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Nonribosomal Peptide Synthetases (nonribosomal + peptide_synthetase)
Selected AbstractsRational Manipulation of Carrier-Domain Geometry in Nonribosomal Peptide SynthetasesCHEMBIOCHEM, Issue 6 2007Ye Liu Changing the assembly line. Nonribosomal peptide synthetases are multidomain enzyme assemblies responsible for the biosynthesis of a wide range of therapeutically important natural products. Here we describe an approach to construct stable, domain-specific conjugates of nonribosomal peptide synthetases by using synthetic analogues of coenzyme A. [source] Type II Thioesterase Restores Activity of a NRPS Module Stalled with an Aminoacyl-S-enzyme that Cannot Be ElongatedCHEMBIOCHEM, Issue 9 2004Ellen Yeh Fidelity and efficiency. Nonribosomal peptide synthetases, which contain domains for the activation (A), thiolation (T), and condensation (C) of amino acids (AA), are high-efficiency, high-fidelity assembly lines for synthesizing peptide natural products. Errors in a single step can have serious consequences for product formation. Type II thioesterases (TEII) might play a critical role in ensuring efficiency and accuracy in these systems, essential features in any attempt to engineer NRPSs to produce novel products. [source] Identification of potentially toxic environmental Microcystis by individual and multiple PCR amplification of specific microcystin synthetase gene regionsENVIRONMENTAL TOXICOLOGY, Issue 3 2005Youness Ouahid Abstract Reliable cyanotoxin monitoring in water reservoirs is difficult because of, among other reasons, unpredictable changes in cyanobacteria biomass, toxin production, and inadequate sampling frequency. Therefore, it would be useful to identify potentially microcystin-producing strains of cyanobacterial populations in field samples. With this aim, we developed a methodology to distinguish microcystin-producing from non-producing Microcystis strains by amplifying six characteristic segments of the microcystin synthetase mcy cluster, three corresponding to the nonribosomal peptide synthetase, genes mcyA, mcyB, and mcyC, and three to the polyketide synthase, genes mcyD, mcyE, and mcyG. For this purpose five new primer sets were designed and tested using purified DNA, cultured cells, and field colonies as DNA sources. Simultaneous amplification of several genes in multipex PCR reactions was performed in this study. The results obtained showed that: (i) the expected specific amplicons were obtained with all microcystin-producing strains but not with nonproducing strains; (ii) cells could be directly used as DNA templates, 2000 cells being a sufficient number in most cases; (iii) simultaneous amplification of several gene regions is feasible both with cultured cells and with field colonies. Our data support the idea that the presence of various mcy genes in Microcystis could be used as a criterion for ascribing potential toxigenicity to field strains, and the possibility of applying whole-cell assays for the simultaneous amplification of various genes may contribute significantly to simplifying toxigenicity testing. © 2005 Wiley Periodicals, Inc. Environ Toxicol 20: 235,242, 2005. [source] Heterologous expression of nonribosomal peptide synthetases in B. subtilis: construction of a bi-functional B. subtilis/E. coli shuttle vector systemFEMS MICROBIOLOGY LETTERS, Issue 2 2002Sascha Doekel Abstract A major obstacle in investigating the biosynthesis of pharmacologically important peptide antibiotics is the heterologous expression of the giant biosynthetic genes. Recently, the genetically engineered strain Bacillus subtilis KE30 has been reported as an excellent surrogate host for the heterologous expression of an entire nonribosomal peptide synthetase (NRPS) gene cluster. In this study, we expand the applicability of this strain, by the development of four Escherichia coli/B. subtilis shuttle expression vectors. Comparative overproduction of hybrid NRPS proteins derived from both organisms revealed a significant beneficial effect of overproducing proteins in B. subtilis KE30 as underlined by the production of stable nondegradative proteins, as well as the formation of active phosphopantetheinylated