Ribosomal Peptide Synthetase Research Articles

Biosynthesis of a clickable pyoverdine via in vivo enzyme engineering of an adenylation domain

The engineering of non ribosomal peptide synthetases (NRPS) for new substrate specificity is a potent strategy to incorporate non-canonical amino acids into peptide sequences, thereby creating peptide diversity and broadening applications. The non-ribosomal peptide pyoverdine is the primary siderophore produced by Pseudomonas aeruginosa and holds biomedical promise in diagnosis, bio-imaging and antibiotic vectorization. We engineered the adenylation domain of PvdD, the terminal NRPS in pyoverdine biosynthesis, to accept a functionalized amino acid. Guided by molecular modeling, we rationally designed mutants of P. aeruginosa with mutations at two positions in the active site. A single amino acid change results in the successful incorporation of an azido-l-homoalanine leading to the synthesis of a new pyoverdine analog, functionalized with an azide function. We further demonstrated that copper free click chemistry is efficient on the functionalized pyoverdine and that the conjugated siderophore retains the iron chelation properties and its capacity to be recognized and transported by P. aeruginosa. The production of clickable pyoverdine holds substantial biotechnological significance, paving the way for numerous downstream applications.

Antimicrobial potential of haloalkaliphilic Nocardiopsis sp. AJ1 isolated from solar salterns in India.

Antagonistic haloalkaliphilic Nocardiopsis sp. AJ1 (GenBank JX575136.1), isolated and identified from the saline soil of Kovalam solar salterns was able to produce antimicrobial secondary metabolites and effectively suppressed several bacterial and fungal pathogens. The metabolite extracted from ethyl acetate precipitation suppressed the bacterial and fungal pathogens to the range between 2.14 and 20.14 mm and also controlled the shrimp killer virus WSSV by 83% than the control and significantly (p < 0.05) differed. GC-MS analysis revealed that, the ethyl acetate precipitation contains pyrrolo (1,2-A(pyrazine-1,4-dione, hexahydro-3-(2-methylpropyl)-) and actinomycin C2. Non ribosomal peptide synthetase (NRPS) was amplified by PCR with the amplicon size of 750-800 bp length and further predicted the secondary structure by Iterative Threading Assembly Refinement (I-TASSER) bioinformatics approach. I-TASSER prediction helped to find out the secondary, 3-D structure, and ligand binding sites. The top ten modelling concluded that, the NRPS gene is closely similar to surfactin synthesizing gene, surfactin A synthetase C (SRFA-C). The findings revealed that, the active compounds from the secondary metabolites effectively suppressed the pathogenic bacteria, fungi, and virus and useful to develop antimicrobials.

Majority of Actinobacterial Strains Isolated from Kashmir Himalaya Soil Are Rich Source of Antimicrobials and Industrially Important Biomolecules

Studies on actinobacterial diversity are immensely important research arenas as they are the major root of antimicrobial, anticancer and immunosuppressant agents. Here we have performed a community analysis and isolated about 135 morphologically different actinobacteria using five different Actinomyces-specific media from an unique unexplored site of Kashmir Himalaya, India, in order to screen different bioactive compounds specially the antimicrobials. Among these actinobacteria, 42 isolates show antimicrobial activity against a set of pathogenic organisms. Secretion of different biomolecules of industrial significance were assessed which resulted 53 xylanase producers, 57 IAA producers, and 63 isolates produce gelatinase. Pectinase, phosphatase, lipase, amylase, and cellulase producing ability have been shown by about 41, 32, 102, 92, and 83 isolates, respectively. Analysis of their 16S rRNA sequence followed by phylogenetic analysis has depicted that most of the isolates belong to the genus of Streptomyces and rests are Streptosporangium, Lentzea, Gordonia, Amycolatopsis and Lechevalieria. We have also screened the presence of secondary metabolite production pathways by PCR amplifying the biosynthetic gene of polyketide synthase (PKS-I) and non ribosomal peptide synthetase (NRPS) from the antibiotic producing strains. Antibiotic sensitivity profile of these isolates has also been studied. The potential antimicrobial compound producing strains have further been studied in order to find their efficacy to kill bacterial and fungal pathogens. Thus the study led to a promising field of antibiotic discovery and bioprospecting actinobacteria through their metabolites of industrial potential in near future.

Molecular basis of methylation and chain-length programming in a fungal iterative highly reducing polyketide synthase.

