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1.
Two important classes of natural products are made by nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs). With most biosynthetic intermediates covalently tethered during biogenesis, protein mass spectrometry (MS) has proven invaluable for their interrogation. New mass spectrometric assay formats (such as selective cofactor ejection and proteomics style LC-MS) are showcased here in the context of functional insights into new breeds of NRPS/PKS enzymes, including the first characterization of an 'iterative' PKS, the biosynthesis of the enediyne antitumor antibiotics, the study of a new strategy for PKS initiation via a GNAT-like mechanism, and the analysis of branching strategies in the so-called 'AT-less' NRPS/PKS hybrid systems. The future of MS analysis of NRPS and PKS biosynthetic pathways lies in adoption and development of methods that continue bridging enzymology with proteomics as both fields continue their post-genomic acceleration. 相似文献
2.
Mohd Zeeshan Ansari Jyoti Sharma Rajesh S Gokhale Debasisa Mohanty 《BMC bioinformatics》2008,9(1):454
Background
Secondary metabolites biosynthesized by polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) family of enzymes constitute several classes of therapeutically important natural products like erythromycin, rapamycin, cyclosporine etc. In view of their relevance for natural product based drug discovery, identification of novel secondary metabolite natural products by genome mining has been an area of active research. A number of different tailoring enzymes catalyze a variety of chemical modifications to the polyketide or nonribosomal peptide backbone of these secondary metabolites to enhance their structural diversity. Therefore, development of powerful bioinformatics methods for identification of these tailoring enzymes and assignment of their substrate specificity is crucial for deciphering novel secondary metabolites by genome mining. 相似文献3.
4.
A proteomic survey of nonribosomal peptide and polyketide biosynthesis in actinobacteria 总被引:1,自引:0,他引:1
Chen Y Ntai I Ju KS Unger M Zamdborg L Robinson SJ Doroghazi JR Labeda DP Metcalf WW Kelleher NL 《Journal of proteome research》2012,11(1):85-94
Actinobacteria such as streptomycetes are renowned for their ability to produce bioactive natural products including nonribosomal peptides (NRPs) and polyketides (PKs). The advent of genome sequencing has revealed an even larger genetic repertoire for secondary metabolism with most of the small molecule products of these gene clusters still unknown. Here, we employed a "protein-first" method called PrISM (Proteomic Investigation of Secondary Metabolism) to screen 26 unsequenced actinomycetes using mass spectrometry-based proteomics for the targeted detection of expressed nonribosomal peptide synthetases or polyketide synthases. Improvements to the original PrISM screening approach (Nat. Biotechnol. 2009, 27, 951-956), for example, improved de novo peptide sequencing, have enabled the discovery of 10 NRPS/PKS gene clusters from 6 strains. Taking advantage of the concurrence of biosynthetic enzymes and the secondary metabolites they generate, two natural products were associated with their previously "orphan" gene clusters. This work has demonstrated the feasibility of a proteomics-based strategy for use in screening for NRP/PK production in actinomycetes (often >8 Mbp, high GC genomes) versus the bacilli (2-4 Mbp genomes) used previously. 相似文献
5.
Ayuso A Clark D González I Salazar O Anderson A Genilloud O 《Applied microbiology and biotechnology》2005,67(6):795-806
The actinomycetes traditionally represent one of the most important sources for the discovery of new metabolites with biological activity; and many of these are described as being produced by polyketide synthases (PKS) and nonribosomal peptide synthetases (NRPS). We present a strain characterization system based on the metabolic potential of microbial strains by targeting these biosynthetic genes. After an initial evaluation of the existing bias derived from the PCR detection in a well defined biosynthetic systems, we developed a new fingerprinting approach based on the restriction analysis of these PKS and NRPS amplified sequences. This method was applied to study the distribution of PKS and NRPS biosynthetic systems in a collection of wild-type actinomycetes isolated from tropical soil samples that were evaluated for the production of antimicrobial activities. We discuss the application of this tool as an alternative characterization approach for actinomycetes and we comment on the relationship observed between the presence of PKS-I, PKS-II and NRPS sequences and the antimicrobial activities observed in some of the microbial groups tested. 相似文献
6.
