scholarly journals Molecular Cloning, Sequence Analysis, and Heterologous Expression of the Phosphinothricin Tripeptide Biosynthetic Gene Cluster from Streptomyces viridochromogenes DSM 40736

2005 ◽  
Vol 49 (1) ◽  
pp. 230-240 ◽  
Author(s):  
Joshua A. V. Blodgett ◽  
Jun Kai Zhang ◽  
William W. Metcalf
Keyword(s):  
Sequence Analysis ◽  
Heterologous Expression ◽  
Gene Cluster ◽  
Genomic Dna ◽  
Biosynthetic Gene Cluster ◽  
Fosmid Library ◽  
Open Reading Frames ◽  
Biosynthetic Gene ◽  
Heterologous Host ◽  
Reading Frames

ABSTRACT A fosmid library from genomic DNA of Streptomyces viridochromogenes DSM 40736 was constructed and screened for the presence of genes known to be involved in the biosynthesis of phosphinothricin tripeptide (PTT). Eight positives were identified, one of which was able to confer PTT biosynthetic capability upon Streptomyces lividans after integration of the fosmid into the chromosome of this heterologous host. Sequence analysis of the 40,241-bp fosmid insert revealed 29 complete open reading frames (ORFs). Deletion analysis demonstrated that a minimum set of 24 ORFs were required for PTT production in the heterologous host. Sequence analysis revealed that most of these PTT genes have been previously identified in either S. viridochromogenes or S. hygroscopicus (or both), although only 11 out of 24 of these ORFs have experimentally defined functions. Three previously unknown genes within the cluster were identified and are likely to have roles in the stepwise production of phosphonoformate from phosphonoacetaldehyde. This is the first report detailing the entire PTT gene cluster from any producing streptomycete.

scholarly journals Identification of the Novobiocin Biosynthetic Gene Cluster of Streptomyces spheroides NCIB 11891

2000 ◽  
Vol 44 (5) ◽  
pp. 1214-1222 ◽  
Author(s):  
Marion Steffensky ◽  
Agnes Mühlenweg ◽  
Zhao-Xin Wang ◽  
Shu-Ming Li ◽  
Lutz Heide
Keyword(s):  
Heterologous Expression ◽  
Gene Cluster ◽  
Biosynthetic Gene Cluster ◽  
Streptomyces Lividans ◽  
Open Reading Frames ◽  
Biosynthetic Gene ◽  
Insertional Inactivation ◽  
Key Enzyme ◽  
Novobiocin Resistance ◽  
Reading Frames

ABSTRACT The novobiocin biosynthetic gene cluster from Streptomyces spheroides NCIB 11891 was cloned by using homologous deoxynucleoside diphosphate (dNDP)-glucose 4,6-dehydratase gene fragments as probes. Double-stranded sequencing of 25.6 kb revealed the presence of 23 putative open reading frames (ORFs), including the gene for novobiocin resistance, gyrB r, and at least 11 further ORFs to which a possible role in novobiocin biosynthesis could be assigned. An insertional inactivation experiment with a dNDP-glucose 4,6-dehydratase fragment resulted in abolishment of novobiocin production, since biosynthesis of the deoxysugar moiety of novobiocin was blocked. Heterologous expression of a key enzyme of novobiocin biosynthesis, i.e., novobiocic acid synthetase, inStreptomyces lividans TK24 further confirmed the involvement of the analyzed genes in the biosynthesis of the antibiotic.

scholarly journals Identification and Analysis of the Biosynthetic Gene Cluster Encoding the Thiopeptide Antibiotic Cyclothiazomycin in Streptomyces hygroscopicus 10-22

2010 ◽  
Vol 76 (7) ◽  
pp. 2335-2344 ◽  
Author(s):  
Jiang Wang ◽  
Yi Yu ◽  
Kexuan Tang ◽  
Wen Liu ◽  
Xinyi He ◽  
...  
Keyword(s):  
Heterologous Expression ◽  
Gene Cluster ◽  
Posttranslational Modifications ◽  
Biosynthetic Gene Cluster ◽  
Open Reading Frames ◽  
Streptomyces Hygroscopicus ◽  
Biosynthetic Gene ◽  
Thiopeptide Antibiotics ◽  
Reading Frames ◽  
Macrocyclic Structure

