scholarly journals Utilization of the Methoxymalonyl-Acyl Carrier Protein Biosynthesis Locus for Cloning of the Tautomycin Biosynthetic Gene Cluster from Streptomyces spiroverticillatus

2006 ◽  
Vol 188 (11) ◽  
pp. 4148-4152 ◽  
Author(s):  
Wenli Li ◽  
Jianhua Ju ◽  
Hiroyuki Osada ◽  
Ben Shen
Keyword(s):  
Gene Cluster ◽  
Protein Phosphatase ◽  
Acyl Carrier Protein ◽  
Protein Biosynthesis ◽  
Acyl Chain ◽  
Biosynthetic Gene Cluster ◽  
Carrier Protein ◽  
Biosynthetic Gene ◽  
Degenerate Primers ◽  
Polyketide Chain

ABSTRACT Tautomycin (TTM), a potent protein phosphatase inhibitor, consists of a polyketide chain containing a spiroketal moiety and an acyl chain bearing a dialkylmaleic anhydride structure. PCR using degenerate primers was used to clone genes from Streptomyces spiroverticillatus for formation of the methoxymalonyl-acyl carrier protein. This locus was found to contain five genes (ttmC, ttmA, ttmD, ttmB, and ttmE), one of which was used as a probe to clone the 110-kb TTM biosynthetic gene cluster. The involvement of the ttmA gene in TTM biosynthesis was confirmed by gene inactivation and mutation complementation experiments.

scholarly journals Utilization of the Methoxymalonyl-Acyl Carrier Protein Biosynthesis Locus for Cloning the Oxazolomycin Biosynthetic Gene Cluster from Streptomyces albus JA3453

2006 ◽  
Vol 188 (11) ◽  
pp. 4142-4147 ◽  
Author(s):  
Chunhua Zhao ◽  
Jianhua Ju ◽  
Steven D. Christenson ◽  
Wyatt C. Smith ◽  
Danfeng Song ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Acyl Carrier Protein ◽  
Protein Biosynthesis ◽  
Biosynthetic Gene Cluster ◽  
Carrier Protein ◽  
Biosynthetic Gene ◽  
Degenerate Primers ◽  
Streptomyces Albus ◽  
Genes Encoding ◽  
Antiviral Activities

ABSTRACT Oxazolomycin (OZM), a hybrid peptide-polyketide antibiotic, exhibits potent antitumor and antiviral activities. Using degenerate primers to clone genes encoding methoxymalonyl-acyl carrier protein (ACP) biosynthesis as probes, a 135-kb DNA region from Streptomyces albus JA3453 was cloned and found to cover the entire OZM biosynthetic gene cluster. The involvement of the cloned genes in OZM biosynthesis was confirmed by deletion of a 12-kb DNA fragment containing six genes for methoxymalonyl-ACP biosynthesis from the specific region of the chromosome, as well as deletion of the ozmC gene within this region, to generate OZM-nonproducing mutants.

Cloning, sequencing and heterologous expression of the medermycin biosynthetic gene cluster of Streptomyces sp. AM-7161: towards comparative analysis of the benzoisochromanequinone gene clusters

Microbiology ◽  
2003 ◽  
Vol 149 (7) ◽  
pp. 1633-1645 ◽  
Author(s):  
Koji Ichinose ◽  
Makoto Ozawa ◽  
Keiko Itou ◽  
Kanako Kunieda ◽  
Yutaka Ebizuka
Keyword(s):  
Heterologous Expression ◽  
Gene Cluster ◽  
Polyketide Synthase ◽  
Acyl Carrier Protein ◽  
Streptomyces Coelicolor ◽  
Gene Clusters ◽  
Biosynthetic Gene Cluster ◽  
Carrier Protein ◽  
Biosynthetic Gene ◽  
Six Genes

