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Non-ribosomal peptide synthetases (NRPSs) are giant enzyme machines that activate amino acids in an assembly line fashion. As NRPSs are not restricted to the incorporation of the 20 proteinogenic amino acids, their efficient manipulation would enable microbial production of a diverse range of peptides; however, the structural requirements for reprogramming NRPSs to facilitate the production of new peptides are not clear. Here we describe a new fusion point inside the condensation domains of NRPSs that results in the development of the exchange unit condensation domain (XUC) concept, which enables the efficient production of peptides, even containing non-natural amino acids, in yields up to 280 mg l . This allows the generation of more specific NRPSs, reducing the number of unwanted peptide derivatives, but also the generation of peptide libraries. The XUC might therefore be suitable for the future optimization of peptide production and the identification of bioactive peptide derivatives for pharmaceutical and other applications.

The modular polyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs) are among the largest and most complicated enzymes in nature. In these biosynthetic systems, independently folding protein domains, which are organized into units called 'modules', operate in assembly-line fashion to construct polymeric chains and tailor their functionalities. Products of PKSs and NRPSs include a number of blockbuster medicines, and this has motivated researchers to understand how they operate so that they can be modified by genetic engineering. Beginning in the 1990s, structural biology has provided a number of key insights. The emerging picture is one of remarkable dynamics and conformational programming in which the chemical states of individual catalytic domains are communicated to the others, configuring the modules for the next stage in the biosynthesis. This unexpected level of complexity most likely accounts for the low success rate of empirical genetic engineering experiments and suggests ways forward for productive megaenzyme synthetic biology.

Here we report a transcription factor decoy strategy for targeted activation of eight large silent polyketide synthase and non-ribosomal peptide synthetase gene clusters, ranging from 50 to 134 kilobases (kb) in multiple streptomycetes, and characterization of a novel oxazole family compound produced by a 98-kb biosynthetic gene cluster. Owing to its simplicity and ease of use, this strategy can be scaled up readily for discovery of natural products in streptomycetes.

Tenuazonic acid (TeA) is a well-known mycotoxin produced by various plant pathogenic fungi. However, its biosynthetic gene has been unknown to date. Here we identify the TeA biosynthetic gene from Magnaporthe oryzae by finding two TeA-inducing conditions of a low-producing strain. We demonstrate that TeA is synthesized from isoleucine and acetoacetyl-coenzyme A by TeA synthetase 1 (TAS1). TAS1 is a unique non-ribosomal peptide synthetase and polyketide synthase (NRPS-PKS) hybrid enzyme that begins with an NRPS module. In contrast to other NRPS/PKS hybrid enzymes, the PKS portion of TAS1 has only a ketosynthase (KS) domain and this domain is indispensable for TAS1 activity. Phylogenetic analysis classifies this KS domain as an independent clade close to type I PKS KS domain. We demonstrate that the TAS1 KS domain conducts the final cyclization step for TeA release. These results indicate that TAS1 is a unique type of NRPS-PKS hybrid enzyme.

Disease-suppressive soils are exceptional ecosystems in which crop plants suffer less from specific soil-borne pathogens than expected owing to the activities of other soil microorganisms. For most disease-suppressive soils, the microbes and mechanisms involved in pathogen control are unknown. By coupling PhyloChip-based metagenomics of the rhizosphere microbiome with culture-dependent functional analyses, we identified key bacterial taxa and genes involved in suppression of a fungal root pathogen. More than 33,000 bacterial and archaeal species were detected, with Proteobacteria, Firmicutes, and Actinobacteria consistently associated with disease suppression. Members of the γ-Proteobacteria were shown to have disease-suppressive activity governed by nonribosomal peptide synthetases. Our data indicate that upon attack by a fungal root pathogen, plants can exploit microbial consortia from soil for protection against infections.

The microtubule (MT) cytoskeleton can transmit mechanical signals and resist compression in contracting cardiomyocytes. How MTs perform these roles remains unclear because of difficulties in observing MTs during the rapid contractile cycle. Here, we used high spatial and temporal resolution imaging to characterize MT behavior in beating mouse myocytes. MTs deformed under contractile load into sinusoidal buckles, a behavior dependent on posttranslational \"detyrosination\" of α-tubulin. Detyrosinated MTs associated with desmin at force-generating sarcomeres. When detyrosination was reduced, MTs uncoupled from sarcomeres and buckled less during contraction, which allowed sarcomeres to shorten and stretch with less resistance. Conversely, increased detyrosination promoted MT buckling, stiffened the myocyte, and correlated with impaired function in cardiomyopathy. Thus, detyrosinated MTs represent tunable, compression-resistant elements that may impair cardiac function in disease.

