram a proposta de biossíntese dessa via mista trans-AT PKS/NRPS. Adicionalmente, o cluster de genes codificando para biossíntese da valinomicina, composto já descrito na literatura e produzido majoritariamente pela linhagem selvagem em diferentes cultivos, foi identificado no genoma e foi também selecionado para triagem de clones na biblioteca genômica. A clonagem e expressão heteróloga levou ao incremento de três vezes a produção do metabólito. Além disso, a associação da estratégia de molecular networking, levou a identificação de pelo menos seis análogos estruturais, entre eles a montanastatina, sendo pela primeira vez associados a mesma via de biossínteseAbstract: Actinobacteria is a phylum of Gram-positive terrestrial or aquatic bacteria of significant importance for academic research and pharmaceutical industries due to their great potential to biosynthesize different types of natural products. Polyketides and nonribosomal peptides are produced by enzymes known as polyketide synthases and nonribosomal synthetase and stand out by combining high structural complexity with a wide range of therapeutic activities, such as antibiotics, antitumor and immunosuppressor. In order to accelerate identification of new therapeutic compounds, the biosynthetic genes encoding to these and other class of enzymes can be mapped by genome sequence and mining of different Streptomyces strains. The endophytic strain Streptomyces sp. CBMAI 2042, isolated from Citrus sinensis, was sequenced and in silico analysis endorsed identification of the orphan biosynthetic genes comprising a type I PKS, trans-AT PKS and NRPS. This study aimed to obtain this biosynthetic gene cluster from genomic libraries constructed in BAC and ESAC vectors and by recombination methodologies l- RED, Gibson Assembly® and TAR- cloning, followed by heterologous expression in S. coelicolor. The orphan cluster obtainen by TAR-cloning and transferred to S. coelicolor M1146 was associated to alpiniamide biosynthesis. Deletion of adenylation domain confirmed the biosynthetic origin of isolated molecule and experiments with labeled precursors embased the proposed biosynthetic steps for assembly of alpiniamide. Additionally, the gene cluster related to biosynthesis of the already described valinomycin was identified from genome annotation and also screened for clones in the genomic library, as it is the major metabolite produced by CBMAI 2042. Cloning and heterologous expression in S. coelicolor M1146 resulted in 3 times increase in production of valinomycin and association with molecular networking allowed the identification of at least six analogues, including montanastatin, related for the first time to the same biosynthetic pathwayDoutoradoQuimica OrganicaDoutora em Ciências2015/01013-4FAPES

Recent advances in combinatorial biosynthesis of polyketides: perspectives and challenges

sem informação4217183Natural products continue to inspire scientists on the search for biological active structures. Recent advances in synthetic biology allow modifying genes to extend structural diversity present on natural products by changing important functional groups guided through genetic information. In this context, polyketides synthases (PKS) represent a class of enzymes from where the first applications and well succeed modifications were achieved. During the last two decades PKSs have been extensively studied, sequenced and deposited in data banks available to scientific community around the world. This newly wide information associated to different methodologies accessible to modify the PKSs genetic circuits including genetic insertion, gene deletion and gene replacement leads to discovery of new natural products. In this review, the recent advances in combinatorial biosynthesis, as well as modern techniques in synthetic biology to promote structural diversity on natural products will be discusse

AVANÇOS RECENTES EM BIOSSÍNTESE COMBINATÓRIA DE POLICETÍDEOS: PERSPECTIVAS E DESAFIOS

Natural products continue to inspire scientists on the search for biological active structures. Recent advances in synthetic biology allow modifying genes to extend structural diversity present on natural products by changing important functional groups guided through genetic information. In this context, polyketides synthases (PKS) represent a class of enzymes from where the first applications and well succeed modifications were achieved. During the last two decades PKSs have been extensively studied, sequenced and deposited in data banks available to scientific community around the world. This newly wide information associated to different methodologies accessible to modify the PKSs genetic circuits including genetic insertion, gene deletion and gene replacement leads to discovery of new natural products. In this review, the recent advances in combinatorial biosynthesis, as well as modern techniques in synthetic biology to promote structural diversity on natural products will be discussed

Whole-genome sequence of the endophytic streptomyces sp. strain CBMAI 2042, isolated from citrus sinensis

The whole-genome sequence of Streptomyces sp. strain CBMAI 2042, an endophytic actinobacterium isolated from Citrus sinensis branches, is described. The strain has the ability to inhibit the growth of Xylella fastidiosa and other human pathogens. In silico analysis highlighted the presence of nonribosomal peptide and polyketide synthases, revealing promising antibiotic assembly lines82CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP140824/2017-0; 313492/2017-42014/50249-8; 2014/12727-5; 2013/12598-8; 2015/01013-