Exchange of 32 different sub-fragments of the C-methyltransferase (C-MeT), pseudo-ketoreductase (ΨKR) and ketoreductase (KR) catalytic domains of the tenellin iterative Type I polyketide synthase non ribosomal peptide synthetase (PKS-NRPS) TENS by homologous fragments from the desmethylbassianin (DMBS) and militarinone (MILS) PKS-NRPS led to the creation of chimeric synthetases in which programming fidelity was altered, resulting in the production of mixtures of products with different methylation patterns and chain lengths. Swap of KR domain subfragments with the homologous fragments from the KR of the heptaketide militarinone synthetase resulted in the synthesis of penta, hexa and heptaketides. The results of these and previous experiments are rationalised by considering the existence of competition for acyl-carrier protein (ACP) bound substrates between different catalytic domains of the PKS. In particular, competition between the C-MeT and ketoreductase domains (KR) can account for methylation programming, and competition between the KR and the off-loading NRPS accounts for chain-length selectivity.

Determination and production of antimicrobial compounds by Aspergillus clavatonanicus strain MJ31, an endophytic fungus from Mirabilis jalapa L. using UPLC-ESI-MS/MS and TD-GC-MS analysis.

Endophytic fungi associated with medicinal plants are reported as potent producers of diverse classes of secondary metabolites. In the present study, an endophytic fungi, Aspergillus clavatonanicus strain MJ31, exhibiting significant antimicrobial activity was isolated from roots of Mirabilis jalapa L., was identified by sequencing three nuclear genes i.e. internal transcribed spacers ribosomal RNA (ITS rRNA), 28S ribosomal RNA (28S rRNA) and translation elongation factor 1- alpha (EF 1α). Ethyl acetate extract of strain MJ31displayed significant antimicrobial potential against Bacillus subtilis, followed by Micrococccus luteus and Staphylococcus aureus with minimum inhibitory concentrations (MIC) of 0.078, 0.156 and 0.312 mg/ml respectively. In addition, the strain was evaluated for its ability to synthesize bioactive compounds by the amplification of polyketide synthase (PKS) and non ribosomal peptide synthetase (NRPS) genes. Further, seven antibiotics (miconazole, ketoconazole, fluconazole, ampicillin, streptomycin, chloramphenicol, and rifampicin) were detected and quantified using UPLC-ESI-MS/MS. Additionally, thermal desorption-gas chromatography mass spectrometry (TD-GC-MS) analysis of strain MJ31 showed the presence of 28 volatile compounds. This is the first report on A. clavatonanicus as an endophyte obtained from M. jalapa. We conclude that A. clavatonanicus strain MJ31 has prolific antimicrobial potential against both plant and human pathogens and can be exploited for the discovery of new antimicrobial compounds and could be an alternate source for the production of secondary metabolites.

Genetic Characteristics of Actinomycetes Isolates (GST, KP, KP11, Kp16, T24, T37 Code) by RFLP of NRPS Genes

Actinomycetes is the one of antibiotic producing microorganisms. Actinomycetes isolates with GST, KP, KP11, KP16, T24, T37 have been isolated from soil. This study aims to determine the diversity of actinomycetes isolates by RFLP (Restriction Fragment Length Polymorphism) of NRPS (Non Ribosomal Peptide Synthetase) genes from GST, KP, KP11, KP16, T24, and T37 isolates. This study is divided into several steps of the DNA isolation from isolate GST, KP16, T24, T37, KP, KP11, PCR of the 16S rRNA and NRPS genes and cutting the NRPS genes fragmens with HaeIII enzyme to determine the diversity of isolates. Results of DNA isolate, PCR of 16S rRNA genes and NRPS genes, and RFLP of NRPS genes performed by agarose gel electrophoresis. The diversity of the isolates were analyzed by multivariate analysis UPGMA with MVSP 2.0 applications. The results showed the diversity of 6 actinomycetes isolates are T37 and GST have 64 % similarity, KP11 and KP16 have 96 % similarity, KP has 76 % similarity with KP11 and KP16, while T24 has 60 % similarity with KP and 12 % similarity with T37 and GST. Based on RFLP analysis of the NRPS genes, among of 6 isolates can divided into 4 groups are T37 and GST isolates as group 1, KP11 and KP16 isolates as group 2, KP isolate as group 3, and T24 isolate as group 4. Keyword : Actinomycetes, diversity, 16S rRNA genes, NRPS genes, RFLP