Medermycin is a Streptomyces aromatic C-glycoside antibiotic classified in the benzoisochromanequinones (BIQs), which presents several interesting biosynthetic problems concerning polyketide synthase (PKS), post-PKS tailoring and deoxysugar pathways. The biosynthetic gene cluster for medermycin (the med cluster) was cloned from Streptomyces sp. AM-7161. Completeness of the clone was proved by the heterologous expression of a cosmid carrying the entire med cluster in Streptomyces coelicolor CH999 to produce medermycin. The DNA sequence of the cosmid (36 202 bp) revealed 34 complete ORFs, with an incomplete ORF at either end. Functional assignment of the deduced products was made for PKS and biosynthetically related enzymes, tailoring steps including strereochemical control, oxidation, angolosamine pathway, C-glycosylation, and regulation. The med cluster was estimated to be about 30 kb long, covering 29 ORFs. An unusual characteristic of the cluster is the disconnected organization of the minimal PKS genes: med-ORF23 encoding the acyl carrier protein is 20 kb apart from med-ORF1 and med-ORF2 for the two ketosynthase components. Secondly, the six genes (med-ORF14, 15, 16, 17, 18 and 20) for the biosynthesis of the deoxysugar, angolosamine, are all contiguous. Finally, the finding of a glycosyltransferase gene, med-ORF8, suggests a possible involvement of conventional C-glycosylation in medermycin biosynthesis. Comparison among the three complete BIQ gene clusters – med and those for actinorhodin (act) and granaticin (gra) – revealed some common genes whose deduced functions are unavailable from database searches (the ‘unknowns’). An example is med-ORF5, a homologue of actVI-ORF3 and gra-ORF18, which was highlighted by a recent proteomic analysis of S. coelicolor A3(2).

scholarly journals Biosynthesis of isonitrile lipopeptides by conserved nonribosomal peptide synthetase gene clusters in Actinobacteria

2017 ◽  
Vol 114 (27) ◽  
pp. 7025-7030 ◽  
Author(s):  
Nicholas C. Harris ◽  
Michio Sato ◽  
Nicolaus A. Herman ◽  
Frederick Twigg ◽  
Wenlong Cai ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Acyl Chain ◽  
Gene Clusters ◽  
Biosynthetic Gene Cluster ◽  
Nonribosomal Peptide Synthetase ◽  
Biosynthetic Gene ◽  
Nonribosomal Peptide ◽  
Peptide Synthetase

A putative lipopeptide biosynthetic gene cluster is conserved in many species of Actinobacteria, including Mycobacterium tuberculosis and M. marinum, but the specific function of the encoding proteins has been elusive. Using both in vivo heterologous reconstitution and in vitro biochemical analyses, we have revealed that the five encoding biosynthetic enzymes are capable of synthesizing a family of isonitrile lipopeptides (INLPs) through a thio-template mechanism. The biosynthesis features the generation of isonitrile from a single precursor Gly promoted by a thioesterase and a nonheme iron(II)-dependent oxidase homolog and the acylation of both amino groups of Lys by the same isonitrile acyl chain facilitated by a single condensation domain of a nonribosomal peptide synthetase. In addition, the deletion of INLP biosynthetic genes in M. marinum has decreased the intracellular metal concentration, suggesting the role of this biosynthetic gene cluster in metal transport.

scholarly journals Biosynthesis of Isonitrile Lipopeptides by Conserved Non-ribosomal Peptide Synthetase Gene Clusters in Actinobacteria

2017 ◽  
Author(s):  
Nicholas C. Harris ◽  
Michio Sato ◽  
Nicolaus A. Herman ◽  
Frederick Twigg ◽  
Wenlong Cai ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Gene Cluster ◽  
Acyl Chain ◽  
Gene Clusters ◽  
Biosynthetic Gene Cluster ◽  
Metal Transport ◽  
Homologous Gene ◽  
Biosynthetic Gene ◽  
New Family ◽  
Peptide Synthetase

AbstractA putative lipopeptide biosynthetic gene cluster is conserved in many species of Actinobacteria, including Mycobacterium tuberculosis and M. marinum, but the specific function of the encoding proteins has been elusive. Using both in vivo heterologous reconstitution and in intro biochemical analyses, we have revealed that the five encoding biosynthetic enzymes are capable of synthesizing a new family of isonitrile lipopeptides (INLPs) through a thio-template mechanism. The biosynthesis features the generation of isonitrile from a single precursor Gly promoted by a thioesterase and a non-heme iron(II)-dependent oxidase homologue, and the acylation of both amino groups of Lys by the same isonitrile acyl chain facilitated by a single condensation domain of a non-ribosomal peptide synthetase (NRPS). In addition, the deletion of INLP biosynthetic genes in M. marinum has decreased the intracellular metal concentration, suggesting the role of this biosynthetic gene cluster in metal transport.Significance StatementMycobacterium tuberculosis is the leading causative agent of tuberculosis (TB), of which millions of deaths occur annually. A putative lipopeptide biosynthetic gene cluster has been shown to be essential for the survival of this pathogen in hosts, and homologous gene clusters have also been found in all pathogenic mycobacteria and other species of Actinobacteria. We have identified the function of these gene clusters in making a new family of isonitrile lipopeptides. The biosynthesis has several unique features, including an unprecedented mechanism for isonitrile synthesis. Our results have further suggested that these biosynthetic gene clusters play a role in metal transport, and thus have shed light on a new metal transport system that is crucial for virulence of pathogenic mycobacteria.