Hybrid peptide-polyketide natural products: biosynthesis and prospects toward engineering novel molecules 总被引:2,自引:0,他引:2
The structural and catalytic similarities between modular nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) inspired us to search for hybrid NRPS-PKS systems. By examining the biochemical and genetic data known to date for the biosynthesis of hybrid peptide-polyketide natural products, we show (1) that the same catalytic sites are conserved between the hybrid NRPS-PKS and normal NRPS or PKS systems, although the ketoacyl synthase domain in NRPS/PKS hybrids is unique, and (2) that specific interpolypeptide linkers exist at both the C- and N-termini of the NRPS and PKS proteins, which presumably play a critical role in facilitating the transfer of the growing peptide or polyketide intermediate between NRPS and PKS modules in hybrid NRPS-PKS systems. These findings provide new insights for intermodular communications in hybrid NRPS-PKS systems and should now be taken into consideration in engineering hybrid peptide-polyketide biosynthetic pathways for making novel "unnatural" natural products. 相似文献
7.
B Shen L Du C Sanchez D J Edwards M Chen J M Murrell 《Journal of industrial microbiology & biotechnology》2001,27(6):378-385
The hybrid peptide–polyketide backbone of bleomycin (BLM) is assembled by the BLM megasynthetase that consists of both nonribosomal
peptide synthetase (NRPS) and polyketide synthase (PKS) modules. BlmIX/BlmVIII/BlmVII constitute a natural hybrid NRPS/PKS/NRPS
system, serving as a model for both hybrid NRPS/PKS and PKS/NRPS systems. Sequence analysis and functional comparison of domains
and modules of BlmIX/BlmVIII/BlmVII with those of nonhybrid NRPS and PKS systems suggest that (1) the same catalytic sites
appear to be conserved in both hybrid NRPS–PKS and nonhybrid NRPS or PKS systems, with the exception of the KS domains in
the hybrid NRPS/PKS systems that are unique; (2) specific interpolypeptide linkers may play a critical role in intermodular
communication to facilitate transfer of the growing intermediates between the interacting NRPS and/or PKS modules; and (3)
posttranslational modification of the BLM megasynthetase has been accomplished by a single PPTase with a broad substrate specificity
toward the apo forms of both acyl carrier proteins (ACPs) and peptidyl carrier proteins (PCPs). Journal of Industrial Microbiology & Biotechnology (2001) 27, 378–385.
Received 08 June 2001/ Accepted in revised form 18 July 2001 相似文献
8.
A large number of antibiotics and other industrially important microbial secondary metabolites are synthesized by polyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs). These multienzymatic complexes provide an enormous flexibility in formation of diverse chemical structures from simple substrates, such as carboxylic acids and amino acids. Modular PKSs and NRPSs, often referred to as megasynthases, have brought about a special interest due to the colinearity between enzymatic domains in the proteins working as an “assembly line” and the chain elongation and modification steps. Extensive efforts toward modified compound biosynthesis by changing organization of PKS and NRPS domains in a combinatorial manner laid good grounds for rational design of new structures and their controllable biosynthesis as proposed by the synthetic biology approach. Despite undeniable progress made in this field, the yield of such “unnatural” natural products is often not satisfactory. Here, we focus on type II thioesterases (TEIIs)—discrete hydrolytic enzymes often encoded within PKS and NRPS gene clusters which can be used to enhance product yield. We review diverse roles of TEIIs (removal of aberrant residues blocking the megasynthase, participation in substrate selection, intermediate, and product release) and discuss their application in new biosynthetic systems utilizing PKS and NRPS parts. 相似文献
9.