ABSTRACT Thiopeptide antibiotics are an important class of natural products resulting from posttranslational modifications of ribosomally synthesized peptides. Cyclothiazomycin is a typical thiopeptide antibiotic that has a unique bridged macrocyclic structure derived from an 18-amino-acid structural peptide. Here we reported cloning, sequencing, and heterologous expression of the cyclothiazomycin biosynthetic gene cluster from Streptomyces hygroscopicus 10-22. Remarkably, successful heterologous expression of a 22.7-kb gene cluster in Streptomyces lividans 1326 suggested that there is a minimum set of 15 open reading frames that includes all of the functional genes required for cyclothiazomycin production. Six genes of these genes, cltBCDEFG flanking the structural gene cltA, were predicted to encode the enzymes required for the main framework of cyclothiazomycin, and two enzymes encoded by a putative operon, cltMN, were hypothesized to participate in the tailoring step to generate the tertiary thioether, leading to the final cyclization of the bridged macrocyclic structure. This rigorous bioinformatics analysis based on heterologous expression of cyclothiazomycin resulted in an ideal biosynthetic model for us to understand the biosynthesis of thiopeptides.

scholarly journals Isolation, Characterization, and Heterologous Expression of the Biosynthesis Gene Cluster for the Antitumor Anthracycline Steffimycin

2006 ◽  
Vol 72 (6) ◽  
pp. 4172-4183 ◽  
Author(s):  
Sonia Gull�n ◽  
Carlos Olano ◽  
Mohamed S. Abdelfattah ◽  
Alfredo F. Bra�a ◽  
J�rgen Rohr ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Biosynthetic Gene Cluster ◽  
Open Reading Frames ◽  
Biosynthetic Gene ◽  
Heterologous Host ◽  
Biosynthesis Gene Cluster ◽  
Polyketide Biosynthesis ◽  
Gene Coding ◽  
Biosynthesis Gene ◽  
Reading Frames

ABSTRACT The biosynthetic gene cluster for the aromatic polyketide steffimycin of the anthracycline family has been cloned and characterized from “Streptomyces steffisburgensis” NRRL 3193. Sequence analysis of a 42.8-kbp DNA region revealed the presence of 36 open reading frames (ORFs) (one of them incomplete), 24 of which, spanning 26.5 kb, are probably involved in steffimycin biosynthesis. They code for all the activities required for polyketide biosynthesis, tailoring, regulation, and resistance but show no evidence of genes involved in l-rhamnose biosynthesis. The involvement of the cluster in steffimycin biosynthesis was confirmed by expression of a region of about 15 kb containing 15 ORFS, 11 of them forming part of the cluster, in the heterologous host Streptomyces albus, allowing the isolation of a biosynthetic intermediate. In addition, the expression in S. albus of the entire cluster, contained in a region of 34.8 kb, combined with the expression of plasmid pRHAM, directing the biosynthesis of l-rhamnose, led to the production of steffimycin. Inactivation of the stfX gene, coding for a putative cyclase, revealed that this enzymatic activity participates in the cyclization of the fourth ring, making the final steps in the biosynthesis of the steffimycin aglycon similar to those in the biosynthesis of jadomycin or rabelomycin. Inactivation of the stfG gene, coding for a putative glycosyltransferase involved in the attachment of l-rhamnose, allowed the production of a new compound corresponding to the steffimycin aglycon compound also observed in S. albus upon expression of the entire cluster.

scholarly journals The granaticin biosynthetic gene cluster of Streptomyces violaceoruber Tü22: sequence analysis and expression in a heterologous host

1998 ◽  
Vol 5 (11) ◽  
pp. 647-659 ◽  
Author(s):  
Koji Ichinose ◽  
David J. Bedford ◽  
Diethild Tornus ◽  
Andreas Bechthold ◽  
Maureen J. Bibb ◽  
...  
Keyword(s):  
Sequence Analysis ◽  
Gene Cluster ◽  
Biosynthetic Gene Cluster ◽  
Biosynthetic Gene ◽  
Heterologous Host ◽  
Streptomyces Violaceoruber

scholarly journals GIP2, a Putative Transcription Factor That Regulates the Aurofusarin Biosynthetic Gene Cluster in Gibberella zeae