holo-proteins. [source] Nonribosomal Peptide Synthesis in Schizosaccharomyces pombe and the Architectures of Ferrichrome-Type Siderophore Synthetases in FungiCHEMBIOCHEM, Issue 4 2006Torsten Schwecke Dr. Abstract A nonribosomal peptide synthetase (NRPS) in Schizosaccharomyces pombe, which possesses an unusual structure incorporating three adenylation domains, six thiolation domains and six condensation domains, has been shown to produce the cyclohexapeptide siderophore ferrichrome. One of the adenylation domains is truncated and contains a distorted key motif. Substrate-binding specificities of the remaining two domains were assigned by molecular modelling to glycine and to N -acetyl- N -hydroxy- L -ornithine. Hexapeptide siderophore synthetase genes of Magnaporthe grisea and Fusarium graminearum were both identified and analyzed with respect to substrate-binding sites, and the predicted product ferricrocin was identified in each. A comparative analysis of these synthetase systems, including those of the basidiomycete Ustilago maydis, the homobasidiomycete Omphalotus olearius and the ascomycetes Aspergillus nidulans, Aspergillus fumigatus, Fusarium graminearum, Cochliobolus heterostrophus, Neurospora crassa and Aureobasidium pullulans, revealed divergent domain compositions with respect to their number and positioning, although all produce similar products by iterative processes. A phylogenetic analysis of both NRPSs and associated L - N5 -ornithine monooxygenases revealed that ferrichrome-type siderophore biosynthesis has coevolved in fungi with varying in trans interactions of NRPS domains. [source] Construction of hybrid peptide synthetases for the production of ,- l -aspartyl- l -phenylalanine, a precursor for the high-intensity sweetener aspartameFEBS JOURNAL, Issue 22 2003Thomas Duerfahrt Microorganisms produce a large number of pharmacologically and biotechnologically important peptides by using nonribosomal peptide synthetases (NRPSs). Due to their modular arrangement and their domain organization NRPSs are particularly suitable for engineering recombinant proteins for the production of novel peptides with interesting properties. In order to compare different strategies of domain assembling and module fusions we focused on the selective construction of a set of peptide synthetases that catalyze the formation of the dipeptide ,- l -aspartyl- l -phenylalanine (Asp-Phe), the precursor of the high-intensity sweetener ,- l -aspartyl- l -phenylalanine methyl ester (aspartame). The de novo design of six different Asp-Phe synthetases was achieved by fusion of Asp and Phe activating modules comprising adenylation, peptidyl carrier protein and condensation domains. Product release was ensured by a C-terminally fused thioesterase domains and quantified by HPLC/MS analysis. Significant differences of enzyme activity caused by the fusion strategies were observed. Two forms of the Asp-Phe dipeptide were detected, the expected ,-Asp-Phe and the by-product ,-Asp-Phe. Dependent on the turnover rates ranging from 0.01,0.7 min,1, the amount of ,-Asp-Phe was between 75 and 100% of overall product, indicating a direct correlation between the turnover numbers and the ratios of ,-Asp-Phe to ,-Asp-Phe. Taken together these results provide useful guidelines for the rational construction of hybrid peptide synthetases. [source] Phylogenetic analysis of condensation domains in the nonribosomal peptide synthetasesFEMS MICROBIOLOGY LETTERS, Issue 1 2005Niran Roongsawang Abstract Condensation (C) domains in the nonribosomal peptide synthetases are capable of catalyzing peptide bond formation between two consecutively bound various amino acids. C-domains coincide in frequency with the number of peptide bonds in the product peptide. In this study, a phylogenetic approach was used to investigate structural diversity of bacterial C-domains. Phylogenetic trees show that the C-domains are clustered into three functional groups according to the types of substrate donor molecules. They are l -peptidyl donors, d -peptidyl donors, and N -acyl donors. The fact that C-domain structure is not subject to optical configuration of amino acid acceptor molecules supports an idea that the conversion from l to