Culturable Endophytes of Medicinal Plants and the Genetic Basis for Their Bioactivity 总被引:1,自引:0,他引:1
The bioactive compounds of medicinal plants are products of the plant itself or of endophytes living inside the plant. Endophytes isolated from eight different anticancer plants collected in Yunnan, China, were characterized by diverse 16S and 18S rRNA gene phylogenies. A functional gene-based molecular screening strategy was used to target nonribosomal peptide synthetase (NRPS) and type I polyketide synthase (PKS) genes in endophytes. Bioinformatic analysis of these biosynthetic pathways facilitated inference of the potential bioactivity of endophyte natural products, suggesting that the isolated endophytes are capable of producing a plethora of secondary metabolites. All of the endophyte culture broth extracts demonstrated antiproliferative effects in at least one test assay, either cytotoxic, antibacterial or antifungal. From the perspective of natural product discovery, this study confirms the potential for endophytes from medicinal plants to produce anticancer, antibacterial and antifungal compounds. In addition, PKS and NRPS gene screening is a valuable method for screening isolates of biosynthetic potential. 相似文献
10.
11.
Recently, considerable insight has been gained into the modular organization of nonribosomal peptide synthetases (NRPS). The three-dimensional structures of domains associated with substrate adenylation and covalent binding have been solved as well as the structure of a priming enzyme required for the post-translational modification of NRPS. Taken together, these studies will help us to understand the architecture of these mega-enzymes. 相似文献
12.
Xiang Li Rostyslav Zvanych Stephanie A. Vanner Wenliang Wang Nathan A. Magarvey 《Bioorganic & medicinal chemistry letters》2013,23(18):5123-5127
Here we report the biosynthetic pathway for the neoantimycin and present three novel neoantimycin analogues, neoantimycin D (1), E (2) and F (3), from this assembly system from Streptoverticillium orinoci. Identification of these novel neoantimycin variants was achieved by selective MS/MS interrogation of natural product extracts using diagnostic fragments of the known neoantimycins. Their structures, including the absolute configurations, were elucidated using a combination of NMR experiments, detailed MS/MS experiments and the advanced Marfey’s method. The biosynthetic pathway of neoantimycin was dissected by genome sequencing data analysis for the first time, which includes a hybrid nonribosomal peptide synthetase (NRPS) and polyketide synthetase (PKS) assembly lines. 相似文献
13.
The high-throughput nature of proteomics mass spectrometry is enabled by a productive combination of data acquisition protocols and the computational tools used to interpret the resulting spectra. One of the key components in mainstream protocols is the generation of tandem mass (MS/MS) spectra by peptide fragmentation using collision induced dissociation, the approach currently used in the large majority of proteomics experiments to routinely identify hundreds to thousands of proteins from single mass spectrometry runs. Complementary to these, alternative peptide fragmentation methods such as electron capture/transfer dissociation and higher-energy collision dissociation have consistently achieved significant improvements in the identification of certain classes of peptides, proteins, and post-translational modifications. Recognizing these advantages, mass spectrometry instruments now conveniently support fine-tuned methods that automatically alternate between peptide fragmentation modes for either different types of peptides or for acquisition of multiple MS/MS spectra from each peptide. But although these developments have the potential to substantially improve peptide identification, their routine application requires corresponding adjustments to the software tools and procedures used for automated downstream processing. This review discusses the computational implications of alternative and alternate modes of MS/MS peptide fragmentation and addresses some practical aspects of using such protocols for identification of peptides and post-translational modifications. 相似文献
14.
Non-ribosomally synthesized peptides have compelling biological activities ranging from antimicrobial to immunosuppressive and from cytostatic to antitumor. The broad spectrum of applications in modern medicine is reflected in the great structural diversity of these natural products. They contain unique building blocks, such as d-amino acids, fatty acids, sugar moieties, and heterocyclic elements, as well as halogenated, methylated, and formylated residues. In the past decades, significant progress has been made toward the understanding of the biosynthesis of these secondary metabolites by nonribosomal peptide synthetases (NRPSs) and their associated tailoring enzymes. Guided by this knowledge, researchers genetically redesigned the NRPS template to synthesize new peptide products. Moreover, chemoenzymatic strategies were developed to rationally engineer nonribosomal peptides products in order to increase or alter their bioactivities. Specifically, chemical synthesis combined with peptide cyclization mediated by nonribosomal thioesterase domains enabled the synthesis of glycosylated cyclopeptides, inhibitors of integrin receptors, peptide/polyketide hybrids, lipopeptide antibiotics, and streptogramin B antibiotics. In addition to the synthetic potential of these cyclization catalysts, which is the main focus of this review, different enzymes for tailoring of peptide scaffolds as well as the manipulation of carrier proteins with reporter-labeled coenzyme A analogs are discussed. 相似文献
15.
