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 Sequence Of Streptomyces Wadayamensis Strain A23, An Endophytic Actinobacterium From Citrus Reticulata.

The actinobacterium Streptomyces wadayamensis A23 is an endophyte of Citrus reticulata that produces the antimycin and mannopeptimycin antibiotics, among others. The strain has the capability to inhibit Xylella fastidiosa growth. The draft genome of S. wadayamensis A23 has ~7.0 Mb and 6,006 protein-coding sequences, with a 73.5% G+C content.

Metabolic screening for PKS and NRPS in endophytic actinobacteria from citrus reticulata

Polyketides and non-ribosomal peptides are natural products widely found in bacteria, fungi and plants. The biological activities associated with these metabolites have attracted special attention in biopharmaceutical studies. Polyketide synthases act similarly to fatty acids synthetases and the whole multi-enzymatic set coordinating precursor and extending unit selection and reduction levels during chain growth. Acting in a similarly orchestrated model, non-ribosomal peptide synthetases biosynthesize NRPs. PKSs-I and NRPSs enzymatic modules and domains are collinearly organized with the parent gene sequence. This arrangement allows the use of degenerated PCR primers to amplify targeted regions in the genes corresponding to specific enzymatic domains such as ketosynthases and acyltransferases in PKSs and adenilation domains in NRPSs. Careful analysis of these short regions allows the classifying of a set of organisms according to their potential to biosynthesize PKs and NRPs. In this work, the biosynthetic potential of a set of 13 endophytic actinobacteria from Citrus reticulata for producing PKs and NRP metabolites was evaluated. The biosynthetic profile was compared to antimicrobial activity. Based on the inhibition promoted, 4 strains were considered for cluster analysis. A PKS/NRPS phylogeny was generated in order to classify some of the representative sequences throughout comparison with homologous genes. Using this approach, a molecular fingerprint was generated to help guide future studies on the most promising strains.v. 38n.333334

Studies in Molecular Recognition: Non-proteogenic Amino Acids for Antibiotic Studies and Chemosensors for Recognition and Reporting of Metal-ions

The field of molecular recognition focuses on the selective and reversible binding of small guest molecules to larger host molecules. This dissertation describes synthesis of small molecules as binding guests for enzymatic substrates as well as molecules as host chemosensors to detect and identify metal ions. Two approaches to new antibiotic drugs have been explored, and an array of sensors for the quantitation of aqueous metals is being commercialized. As strains of deadly bacteria emerge with evolved resistance to known antibiotics, new drugs are needed with novel mechanisms of action. Natural product antibiotics containing enduracididine, a non-canonical amino acid derived from arginine, have been found effective against resistant organisms. Recently, the biosynthesis of enduracididine has been elucidated by the Silvaggi group. Various derivatives of arginine are of interest as guest molecules for the Mpp family of proteins. We have developed a particularly succinct route to γ-hydroxy-arginine, which has also been used as a precursor to other oxidized arginine derivatives. Our route provides quantities of arginine derivatives which have been synthesized via a four step route utilizing an isoxazoline intermediate. The synthetic methods for formation and subsequent reduction of the isoxazoline have been studied extensively; and this succinct and versatile synthesis yields either γ-hydroxy-arginine or the keto acid derived from it by changing the conditions of the reduction. iii In another approach to developing new antibiotic treatment, we have pursued the inhibition of the β-barrel assembly machine (BAM), through a small molecule scaffold that binds β-sheets. BAMa is the only known β-barrel protein that spontaneously folds, while all others require the help of BAMa. The β-barrel membrane proteins include efflux pumps, proteins for active transport that allow bacterial survival by ejection of antibiotic drugs. Inhibition of BAMa may itself prove bactericidal, or used in combination therapy increase efficacy of drugs rendered previously ineffective due to acquired resistance. We have also developed metal ion chemosensors for simulataneous identification and quantitation of multiple metals. These are useful for monitoring metal ion concentrations in industrial wastewater. Current limitations in measuring metal concentration in wastewater can lead to increased costs and excess solid waste in order to meet compliance standards. Our goal is a simple system to allow continuous, real-time measurement of multiple metals on site to decrease over-treatment and detect spikes in pollutant metals. Utilizing UV/VIS absorption an array of semi-selective sensors each with its own spectral response to metal ions allows identification and concentration of pollutants to be determined. Toward this end, we have prepared chemosensors, demonstrated their sensing ability, and covalently attached them to transparent polymers and transparent supports in several ways that allow repeated use for metal-ion measurement. These dyes have been studied in solution and when covalently bound to polymers. Dyes with complementary metal-selectivity allow for high information from a few sensors; a model using three sensors has been demonstrated to simultaneously measure the concentration of seven metals in solution