2006 ◽  
Vol 72 (2) ◽  
pp. 1645-1652 ◽  
Author(s):  
Jung-Eun Kim ◽  
Jianming Jin ◽  
Hun Kim ◽  
Jin-Cheol Kim ◽  
Sung-Hwan Yun ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Biosynthetic Gene Cluster ◽  
Gibberella Zeae ◽  
Open Reading Frames ◽  
Biosynthetic Gene ◽  
Targeted Gene Deletion ◽  
Binuclear Cluster ◽  
Putative Transcription Factor ◽  
Type Strains ◽  
Reading Frames

ABSTRACT Gibberella zeae (anamorph: Fusarium graminearum) is an important pathogen of maize, wheat, and rice. Colonies of G. zeae produce yellow-to-tan mycelia with the white-to-carmine red margins. In this study, we focused on nine putative open reading frames (ORFs) closely linked to PKS12 and GIP1, which are required for aurofusarin biosynthesis in G. zeae. Among them is an ORF designated GIP2 (for Gibberella zeae pigment gene 2), which encodes a putative protein of 398 amino acids that carries a Zn(II)2Cys6 binuclear cluster DNA-binding domain commonly found in transcription factors of yeasts and filamentous fungi. Targeted gene deletion and complementation analyses confirmed that GIP2 is required for aurofusarin biosynthesis. Expression of GIP2 in carrot medium correlated with aurofusarin production by G. zeae and was restricted to vegetative mycelia. Inactivation of the 10 contiguous genes in the ΔGIP2 strain delineates an aurofusarin biosynthetic gene cluster. Overexpression of GIP2 in both the ΔGIP2 and the wild-type strains increases aurofusarin production and reduces mycelial growth. Thus, GIP2 is a putative positive regulator of the aurofusarin biosynthetic gene cluster, and aurofusarin production is negatively correlated with vegetative growth by G. zeae.

scholarly journals Analysis of Genes Involved in Biosynthesis of Coronafacic Acid, the Polyketide Component of the Phytotoxin Coronatine

1998 ◽  
Vol 180 (13) ◽  
pp. 3330-3338 ◽  
Author(s):  
Vidhya Rangaswamy ◽  
Robin Mitchell ◽  
Matthias Ullrich ◽  
Carol Bender
Keyword(s):  
Gene Cluster ◽  
Pseudomonas Syringae ◽  
Biosynthetic Gene Cluster ◽  
Open Reading Frames ◽  
Plant Pathogenic Bacterium ◽  
Biosynthetic Gene ◽  
Future Studies ◽  
Coronafacic Acid ◽  
Single Transcript ◽  
Reading Frames

ABSTRACT Coronafacic acid (CFA) is the polyketide component of coronatine (COR), a phytotoxin produced by the plant-pathogenic bacteriumPseudomonas syringae. The genes involved in CFA biosynthesis are encoded by a single transcript which encompasses 19 kb of the COR gene cluster. In the present study, the nucleotide sequence was determined for a 4-kb region located at the 3′ end of the CFA biosynthetic gene cluster. Three open reading frames were identified and designated cfa8, cfa9, andtnp1; the predicted translation products of these genes showed relatedness to oxidoreductases, thioesterases, and transposases, respectively. The translational products of cfa8 andcfa9 were overproduced in Escherichia coliBL21; however, tnp1 was not translated in these experiments. Mutagenesis and complementation analysis indicated thatcfa8 is required for the production of CFA and COR. Analysis of a cfa9 mutant indicated that this gene is dispensable for CFA and COR production but may increase the release of enzyme-bound products from the COR pathway; tnp1, however, had no obvious function in CFA or COR biosynthesis. A genetic strategy was used to produce CFA in a P. syringae strain which lacks the COR gene cluster; this approach will be useful in future studies designed to investigate biosynthetic products of the CFA gene cluster.

scholarly journals Biosynthetic Gene Cluster for the Polyenoyltetramic Acid α-Lipomycin

