scholarly journals Characterization of the Noncanonical Regulatory and Transporter Genes in Atratumycin Biosynthesis and Production in a Heterologous Host

Marine Drugs ◽  
2019 ◽  
Vol 17 (10) ◽  
pp. 560 ◽  
Author(s):  
Zhijie Yang ◽  
Xin Wei ◽  
Jianqiao He ◽  
Changli Sun ◽  
Jianhua Ju ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Streptomyces Coelicolor ◽  
Regulatory Gene ◽  
Regulatory Protein ◽  
Biosynthetic Gene Cluster ◽  
Negative Regulator ◽  
Over Expression ◽  
Lead Compounds ◽  
Mycobacteria Tuberculosis ◽  
Abc Transporter Genes

Atratumycin is a cyclodepsipeptide with activity against Mycobacteria tuberculosis isolated from deep-sea derived Streptomyces atratus SCSIO ZH16NS-80S. Analysis of the atratumycin biosynthetic gene cluster (atr) revealed that its biosynthesis is regulated by multiple factors, including two LuxR regulatory genes (atr1 and atr2), two ABC transporter genes (atr29 and atr30) and one Streptomyces antibiotic regulatory gene (atr32). In this work, three regulatory and two transporter genes were unambiguously determined to provide positive, negative and self-protective roles during biosynthesis of atratumycin through bioinformatic analyses, gene inactivations and trans-complementation studies. Notably, an unusual Streptomyces antibiotic regulatory protein Atr32 was characterized as a negative regulator; the function of Atr32 is distinct from previous studies. Five over-expression mutant strains were constructed by rational application of the regulatory and transporter genes; the resulting strains produced significantly improved titers of atratumycin that were ca. 1.7–2.3 fold greater than wild-type (WT) producer. Furthermore, the atratumycin gene cluster was successfully expressed in Streptomyces coelicolor M1154, thus paving the way for the transfer and recombination of large DNA fragments. Overall, this finding sets the stage for understanding the unique biosynthesis of pharmaceutically important atratumycin and lays the foundation for generating anti-tuberculosis lead compounds possessing novel structures.

scholarly journals Regulation of valanimycin biosynthesis in Streptomyces viridifaciens: characterization of VlmI as a Streptomyces antibiotic regulatory protein (SARP)

Microbiology ◽  
2010 ◽  
Vol 156 (2) ◽  
pp. 472-483 ◽  
Author(s):  
Ram P. Garg ◽  
Ronald J. Parry
Keyword(s):  
Gene Cluster ◽  
Streptomyces Coelicolor ◽  
Regulatory Gene ◽  
Regulatory Protein ◽  
Biosynthetic Gene Cluster ◽  
Positive Control ◽  
Biosynthetic Gene ◽  
Direct Repeats ◽  
Expression Plasmid ◽  
Intergenic Regions

Streptomyces antibiotic regulatory proteins (SARPs) have been shown to activate transcription by binding to a tandemly arrayed set of heptameric direct repeats located around the −35 region of their cognate promoters. Experimental evidence is presented here showing that vlmI is a regulatory gene in the valanimycin biosynthetic gene cluster of Streptomyces viridifaciens and encodes a protein belonging to the SARP family. The organization of the valanimycin biosynthetic gene cluster suggests that the valanimycin biosynthetic genes are located on three potential transcripts, vlmHORBCD, vlmJKL and vlmA. Disruption of vlmI abolished valanimycin biosynthesis. Western blot analyses showed that VlmR and VlmA are absent from the vlmI mutant and that the production of VlmK is severely diminished. These results demonstrate that the expression of these genes from the three potential transcripts is under the positive control of VlmI. The vlmA–vlmH and vlmI–vlmJ intergenic regions both exhibit a pattern of heptameric direct repeats. Gel shift assays with VlmI overproduced in Escherichia coli as a C-terminal FLAG-tagged protein clearly demonstrated that VlmI binds to DNA fragments from both regions that contain these heptameric repeats. When a high-copy-number vlmI expression plasmid was introduced into Streptomyces coelicolor M512, which contains mutations in the undecylprodigiosin and actinorhodin activators redD and actII-orf4, undecylprodigiosin production was restored, showing that vlmI can complement a redD mutation. Introduction of the same vlmI expression plasmid into an S. viridifaciens vlmI mutant restored valanimycin production to wild-type levels.