Nonribosomal peptides are processed on multifunctional enzymes called nonribosomal peptide synthetases (NRPSs), whose modular multidomain arrangement allowed the rational design of new peptide products. However, the lack of natural competence and efficient transformation methods for most of nonribosomal peptide producer strains prevented the in vivo manipulation of these biosynthetic gene clusters. In this study, we present methods for the construction of a genetically engineered Bacillus subtilis surrogate host for the integration and heterologous expression of foreign NRPS genes. In the B. subtilis surrogate host, we deleted the resident 26-kilobase srfA gene cluster encoding the surfactin synthetases and subsequently used the same chromosomal location for integration of the entire 49-kilobase bacitracin biosynthetic gene cluster from Bacillus licheniformis by a stepwise homologous recombination method. Synthesis of the branched cyclic peptide antibiotic bacitracin in the engineered B. subtilis strain was achieved at high level, indicating a functional production and proper posttranslational modification of the bacitracin synthetases BacABC, as well as the expression of the associated bacitracin self-resistance genes. This engineered and genetically amenable B. subtilis strain will facilitate the rational design of new bacitracin derivatives. 相似文献
16.
Baltz RH 《Journal of industrial microbiology & biotechnology》2011,38(11):1747-1760
Mycobacterium tuberculosis encodes mycobactin, a peptide siderophore that is biosynthesized by a nonribosomal peptide synthetase (NRPS) mechanism. Within
the mycobactin biosynthetic gene cluster is a gene that encodes a 71-amino-acid protein MbtH. Many other NRPS gene clusters
harbor mbtH homologs, and recent genetic, biochemical, and structural studies have begun to shed light on the function(s) of these proteins.
In some cases, MbtH-like proteins are required for biosynthesis of their cognate peptides, and non-cognate MbtH-like proteins
have been shown to be partially complementary. Biochemical studies revealed that certain MbtH-like proteins participate in
tight binding to NRPS proteins containing adenylation (A) domains where they stimulate adenylation reactions. Expression of
MbtH-like proteins is important for a number of applications, including optimal production of native and genetically engineered
secondary metabolites produced by mechanisms that employ NRPS enzymes. They also may serve as beacons to identify gifted actinomycetes
and possibly other bacteria that encode multiple functional NRPS pathways for discovery of novel secondary metabolites by
genome mining. 相似文献
17.
Xiang Li Rostyslav Zvanych Jonathon Torchia Nathan A. Magarvey 《Bioorganic & medicinal chemistry letters》2013,23(14):4150-4153
Two novel depsipeptides (1–2) were isolated from Streptomyces sp. ML55 together with two known analogues (3–4). Their structures were elucidated using a combination of NMR experiments, as well as detailed MS/MS experiments. The biosynthetic pathway of isolated compounds was dissected by genome sequencing data analysis for a hybrid nonribosomal peptide synthetase (NRPS) and polyketide synthetase (PKS) assembly line. 相似文献
18.