2006 ◽  
Vol 50 (6) ◽  
pp. 2113-2121 ◽  
Author(s):  
C. Bihlmaier ◽  
E. Welle ◽  
C. Hofmann ◽  
K. Welzel ◽  
A. Vente ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Polyketide Synthase ◽  
Biosynthetic Gene Cluster ◽  
Open Reading Frames ◽  
Biosynthetic Gene ◽  
Peptide Synthetases ◽  
Insight Into ◽  
Reading Frames ◽  
Module System

ABSTRACT The gram-positive bacterium Streptomyces aureofaciens Tü117 produces the acyclic polyene antibiotic α-lipomycin. The entire biosynthetic gene cluster (lip gene cluster) was cloned and characterized. DNA sequence analysis of a 74-kb region revealed the presence of 28 complete open reading frames (ORFs), 22 of them belonging to the biosynthetic gene cluster. Central to the cluster is a polyketide synthase locus that encodes an eight-module system comprised of four multifunctional proteins. In addition, one ORF shows homology to those for nonribosomal peptide synthetases, indicating that α-lipomycin belongs to the classification of hybrid peptide-polyketide natural products. Furthermore, the lip cluster includes genes responsible for the formation and attachment of d-digitoxose as well as ORFs that resemble those for putative regulatory and export functions. We generated biosynthetic mutants by insertional gene inactivation. By analysis of culture extracts of these mutants, we could prove that, indeed, the genes involved in the biosynthesis of lipomycin had been cloned, and additionally we gained insight into an unusual biosynthesis pathway.

scholarly journals Heterologous expression of the atypical tetracycline chelocardin reveals the full set of genes required for its biosynthesis

2020 ◽  
Vol 19 (1) ◽  
Author(s):  
Tadeja Lukežič ◽  
Špela Pikl ◽  
Nestor Zaburannyi ◽  
Maja Remškar ◽  
Hrvoje Petković ◽  
...  
Keyword(s):  
Heterologous Expression ◽  
Gene Cluster ◽  
Regulatory Protein ◽  
Model Organism ◽  
Gene Clusters ◽  
Biosynthetic Gene Cluster ◽  
Biosynthetic Gene ◽  
Heterologous Host ◽  
Yield Increase ◽  
Tract Infections

Abstract Background Chelocardin (CHD) exhibits a broad-spectrum antibiotic activity and showed promising results in a small phase II clinical study conducted on patients with urinary tract infections. Importantly, CHD was shown to be active also against tetracycline-resistant Gram-negative pathogens, which is gaining even more importance in today’s antibiotic crisis. We have demonstrated that modifications of CHD through genetic engineering of its producer, the actinomycete Amycolatopsis sulphurea, are not only possible but yielded even more potent antibiotics than CHD itself, like 2-carboxamido-2-deacetyl-chelocardin (CD-CHD), which is currently in preclinical evaluation. A. sulphurea is difficult to genetically manipulate and therefore manipulation of the chd biosynthetic gene cluster in a genetically amenable heterologous host would be of high importance for further drug-discovery efforts. Results We report heterologous expression of the CHD biosynthetic gene cluster in the model organism Streptomyces albus del14 strain. Unexpectedly, we found that the originally defined CHD gene cluster fails to provide all genes required for CHD formation, including an additional cyclase and two regulatory genes. Overexpression of the putative pathway-specific streptomyces antibiotic regulatory protein chdB in A. sulphurea resulted in an increase of both, CHD and CD-CHD production. Applying a metabolic-engineering approach, it was also possible to generate the potent CHD analogue, CD-CHD in S. albus. Finally, an additional yield increase was achieved in S. albus del14 by in-trans overexpression of the chdR exporter gene, which provides resistance to CHD and CDCHD. Conclusions We identified previously unknown genes in the CHD cluster, which were shown to be essential for chelocardin biosynthesis by expression of the full biosynthetic gene cluster in S. albus as heterologous host. When comparing to oxytetracycline biosynthesis, we observed that the CHD gene cluster contains additional enzymes not found in gene clusters encoding the biosynthesis of typical tetracyclines (such as oxytetracycline). This finding probably explains the different chemistries and modes of action, which make CHD/CD-CHD valuable lead structures for clinical candidates. Even though the CHD genes are derived from a rare actinomycete A. sulphurea, the yield of CHD in the heterologous host was very good. The corrected nucleotide sequence of the CHD gene cluster now contains all gene products required for the production of CHD in a genetically amenable heterologous host, thus opening new possibilities towards production of novel and potent tetracycline analogues with a new mode of action.