scholarly journals Transcriptional regulation of the novobiocin biosynthetic gene cluster

Microbiology ◽  
2009 ◽  
Vol 155 (12) ◽  
pp. 4025-4035 ◽  
Author(s):  
Volker Dangel ◽  
Johannes Härle ◽  
Christiane Goerke ◽  
Christiane Wolz ◽  
Bertolt Gust ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Regulatory Gene ◽  
Regulatory Protein ◽  
Biosynthetic Gene Cluster ◽  
Regulatory Genes ◽  
Pcr Analysis ◽  
Minor Importance ◽  
Biosynthetic Gene ◽  
Chromosomal Dna ◽  
Experimental Conditions

The aminocoumarin antibiotic novobiocin is a gyrase inhibitor formed by a Streptomyces strain. The biosynthetic gene cluster of novobiocin spans 23.4 kb and contains 20 coding sequences, among them the two regulatory genes novE and novG. We investigated the location of transcriptional promoters within this cluster by insertion of transcriptional terminator cassettes and RT-PCR analysis of the resulting mutants. The cluster was found to contain eight DNA regions with promoter activity. The regulatory protein NovG binds to a previously identified binding site within the promoter region located upstream of novH, but apparently not to any of the other seven promoters. Quantitative real-time PCR was used to compare the number of transcripts in a strain carrying an intact novobiocin cluster with strains carrying mutated clusters. Both in-frame deletion of the regulatory gene novG and insertion of a terminator cassette into the biosynthetic gene novH led to a strong reduction of the number of transcripts of the genes located between novH and novW. This suggested that these 16 biosynthetic genes form a single operon. Three internal promoters are located within this operon but appear to be of minor importance, if any, under our experimental conditions. Transcription of novG was found to depend on the presence of NovE, suggesting that the two regulatory genes, novE and novG, act in a cascade-like mechanism. The resistance gene gyrBR , encoding an aminocoumarin-resistant gyrase B subunit, may initially be co-transcribed with the genes from novH to novW. However, when the gyrase inhibitor novobiocin accumulates in the cultures, gyrBR is transcribed from its own promoter. Previous work has suggested that this promoter is controlled by the superhelical density of chromosomal DNA.

scholarly journals The Mithramycin Gene Cluster of Streptomyces argillaceus Contains a Positive Regulatory Gene and Two Repeated DNA Sequences That Are Located at Both Ends of the Cluster

1999 ◽  
Vol 181 (2) ◽  
pp. 642-647 ◽  
Author(s):  
Felipe Lombó ◽  
Alfredo F. Braña ◽  
Carmen Méndez ◽  
José A. Salas
Keyword(s):  
Gene Cluster ◽  
Dna Sequences ◽  
Streptomyces Coelicolor ◽  
Regulatory Gene ◽  
Regulatory Protein ◽  
Fold Increase ◽  
Open Reading Frames ◽  
Antibiotic Biosynthesis ◽  
Positive Regulators ◽  
Actinorhodin Production

ABSTRACT Sequencing of a 4.3-kb DNA region from the chromosome ofStreptomyces argillaceus, a mithramycin producer, revealed the presence of two open reading frames (ORFs). The first one (orfA) codes for a protein that resembles several transport proteins. The second one (mtmR) codes for a protein similar to positive regulators involved in antibiotic biosynthesis (DnrI, SnoA, ActII-orf4, CcaR, and RedD) belonging to the Streptomycesantibiotic regulatory protein (SARP) family. Both ORFs are separated by a 1.9-kb, apparently noncoding region. Replacement of themtmR region by an antibiotic resistance cassette completely abolished mithramycin biosynthesis. Expression of mtmR in a high-copy-number vector in S. argillaceus caused a 16-fold increase in mithramycin production. The mtmR gene restored actinorhodin production in Streptomyces coelicolor JF1 mutant, in which the actinorhodin-specific activator ActII-orf4 is inactive, and also stimulated actinorhodin production byStreptomyces lividans TK21. A 241-bp region located 1.9 kb upstream of mtmR was found to be repeated approximately 50 kb downstream of mtmR at the other end of the mithramycin gene cluster. A model to explain a possible route for the acquisition of the mithramycin gene cluster by S. argillaceus is proposed.