New bioinformatic tools are needed to analyze the growing volume of DNA sequence data. This is especially true in the case of secondary metabolite biosynthesis, where the highly repetitive nature of the associated genes creates major challenges for accurate sequence assembly and analysis. Here we introduce the web tool Natural Product Domain Seeker (NaPDoS), which provides an automated method to assess the secondary metabolite biosynthetic gene diversity and novelty of strains or environments. NaPDoS analyses are based on the phylogenetic relationships of sequence tags derived from polyketide synthase (PKS) and non-ribosomal peptide synthetase (NRPS) genes, respectively. The sequence tags correspond to PKS-derived ketosynthase domains and NRPS-derived condensation domains and are compared to an internal database of experimentally characterized biosynthetic genes. NaPDoS provides a rapid mechanism to extract and classify ketosynthase and condensation domains from PCR products, genomes, and metagenomic datasets. Close database matches provide a mechanism to infer the generalized structures of secondary metabolites while new phylogenetic lineages provide targets for the discovery of new enzyme architectures or mechanisms of secondary metabolite assembly. Here we outline the main features of NaPDoS and test it on four draft genome sequences and two metagenomic datasets. The results provide a rapid method to assess secondary metabolite biosynthetic gene diversity and richness in organisms or environments and a mechanism to identify genes that may be associated with uncharacterized biochemistry.

Reprogramming of the NRPS/PKS assembly line is an attractive method for the production of new bioactive molecules. However, it is usually hampered by the loss of intimate domain/module interactions required for the precise control of chain transfer and elongation reactions. In this study, we first establish heterologous expression systems of the unique antimycin-type cyclic depsipeptides: JBIR-06 (tri-lactone) and neoantimycin (tetra-lactone), and engineer their biosyntheses by taking advantage of bioinformatic analyses and evolutionary insights. As a result, we successfully accomplish three manipulations: (i) ring contraction of neoantimycin (from tetra-lactone to tri-lactone), (ii) ring expansion of JBIR-06 (from tri-lactone to tetra-lactone), and (iii) alkyl chain diversification of JBIR-06 by the incorporation of various alkylmalonyl-CoA extender units, to generate a set of unnatural derivatives in practical yields. This study presents a useful strategy for engineering NRPS-PKS module enzymes, based on nature's diversification of the domain and module organizations.

Cyanobacteria are an ancient lineage of photosynthetic bacteria from which hundreds of natural products have been described, including many notorious toxins but also potent natural products of interest to the pharmaceutical and biotechnological industries. Many of these compounds are the products of non-ribosomal peptide synthetase (NRPS) or polyketide synthase (PKS) pathways. However, current understanding of the diversification of these pathways is largely based on the chemical structure of the bioactive compounds, while the evolutionary forces driving their remarkable chemical diversity are poorly understood. We carried out a phylum-wide investigation of genetic diversification of the cyanobacterial NRPS and PKS pathways for the production of bioactive compounds. 452 NRPS and PKS gene clusters were identified from 89 cyanobacterial genomes, revealing a clear burst in late-branching lineages. Our genomic analysis further grouped the clusters into 286 highly diversified cluster families (CF) of pathways. Some CFs appeared vertically inherited, while others presented a more complex evolutionary history. Only a few horizontal gene transfers were evidenced amongst strongly conserved CFs in the phylum, while several others have undergone drastic gene shuffling events, which could result in the observed diversification of the pathways. Therefore, in addition to toxin production, several NRPS and PKS gene clusters are devoted to important cellular processes of these bacteria such as nitrogen fixation and iron uptake. The majority of the biosynthetic clusters identified here have unknown end products, highlighting the power of genome mining for the discovery of new natural products.

Polycyclic tetramate macrolactams (PTMs) are a widely distributed class of natural products with important biological activities. However, many of these PTMs have not been characterized. Here we apply a plug-and-play synthetic biology strategy to activate a cryptic PTM biosynthetic gene cluster SGR810-815 from Streptomyces griseus and discover three new PTMs. This gene cluster is highly conserved in phylogenetically diverse bacterial strains and contains an unusual hybrid polyketide synthase-nonribosomal peptide synthetase, which resembles iterative polyketide synthases known in fungi. To further characterize this gene cluster, we use the same synthetic biology approach to create a series of gene deletion constructs and elucidate the biosynthetic steps for the formation of the polycyclic system. The strategy we employ bypasses the traditional laborious processes to elicit gene cluster expression and should be generally applicable to many other silent or cryptic gene clusters for discovery and characterization of new natural products.