Metabolic screening for PKS and NRPS in endophytic actinobacteria from citrus reticulata

Polyketides and non-ribosomal peptides are natural products widely found in bacteria, fungi and plants. The biological activities associated with these metabolites have attracted special attention in biopharmaceutical studies. Polyketide synthases act similarly to fatty acids synthetases and the whole multi-enzymatic set coordinating precursor and extending unit selection and reduction levels during chain growth. Acting in a similarly orchestrated model, non-ribosomal peptide synthetases biosynthesize NRPs. PKSs-I and NRPSs enzymatic modules and domains are collinearly organized with the parent gene sequence. This arrangement allows the use of degenerated PCR primers to amplify targeted regions in the genes corresponding to specific enzymatic domains such as ketosynthases and acyltransferases in PKSs and adenilation domains in NRPSs. Careful analysis of these short regions allows the classifying of a set of organisms according to their potential to biosynthesize PKs and NRPs. In this work, the biosynthetic potential of a set of 13 endophytic actinobacteria from Citrus reticulata for producing PKs and NRP metabolites was evaluated. The biosynthetic profile was compared to antimicrobial activity. Based on the inhibition promoted, 4 strains were considered for cluster analysis. A PKS/NRPS phylogeny was generated in order to classify some of the representative sequences throughout comparison with homologous genes. Using this approach, a molecular fingerprint was generated to help guide future studies on the most promising strains383333341CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESPPQ 306932/2009-12008/00605-1; 2008/06097-8; 2009/03793- 6; 2010/16798-

Studies on the production of antimycin and mannopeptimycin in Streptomyces sp. CBMAI 2043

Orientador: Luciana Gonzaga de OliveiraDissertação (mestrado) - Universidade Estadual de Campinas, Instituto de QuímicaResumo: Streptomyces são bactérias que habitam predominantemente o solo, porém podem ser encontradas nos mais diversos ambientes. O metabolismo secundário desses micro-organismos é extensamente estudado, pois é rico em moléculas com diferentes aplicações na indústria farmacêutica. Um exemplo desse vasto metabolismo e suas aplicações pode ser reconhecido na descoberta de antibióticos no século XX, os quais foram, em sua maioria, isolados de diferentes cepas de Streptomyces. Dentre a variedade de metabólitos produzidos por esse gênero estão as antimicinas, um inibidor da cadeia respiratória, que é empregado como piscicida, e as manopeptimicinas, glicopeptídeo isolado inicialmente de S. hygroscopicus, que possuem ação contra cepas resistentes aos antibióticos mais potentes, tais como metilciclinas e vancomicina. Nesse sentido, esse projeto teve como objetivo estudar a produção desses metabólitos, codificados no genoma da linhagem da Streptomyces sp. CBMAI 2043 realizando mutações na linhagem selvagem e ainda expressando os agrupamentos de genes relacionados à biossíntese desses metabólitos em linhagem heteróloga (S. coelicolor), de forma a aprofundar o conhecimento sobre o maquinário enzimático produtor destas moléculas e outras análogasAbstract: Streptomyces are bacteria that inhabit predominantly the soil but the secondary metabolism of these microorganisms is extensively studied as it is rich in molecules with different applications in the pharmaceutical industry. An example of this vast metabolism and its functions can be recognized in the discovery of antibiotics in the twentieth century; these entities were mostly isolated from different strains of Streptomyces. Among the variety of metabolites produced by these microorganisms are antimycins, a respiratory chain inhibitor, which is used as a piscicide, and the mannopeptimycins, a glycopeptide, isolated primarily from S. hygroscopicus, that act against strains resistant to the most potent antibiotics, such as methylcyclines and vancomycin. In this sense, this project aimed to study the production of these metabolites, encoded in Streptomyces sp. CBMAI 2043 genome performing mutations in the wild strain and expressing genes clusters related to the biosynthesis of these metabolites in a heterologous strain (S. coelicolor) in order to expand the knowledge about the enzymatic machinery producing these molecules and possible analoguesMestradoQuimica OrganicaMestra em Química2014/12727-5, 2014/50249-8CAPESFAPES