English

Forty-nine Streptomyces isolates were recovered from sediment samples in the gulf of Aqaba/Jordan. All isolates were tested for antimicrobial activity against Gram positive bacteria, Gram negative bacteria, and yeast. Twenty eight Streptomyces isolates were active against at least one of the tested strains. The majority of the isolates showed activity against Gram positive bacteria: Streptomyces aureus (89%), Streptomyces epidermidis (64%) and Bacillus Subtilis (50 %). Lower activity was observed toward Gram negative bacteria with only 25% active against Pseudomonas aeruginosa , whereas only 17% were active against the yeast Candida albicans . Isolate S34 showed best activity. It produced heat stable antimicrobial activity at both acidic and alkaline pH (5 to 5.5 and 8 to 9.5). S34 was found to be related to Streptomyces rochei . Forty-nine Streptomyces isolates were screened for genes encoding non ribosomal peptide synthetases (NRPS) and polyketides synthases (PKS; types I and II). NRPS sequences were widely distributed and detected in 81% of Streptomyces isolates. PKS types I and II were detected in 63.2 and 65.3% of isolates, respectively. Additionally, the relationship between the occurrences of biosynthetic gene sequences (NPRS and PKS sequences) and the production of antimicrobial activities was determined. The above results reveal that the marine Streptomycetes are a promising source of novel and unique products. Keywords: Marine Streptomyces , antimicrobial activity, non ribosomal peptide synthetases (NRPS), polyketides synthases (PKS), enzymes, gulf of Aqaba, Jordan African Journal of Biotechnology , Vol 13(31) 3505-3515

KERAGAMAN ISOLAT ACTINOMYCETES BERDASARKAN ANALISIS RFLP TERHADAP GEN NRPS

Actinomycetes is one of the types of microorganisms that is essential for producing the active compounds, one of which is an antibiotic. Isolation of Actinomycetes from the rhizosphere of rice plant (Oryza sativa L.) and tin plant (Ficus carica L.) has yielded 13 isolates of antibiotic-producing Actinomycetes. This study aims to determine the diversity of isolates by RFLP analysis (Restriction Fragment Length Polymorphism) of the NRPS (Non Ribosomal Peptide Synthetase) gene. The study was carried out by isolating Actinomycetes isolate DNA and performed PCR on the 16SrRNA and NRPS genes. The RFLP analysis of the NRPS gene PCR products is then performed using the enzyme HaeIII. Identify the presence of DNA, the PCR product and RFLP results was performed by agarose gel electrophoresis. The diversity of isolates was analyzed by multivariate analysis. The results showed that the diversity of 13 isolates of Actinomycetes can be grouped into 5 groups: isolates 5 (group 1), isolates 8 (group 2), isolates 11 (group 3), isolates 6,7,9 (group 4) and isolates 1,2,3,4,10,12,13 (group 5).

The Nocardia cyriacigeorgica GUH-2 genome shows ongoing adaptation of an environmental Actinobacteria to a pathogen’s lifestyle

BackgroundNocardia cyriacigeorgica is recognized as one of the most prevalent etiological agents of human nocardiosis. Human exposure to these Actinobacteria stems from direct contact with contaminated environmental matrices. The full genome sequence of N. cyriacigeorgica strain GUH-2 was studied to infer major trends in its evolution, including the acquisition of novel genetic elements that could explain its ability to thrive in multiple habitats.ResultsN. cyriacigeorgica strain GUH-2 genome size is 6.19 Mb-long, 82.7% of its CDS have homologs in at least another actinobacterial genome, and 74.5% of these are found in N. farcinica. Among N. cyriacigeorgica specific CDS, some are likely implicated in niche specialization such as those involved in denitrification and RuBisCO production, and are found in regions of genomic plasticity (RGP). Overall, 22 RGP were identified in this genome, representing 11.4% of its content. Some of these RGP encode a recombinase and IS elements which are indicative of genomic instability. CDS playing part in virulence were identified in this genome such as those involved in mammalian cell entry or encoding a superoxide dismutase. CDS encoding non ribosomal peptide synthetases (NRPS) and polyketide synthases (PKS) were identified, with some being likely involved in the synthesis of siderophores and toxins. COG analyses showed this genome to have an organization similar to environmental Actinobacteria.ConclusionN. cyriacigeorgica GUH-2 genome shows features suggesting a diversification from an ancestral saprophytic state. GUH-2 ability at acquiring foreign DNA was found significant and to have led to functional changes likely beneficial for its environmental cycle and opportunistic colonization of a human host.

Biosynthesis of natural products by microbial iterative hybrid PKS–NRPS

This review discusses the biosynthesis of natural products generated by iterative hybrid polyketide synthases–non ribosomal peptide synthetases (PKS–NRPS) from fungi and bacteria, the programming of the enzymes and bioengineering approaches.