d -form of incorporating amino acid acceptor occurs during or after peptide bond formation. l -peptidyl donors and d -peptidyl donors are suggested to separate before separating the lineage of Gram-positive and Gram-negative bacteria in the evolution process. [source] Heterologous expression of nonribosomal peptide synthetases in B. subtilis: construction of a bi-functional B. subtilis/E. coli shuttle vector systemFEMS MICROBIOLOGY LETTERS, Issue 2 2002Sascha Doekel Abstract A major obstacle in investigating the biosynthesis of pharmacologically important peptide antibiotics is the heterologous expression of the giant biosynthetic genes. Recently, the genetically engineered strain Bacillus subtilis KE30 has been reported as an excellent surrogate host for the heterologous expression of an entire nonribosomal peptide synthetase (NRPS) gene cluster. In this study, we expand the applicability of this strain, by the development of four Escherichia coli/B. subtilis shuttle expression vectors. Comparative overproduction of hybrid NRPS proteins derived from both organisms revealed a significant beneficial effect of overproducing proteins in B. subtilis KE30 as underlined by the production of stable nondegradative proteins, as well as the formation of active phosphopantetheinylated holo-proteins. [source] Functional, genetic and chemical characterization of biosurfactants produced by plant growth-promoting Pseudomonas putida 267JOURNAL OF APPLIED MICROBIOLOGY, Issue 2 2009Marco Kruijt Abstract Aims:, Plant growth-promoting Pseudomonas putida strain 267, originally isolated from the rhizosphere of black pepper, produces biosurfactants that cause lysis of zoospores of the oomycete pathogen Phytophthora capsici. The biosurfactants were characterized, the biosynthesis gene(s) partially identified, and their role in control of Phytophthora damping-off of cucumber evaluated. Methods and Results:, The biosurfactants were shown to lyse zoospores of Phy. capsici and inhibit growth of the fungal pathogens Botrytis cinerea and Rhizoctonia solani. In vitro assays further showed that the biosurfactants of strain 267 are essential in swarming motility and biofilm formation. In spite of the zoosporicidal activity, the biosurfactants did not play a significant role in control of Phytophthora damping-off of cucumber, since both wild type strain 267 and its biosurfactant-deficient mutant were equally effective, and addition of the biosurfactants did not provide control. Genetic characterization revealed that surfactant biosynthesis in strain 267 is governed by homologues of PsoA and PsoB, two nonribosomal peptide synthetases involved in production of the cyclic lipopeptides (CLPs) putisolvin I and II. The structural relatedness of the biosurfactants of strain 267 to putisolvins I and II was supported by LC-MS and MS-MS analyses. Conclusions:, The biosurfactants produced by Ps. putida 267 were identified as putisolvin-like CLPs; they are essential in swarming motility and biofilm formation, and have zoosporicidal and antifungal activities. Strain 267 provides excellent biocontrol activity against Phytophthora damping-off of cucumber, but the lipopeptide surfactants are not involved in disease suppression. Significance and Impact of the Study:,Pseudomonas putida 267 suppresses Phy. capsici damping-off of cucumber and provides a potential supplementary strategy to control this economically important oomycete pathogen. The putisolvin-like biosurfactants exhibit zoosporicidal and antifungal activities, yet they do not contribute to biocontrol of Phy. capsici and colonization of cucumber roots by Ps. putida 267. These results suggest that Ps. putida 267 employs other, yet uncharacterized, mechanisms to suppress Phy. capsici. [source] Rational Manipulation of Carrier-Domain Geometry in Nonribosomal Peptide SynthetasesCHEMBIOCHEM, Issue 6 2007Ye Liu Changing the assembly line. Nonribosomal peptide synthetases are multidomain enzyme assemblies responsible for the biosynthesis of a wide range of therapeutically important natural products. Here we describe an approach to construct stable, domain-specific conjugates of nonribosomal peptide synthetases by using synthetic analogues of coenzyme A. [source] |