scholarly journals Cloning and Characterization of the Pyrrolomycin Biosynthetic Gene Clusters from Actinosporangium vitaminophilum ATCC 31673 and Streptomyces sp. Strain UC 11065

2006 ◽  
Vol 51 (3) ◽  
pp. 946-957 ◽  
Author(s):  
Xiujun Zhang ◽  
Ronald J. Parry
Keyword(s):  
Sequence Analysis ◽  
Gene Cluster ◽  
Genetic Locus ◽  
Gene Clusters ◽  
Biosynthetic Gene Cluster ◽  
Open Reading Frames ◽  
Biosynthetic Gene ◽  
Genes Encoding ◽  
Cloned Gene

ABSTRACT The pyrrolomycins are a family of polyketide antibiotics, some of which contain a nitro group. To gain insight into the nitration mechanism associated with the formation of these antibiotics, the pyrrolomycin biosynthetic gene cluster from Actinosporangium vitaminophilum was cloned. Sequencing of ca. 56 kb of A. vitaminophilum DNA revealed 35 open reading frames (ORFs). Sequence analysis revealed a clear relationship between some of these ORFs and the biosynthetic gene cluster for pyoluteorin, a structurally related antibiotic. Since a gene transfer system could not be devised for A. vitaminophilum, additional proof for the identity of the cloned gene cluster was sought by cloning the pyrrolomycin gene cluster from Streptomyces sp. strain UC 11065, a transformable pyrrolomycin producer. Sequencing of ca. 26 kb of UC 11065 DNA revealed the presence of 17 ORFs, 15 of which exhibit strong similarity to ORFs in the A. vitaminophilum cluster as well as a nearly identical organization. Single-crossover disruption of two genes in the UC 11065 cluster abolished pyrrolomycin production in both cases. These results confirm that the genetic locus cloned from UC 11065 is essential for pyrrolomycin production, and they also confirm that the highly similar locus in A. vitaminophilum encodes pyrrolomycin biosynthetic genes. Sequence analysis revealed that both clusters contain genes encoding the two components of an assimilatory nitrate reductase. This finding suggests that nitrite is required for the formation of the nitrated pyrrolomycins. However, sequence analysis did not provide additional insights into the nitration process, suggesting the operation of a novel nitration mechanism.

scholarly journals Cloning of the Bisucaberin B Biosynthetic Gene Cluster from the Marine Bacterium Tenacibaculum mesophilum, and Heterologous Production of Bisucaberin B

Marine Drugs ◽  
2018 ◽  
Vol 16 (9) ◽  
pp. 342 ◽  
Author(s):  
Masaki Fujita ◽  
Yusuke Goto ◽  
Ryuichi Sakai
Keyword(s):  
Gene Cluster ◽  
Marine Bacterium ◽  
Gene Clusters ◽  
Biosynthetic Gene Cluster ◽  
Open Reading Frames ◽  
Linear Molecule ◽  
Biosynthetic Gene ◽  
Biosynthetic Gene Clusters ◽  
Key Enzyme ◽  
Reading Frames

The biosynthetic gene cluster for bisucaberin B (1, bsb gene cluster), an N-hydroxy-N-succinyl diamine (HSD)-based siderophore, was cloned from the marine bacterium Tenacibaculum mesophilum, originated from a marine sponge. The bsb gene cluster consists of six open reading frames (ORFs), in contrast to the four ORFs typically seen in biosynthetic gene clusters of the related molecules. Heterologous expression of the key enzyme, BsbD2, which is responsible for the final biosynthetic step of 1 resulted in production of bisucaberin B (1), but not bisucaberin (2) a macrocyclic counterpart of 1. To date, numbers of related enzymes producing macrocyclic analogues have been reported, but this work represents the first example of the HSD-based siderophore biosynthetic enzyme which exclusively produces a linear molecule rather than macrocyclic counterparts.

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