Overexpression of NRPS4 leads to increased surface hydrophobicity in fusarium graminearum

The plant pathogen Fusarium graminearum is the infamous cause of Fusarium head blight worldwide resulting in significant losses of yield and reduced grain feed quality. It also has the potential to produce a range of small bioactive peptides produced by the non ribosomal peptide synthetases (NRPSs). Most of these are unknown as F. graminearum contains 19 NRPS encoding genes, but only three have been assigned products. For the first time, we use deletion and overexpression mutants to investigate the functions and product of NRPS4 in F. graminearum. Deletion of NRPS4 homologues in Alternaria brassicicola and Cochloibolus heterostrophus has been shown to result in mutants unable to repel water. In a time study of surface hydrophobicity we observed that water droplets could penetrate 7d old colonies of the NRPS4 deletion mutants. Loss in ability to repel water was first observed on 13d old cultures of the wild type strain, whereas the overexpression strain remained water repellant throughout the 38d time study. The conidia of both mutants were examined and those of the overexpression mutant showed distinct morphological differences in form of collapsed cells. These observations might suggest that the peptide product of NRPS4 could be an architectural factor in the cell walls of Fusarium or an indirect regulator of hydrophobicity.

1H, 13C and 15N NMR assignment of CGC-19, a single domain proteic constituent of a non ribosomal peptide synthetase

CGC-19, a 14 kDa proteic constituent of a non ribosomal peptide synthetase implicated in the biosynthesis of a secondary metabolite in Streptomyces ambofaciens, has been isotopically enriched and recombinantly expressed. Its nearly complete (1)H, (13)C and (15)N resonance assignment is reported hereunder.

Identification of surfactin producing strains in Soumbala and Bikalga fermented condiments using Polymerase Chain Reaction and Matrix Assisted Laser Desorption/Ionization-Mass Spectrometry methods

In this study, 67 strains were isolated from two fermented condiments from Burkina Faso: Soumbala and Bikalga. Phenotypical methods, biochemical tests and molecular approaches were used to determinate their genus or species. Twenty-two of them belong to the Bacillus genus. Six strains were selected for their antibacterial or antifungal properties. Their ability to produce lipopeptides synthesized by Non Ribosomal Peptide Synthetases was investigated using two different approaches: PCR with specific degenerated primers and Matrix-assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-ToF MS) performed on whole cells cultivated on a solid medium. PCR revealed that the six strains contain genes involved in the biosynthesis of surfactins whereas surfactins C₁₄ and C₁₅ were only detected by MALDI-ToF MS in two of the six strains. For the first time, the presence of surfactins C₁₄ and C₁₅ was also identified by MALDI-ToF MS analyses directly performed on Soumbala methanolic crude extracts. The structure of these compounds was confirmed by +MS2 and +MS3 of sample and reference surfactins.

Characterization and Localization of a Hybrid Non-ribosomal Peptide Synthetase and Polyketide Synthase Gene from the Toxic Dinoflagellate Karenia brevis

The toxic dinoflagellate Karenia brevis, a causative agent of the red tides in Florida, produces a series of toxic compounds known as brevetoxins and their derivatives. Recently, several putative genes encoding polyketide synthase (PKS) were identified from K. brevis in an effort to elucidate the genetic systems involved in brevetoxin production. In this study, novel PKS sequences were isolated from three clones of K. brevis. Eighteen unique sequences were obtained for the PKS ketosynthase (KS) domain of K. brevis. Phylogenetic comparison with closely related PKS genes revealed that 16 grouped with cyanobacteria sequences, while the remaining two grouped with Apicomplexa and previously reported sequences for K. brevis. A fosmid library was also constructed to further characterize PKS genes detected in K. brevis Wilson clone. Several fosmid clones were positive for the presence of PKS genes, and one was fully sequenced to determine the full structure of the PKS cluster. A hybrid non ribosomal peptide synthetase and PKS (NRPS-PKS) gene cluster of 16,061 bp was isolated. In addition, we assessed whether the isolated gene was being actively expressed using reverse transcription polymerase chain reaction (RT-PCR) and determined its localization at the cellular level by chloroplast isolation. RT-PCR analyses revealed that this gene was actively expressed in K. brevis cultures. The hybrid NRPS-PKS gene cluster was located in the chloroplast, suggesting that K. brevis acquired the ability to produce some of its secondary metabolites through endosymbiosis with ancestral cyanobacteria. Further work is needed to determine the compound produced by the NRPS-PKS hybrid, to find other PKS gene sequences, and to assess their role in K. brevis toxin biosynthetic pathway.