Structure-Function Relationships in Bacterial Regulatory Proteins and an Enzyme Involved in Antibiotic Biosynthesis

The first part of my thesis is focused on a new family of two-component response regulator proteins: Aspartate-Less Regulators (ALR). They lack the catalytic aspartate residue required for the phosphorylation mechanism of typical two component response regulators. We are using biophysical tools to characterize two proteins with redox-sensitive ALR domains: repressor of iron transport regulator (RitR) from Streptococcus pneumonia R6 and diguanylate cyclase Q15Z91 from Pseudoalteromonas atalantica. The structure of inactive RitRC128S monomer showed that the ALR domain and the DNA-binding domain are linked by an α-helix that runs the length of the entire protein, with C128 near the C-terminal end. Bioinformatic analysis of all streptococcal RitR homologs showed that Cys128 is strictly conserved, suggesting that RitR may be a novel redox sensor. Hydrogen peroxide was used to oxidize the cysteine thiol group to determine the structure of the oxidized, dimeric form of RitR. Oxidation of C128 to the disulfide caused a conformational change that caused the DBD to release from the ALR domain. Surprisingly, the freed DBD was observed bound to the ALR domain of the other, disulfide-linked molecule of RitR, recapitulating almost exactly the structure of the inactive, monomeric protein. An extended dimeric conformation was found in the RitRL86A/V93A variant. It binds to the target DNA according to gel filtration and differential scanning fluorimetry. The crystal structure of the RitRL86A/V93A ALR domain showed an unprecedented conformational change for a response regulator protein, where helix α4 is disordered and the two protomers swap their α5 helices to form the dimer. Combined with the C128D mutant in vivo studies, it seems that oxidation of C128 is part of the activation mechanism, but there must be an additional factor that leads to dimerization of the ALR domains. The second ALR protein Q15Z91 has R61 replacing the phosphorylatable aspartate residue in the ALR domain. According to the structure of Q15Z91 with GTP and c-di-GMP, purified Q15Z91 is an activated but product-inhibited dimer. C142 is conserved in the same position as C128 in RitR, and substitution demonstrated that C142 residue is also a redox sensor that involved in Q15Z91 activity regulation. The second part is a mechanistic enzymology project aimed at understanding the structure and mechanism of the novel pyridoxal-5’-phosphate (PLP)-dependent L-arginine hydroxylase/deaminase, MppP, from Streptomyces wadayamensis (SwMppP). SwMppP is predicted to be a type I/II aminotransferase based on primary sequence identity. However, NMR and ESI-MS results showed that SwMppP is not an aminotransferase, but rather a hydroxylase. The enzyme catalyzes the oxygen-dependent hydroxylation of L-arginine, forming 4-hydroxy-2-ketoarginine and the abortive side-product 2-ketoargine in a ratio of 1.7:1. This is exciting because SwMppP is the first PLP-dependent enzyme to react with oxygen in any context other than oxidative decarboxylation. The discovery of this new activity is especially surprising given that the tertiary structure of SwMppP is very similar to that of the prototypical aminotransferase, the E. coli aspartate aminotransferase (PDB entry 1ARS; RMSD of aligned Cα atoms is 3.7 Å). The major differences between the two enzymes are the disordered N terminus of SwMppP, and changes of a limited number of amino acids near the PLP cofactor. The N-terminal helix transitions from a disordered, random-coil state to a helical conformation covering the active site only if/when the substrate is bound. Specific roles of the un-conserved residues in the active site are being studied by mutagenesis. So far, most of the SwMppP mutants have lost the hydroxylase activity and only produce abortive side product 2-ketoarginine. Our mechanistic studies have revealed that formation of the fully oxidized (hydroxylated) product requires 2 equivalents of dioxygen, while formation of 2-ketoarginine requires only one equivalent of dioxygen. Interestingly, the hydroxyl group of 4-hydroxy-2-ketoarginine comes from H2O, not dioxygen. Mutagenesis, structural and kinetic studies were used to understand how the residues in the active site stabilize the quinonoid form of the L-arginine-PLP complex to promote the reaction with dioxygen. Our structural and kinetic characterization of the wild-type and variant forms of SwMppP have allowed us to propose a model where the oxygen incorporated in the hydroxy-arginine product is derived from water rather than from dioxygen. In addition, SwMppP exhibits very high substrate specificity. Either change on the substrate length or guanidine group would result in no binding affinity or little activity