The modular polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) have been found to be involved in natural
product synthesis in many microorganisms. Study on their diversities in natural environment may provide important ecological
insights, in addition to opportunities for antibacterial drugs development. In this study, the PKS and NRPS gene diversities
in two coast sediments near China Zhongshan Station were studied. The phylogenetic analysis of amino acid (AA) sequences indicated
that the identified ketosynthase (KS) domains were clustered with those from diverse bacterial groups, including Proteobacteria, Firmicutes, Planctomycetes, Cyanobacteria, Actinobacteria, and some uncultured symbiotic bacteria. One new branch belonging to hybrid PKS/NRPS enzyme complexes and five independent
clades were found on the phylogenetic tree. The obtained adenylation (A) domains were mainly clustered within the Cyanobacteria and Proteobacteria group. Most of the identified KS and A domains showed below 80 and 60% identities at the AA level to their closest matches
in GenBank, respectively. The diversities of both KS and A domains in natural environmental sample were different from those
in sewage-contaminated sample. These results revealed the great diversity and novelty of both PKS and NRPS genes in Antarctic
sediment. 相似文献
19.
Martens T Gram L Grossart HP Kessler D Müller R Simon M Wenzel SC Brinkhoff T 《Microbial ecology》2007,54(1):31-42
Members of the Roseobacter clade are abundant and widespread in marine habitats and have very diverse metabolisms. Production of acylated homoserine
lactones (AHL) and secondary metabolites, e.g., antibiotics has been described sporadically. This prompted us to screen 22
strains of this group for production of signaling molecules, antagonistic activity against bacteria of different phylogenetic
groups, and the presence of genes encoding for nonribosomal peptide synthetases (NRPS) and polyketide synthases (PKS), representing
enzymes involved in the synthesis of various pharmaceutically important natural products. The screening approach for NRPS
and PKS genes was based on polymerase chain reaction (PCR) with degenerate primers specific for conserved sequence motifs.
Additionally, sequences from whole genome sequencing projects of organisms of the Roseobacter clade were considered. Obtained PCR products were cloned, sequenced, and compared with genes of known function. With the
PCR approach genes showing similarity to known NRPS and PKS genes were found in seven and five strains, respectively, and
three PKS and NRPS sequences from genome sequencing projects were obtained. Three strains exhibited antagonistic activity
and also showed production of AHL. Overall production of AHL was found in 10 isolates. Phylogenetic analysis of the 16S rRNA
gene sequences of the tested organisms showed that several of the AHL-positive strains clustered together. Three strains were
positive for three or four categories tested, and were found to be closely related within the genus Phaeobacter. The presence of a highly similar hybrid PKS/NRPS gene locus of unknown function in sequenced genomes of the Roseobacter clade plus the significant similarity of gene fragments from the strains studied to these genes argues for the functional
requirement of the encoded hybrid PKS/NRPS complex. Our screening results therefore suggest that the Roseobacter clade is indeed employing PKS/NRPS biochemistry and should thus be further studied as a potential and largely untapped source
of secondary metabolites. 相似文献
20.
Natural products are a functionally diverse class of biochemically synthesized compounds, which include antibiotics, toxins, and siderophores. In this paper, we describe both the detection of natural product activities and the sequence identification of gene fragments from two molecular systems that have previously been implicated in natural product production, i.e., nonribosomal peptide synthetases (NRPSs) and modular polyketide synthases (PKSs), in diverse marine and freshwater cyanobacterial cultures. Using degenerate PCR and the sequencing of cloned products, we show that NRPSs and PKSs are common among the cyanobacteria tested. Our molecular data, when combined with genomic searches of finished and progressing cyanobacterial genomes, demonstrate that not all cyanobacteria contain NRPS and PKS genes and that the filamentous and heterocystous cyanobacteria are the richest sources of these genes and the most likely sources of novel natural products within the phylum. In addition to validating the use of degenerate primers for the identification of PKS and NRPS genes in cyanobacteria, this study also defines numerous gene fragments that will be useful as probes for future studies of the synthesis of natural products in cyanobacteria. Phylogenetic analyses of the cyanobacterial NRPS and PKS fragments sequenced in this study, as well as those from the cyanobacterial genome projects, demonstrate that there is remarkable diversity and likely novelty of these genes within the cyanobacteria. These results underscore the potential variety of novel products being produced by these ubiquitous organisms. 相似文献