scholarly journals New Insights into Chloramphenicol Biosynthesis in Streptomyces venezuelae ATCC 10712

2014 ◽  
Vol 58 (12) ◽  
pp. 7441-7450 ◽  
Author(s):  
Lorena T. Fernández-Martínez ◽  
Chiara Borsetto ◽  
Juan Pablo Gomez-Escribano ◽  
Maureen J. Bibb ◽  
Mahmoud M. Al-Bassam ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Acyl Carrier Protein ◽  
Bioinformatic Analysis ◽  
Biosynthetic Gene Cluster ◽  
Transcriptional Analysis ◽  
Biosynthetic Gene ◽  
Phosphopantetheinyl Transferase ◽  
Streptomyces Venezuelae ◽  
Content Type ◽  
New Genes

ABSTRACTComparative genome analysis revealed seven uncharacterized genes,sven0909tosven0915, adjacent to the previously identified chloramphenicol biosynthetic gene cluster (sven0916–sven0928) ofStreptomyces venezuelaestrain ATCC 10712 that was absent in a closely relatedStreptomycesstrain that does not produce chloramphenicol. Transcriptional analysis suggested that three of these genes might be involved in chloramphenicol production, a prediction confirmed by the construction of deletion mutants. These three genes encode a cluster-associated transcriptional activator (Sven0913), a phosphopantetheinyl transferase (Sven0914), and a Na+/H+antiporter (Sven0915). Bioinformatic analysis also revealed the presence of a previously undetected gene,sven0925, embedded within the chloramphenicol biosynthetic gene cluster that appears to encode an acyl carrier protein, bringing the number of new genes likely to be involved in chloramphenicol production to four. Microarray experiments and synteny comparisons also suggest thatsven0929is part of the biosynthetic gene cluster. This has allowed us to propose an updated and revised version of the chloramphenicol biosynthetic pathway.

Anacyclamide D8P, a prenylated cyanobactin from a Sphaerospermopsis sp. cyanobacterium

2017 ◽  
Author(s):  
Joana Martins ◽  
Niina Leikoski ◽  
Matti Wahlsten ◽  
Joana Azevedo ◽  
Jorge Antunes ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Cyclic Peptides ◽  
Biosynthetic Gene Cluster ◽  
The Novel ◽  
Tyrosine Residue ◽  
Biosynthetic Gene ◽  
Post Translational Modification ◽  
Biological Assays ◽  
Mass Spectrometric Data ◽  
Spectrometric Data

Cyanobactins are a family of linear and cyclic peptides produced through the post-translational modification of short precursor peptides. Anacyclamides are macrocyclic cyanobactins with a highly diverse sequence that are common in the genus <i>Anabaena</i>. A mass spectrometry-based screening of potential cyanobactin producers led to the discovery of a new prenylated member of this family of compounds, anacyclamide D8P (<b>1</b>), from <i>Sphaerospermopsis</i> sp. LEGE 00249. The anacyclamide biosynthetic gene cluster (<i>acy</i>) encoding the novel macrocyclic prenylated cyanobactin, was sequenced. Heterologous expression of the acy gene cluster in <i>Escherichia</i> <i>coli</i> established the connection between genomic and mass spectrometric data. Unambiguous establishment of the type and site of prenylation required the full structural elucidation of <b>1</b> using Nuclear Magnetic Resonance (NMR), which demonstrated that a forward prenylation occurred on the tyrosine residue. Compound <b>1</b> was tested in pharmacologically or ecologically relevant biological assays and revealed moderate antimicrobial activity towards the fouling bacterium <i>Halomonas aquamarina</i> CECT 5000.<br>

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