A phylogenetic and evolutionary analysis of antimycin biosynthesis

Streptomyces species and other Actinobacteria are ubiquitous in diverse environments worldwide and are the source of, or inspiration for, the majority of antibiotics. The genomic era has enhanced biosynthetic understanding of these valuable chemical entities and has also provided a window into the diversity and distribution of natural product biosynthetic gene clusters. Antimycin is an inhibitor of mitochondrial cytochrome c reductase and more recently was shown to inhibit Bcl-2/Bcl-XL-related anti-apoptotic proteins commonly overproduced by cancerous cells. Here we identify 73 putative antimycin biosynthetic gene clusters (BGCs) in publicly available genome sequences of Actinobacteria and classify them based on the presence or absence of cluster-situated genes antP and antQ, which encode a kynureninase and a phosphopantetheinyl transferase (PPTase), respectively. The majority of BGCs possess either both antP and antQ (L-form) or neither (S-form), while a minority of them lack either antP or antQ (IQ- or IP-form, respectively). We also evaluate the biogeographical distribution and phylogenetic relationships of antimycin producers and BGCs. We show that antimycin BGCs occur on five of the seven continents and are frequently isolated from plants and other higher organisms. We also provide evidence for two distinct phylogenetic clades of antimycin producers and gene clusters, which delineate S-form from L- and I-form BGCs. Finally, our findings suggest that the ancestral antimycin producer harboured an L-form gene cluster which was primarily propagated by vertical transmission and subsequently diversified into S-, IQ- and IP-form biosynthetic pathways

A trans-acting cyclase off-loading strategy for non-ribosomal peptide synthetases

The terminal step in the biosynthesis of nonribosomal peptides is the hydrolytic release and, frequently, macrocyclization of an aminoacyl-S-thioester by an embedded thioesterase. The surugamide biosynthetic pathway is composed of two nonribosomal peptide synthetase (NRPS) assembly lines in which one produces surugamide A, which is a cyclic octapeptide, and the other produces surugamide F, a linear decapeptide. The terminal module of each system lacks an embedded thioesterase, which led us to question how the peptides are released from the assembly line (and cyclized in the case of surugamide A). We characterized a cyclase belonging to the β-lactamase superfamily in vivo, established that it is a trans-acting release factor for both compounds, and verified this functionality in vitro with a thioester mimic of linear surugamide A. Using bioinformatics, we estimate that ∼11% of filamentous Actinobacteria harbor an NRPS system lacking an embedded thioesterase and instead employ a trans-acting cyclase. This study improves the paradigmatic understanding of how nonribosomal peptides are released from the terminal peptidyl carrier protein and adds a new dimension to the synthetic biology toolkit

Genome Mining Revealed a High Biosynthetic Potential for Antifungal Streptomyces sp. S-2 Isolated from Black Soot

The increasing resistance of fungal pathogens has heightened the necessity of searching for new organisms and compounds to combat their spread. Streptomyces are bacteria that are well-known for the production of many antibiotics. To find novel antibiotic agents, researchers have turned to previously neglected and extreme environments. Here, we isolated a new strain, Streptomyces sp. S-2, for the first time, from black soot after hard coal combustion (collected from an in-use household chimney). We examined its antifungal properties against plant pathogens and against fungi that potentially pose threat to human health (Fusarium avenaceum, Aspergillus niger and the environmental isolates Trichoderma citrinoviridae Cin-9, Nigrospora oryzae sp. roseF7, and Curvularia coatesieae sp. junF9). Furthermore, we obtained the genome sequence of S-2 and examined its potential for secondary metabolites production using anti-SMASH software. The S-2 strain shows activity against all of the tested fungi. Genome mining elucidated a vast number of biosynthetic gene clusters (55), which distinguish this strain from closely related strains. The majority of the predicted clusters were assigned to non-ribosomal peptide synthetases or type 1 polyketide synthetases, groups known to produce compounds with antimicrobial activity