New tricks from ancient algae: natural products biosynthesis in marine cyanobacteria

Cyanobacteria, among Earth's oldest organisms, have evolved sophisticated biosynthetic pathways to produce a rich arsenal of bioactive natural products. In consequence, cyanobacterial secondary metabolites have been an incredibly fruitful source of lead compounds in drug discovery efforts. Investigations into the biochemistry responsible for the creation of these compounds, complemented by genome sequencing efforts, are revealing unique enzymatic mechanisms not described or rarely described elsewhere in the natural world. Herein, we discuss recent advances in understanding the biosynthesis of three cyanobacterial classes of natural product: mixed polyketide synthase/non ribosomal peptide synthetase (PKS/NRPS) metabolites, aromatic amino acid-derived alkaloids, and ribosomally encoded cyclic peptides. The unique biosynthetic mechanisms employed by cyanobacteria are inspiring new developments in heterologous gene expression and biotechnology.

Combination Methods for Screening Marine Actinomycetes Producing Potential Compounds as Anticancer

Marine actinomycetes is a robust source of secondary metabolites including anticancer compounds . The objective of this research was to select marine actinomycetes producing potential compounds as anticancer used combination methods that consist of amplification PKS I (polyketide synthases type I) and NRPS (non ribosomal peptide synthetases) genes, analysis the diversity of secondary metabolites and genetic. Selected isolates were used for cytotoxicity assay. PKS I and NRPS genes were amplified using sets of degenerate primers. K1F and M6R were used for amplify ketosynthase and methyl-malonyl-CoA transferase modules of PKS I gene which targeted sequences 1200-1400 bp. A3F and A7R were used for amplify adenilation domains of NRPS gene which targeted sequences 700-800 bp. The diversity of secondary metabolites was analized by TLC and densitometry of ethyl acetate extracts. Genetic diversity was analized by repetitive DNA fingerprinting using BOXA1R primers. The cytotoxicity of secondary metabolites on T47D and MCF7 breast cell lines cancer was measured by MTT assay method. Fifty two marine actinomycetes isolates were screened using combination methods. Ten isolates were detected encoding both PKS I and NRPS genes, whereas 11 isolates were detected encoding the NRPS gene. The screening by analysis of secondary metabolites and genetic diversity methods were obtained 6 selected isolates for cytotoxicity assay, which consist of 3 isolates encoding both PKS I and NRPS genes and 3 isolates encoding NRPS gene.Isolate 1 had high cytotoxicity with the IC50 on T47D cell was 19 mg/ml and the IC50 on MCF7 cell was 7 g/ml. This findings suggests that combination methods were effective and efficient way to select marine actinomycetes producing potential compounds as anticancer.

The Albonoursin Gene Cluster of S. noursei: Biosynthesis of Diketopiperazine Metabolites Independent of Nonribosomal Peptide Synthetases

Albonoursin [cyclo(ΔPhe-ΔLeu)], an antibacterial peptide produced by Streptomyces noursei, is one of the simplest representatives of the large diketopiperazine (DKP) family. Formation of α,β unsaturations was previously shown to occur on cyclo( L- Phe- L- Leu), catalyzed by the cyclic dipeptide oxidase (CDO). We used CDO peptide sequence information to isolate a 3.8 kb S. noursei DNA fragment that directs albonoursin biosynthesis in Streptomyces lividans. This fragment encompasses four complete genes: albA and albB, necessary for CDO activity; albC, sufficient for cyclic dipeptide precursor formation, although displaying no similarity to non ribosomal peptide synthetase (NRPS) genes; and albD, encoding a putative membrane protein. This first isolated DKP biosynthetic gene cluster should help to elucidate the mechanism of DKP formation, totally independent of NRPS, and to characterize novel DKP biosynthetic pathways that could be engineered to increase the molecular diversity of DKP derivatives.

Intraribosomal Environment of the Nascent Peptide Chain

This chapter discusses the chain of events that occurs during protein synthesis. The general reaction scheme is presented: the smaller subunits of ribosomes (30 S or 40 S subunits) attach to the initiation codon together with initiator tRNA (formyl methionyl tRNA for bacterial systems and methionyl tRNA for higher organisms). To this complex the larger subunits (50 S or 60 S subunit) bind to form the initiation complex of 70 S (or 80 S) ribosomes, initiation codon, and initiator tRNA. In this initiation process at least three initiation factors and GTP are involved. The chain then elongates as a result of the concerted action of three soluble factors, ribosomal peptide synthetase, aminoacyl tRNA, and 2 GTP molecules. When ribosomes carrying peptidyl tRNA reach the termination codon in mRNA, the termination factor binds to the ribosome–codon complex with the help of another soluble factor. At this point the chain is released from tRNA and the complex of mRNA, ribosome, and tRNA remains behind. The chapter also discusses the formation and structure of ribosomes and the formation of aminoacyl tRNA.