scholarly journals New Insights into the Genetic Organization of the FK228 Biosynthetic Gene Cluster inChromobacterium violaceumNo. 968

2010 ◽  
Vol 77 (4) ◽  
pp. 1508-1511 ◽  
Author(s):  
Vishwakanth Y. Potharla ◽  
Shane R. Wesener ◽  
Yi-Qiang Cheng
Keyword(s):  
Natural Product ◽  
Gene Cluster ◽  
Gene Deletion ◽  
Regulatory Gene ◽  
Biosynthetic Gene Cluster ◽  
Transcriptional Analysis ◽  
Biosynthetic Gene ◽  
Genetic Organization

ABSTRACTThe biosynthetic gene cluster of FK228, an FDA-approved anticancer natural product, was identified and sequenced previously. The genetic organization of this gene cluster has now been delineated through systematic gene deletion and transcriptional analysis. As a result, the gene cluster is redefined to contain 12 genes:depAthroughdepJ,depM, and a newly identified pathway regulatory gene,depR.

Cloning and characterization of the histidine biosynthetic gene cluster of Streptomyces coelicolor A3(2)

Gene ◽  
1990 ◽  
Vol 90 (1) ◽  
pp. 31-41 ◽  
Author(s):  
Danila Limauro ◽  
Alessandra Avitabile ◽  
Carmela Cappellano ◽  
Anna Maria Puglia ◽  
Carmelo B. Bruni
Keyword(s):  
Gene Cluster ◽  
Streptomyces Coelicolor ◽  
Biosynthetic Gene Cluster ◽  
Biosynthetic Gene

scholarly journals Two Master Switch Regulators Trigger A40926 Biosynthesis in Nonomuraea sp. Strain ATCC 39727

2015 ◽  
Vol 197 (15) ◽  
pp. 2536-2544 ◽  
Author(s):  
Letizia Lo Grasso ◽  
Sonia Maffioli ◽  
Margherita Sosio ◽  
Mervyn Bibb ◽  
Anna Maria Puglia ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Antibiotic Production ◽  
Regulatory Protein ◽  
Gene Clusters ◽  
Biosynthetic Gene Cluster ◽  
Regulatory Mechanisms ◽  
Antibiotic Biosynthesis ◽  
Strain Atcc ◽  
Protein Coding ◽  
Content Type

ABSTRACTThe actinomyceteNonomuraeasp. strain ATCC 39727 produces the glycopeptide A40926, the precursor of dalbavancin. Biosynthesis of A40926 is encoded by thedbvgene cluster, which contains 37 protein-coding sequences that participate in antibiotic biosynthesis, regulation, immunity, and export. In addition to the positive regulatory protein Dbv4, the A40926-biosynthetic gene cluster encodes two additional putative regulators, Dbv3 and Dbv6. Independent mutations in these genes, combined with bioassays and liquid chromatography-mass spectrometry (LC-MS) analyses, demonstrated that Dbv3 and Dbv4 are both required for antibiotic production, while inactivation ofdbv6had no effect. In addition, overexpression ofdbv3led to higher levels of A40926 production. Transcriptional and quantitative reverse transcription (RT)-PCR analyses showed that Dbv4 is essential for the transcription of two operons,dbv14-dbv8anddbv30-dbv35, while Dbv3 positively controls the expression of four monocistronic transcription units (dbv4,dbv29,dbv36, anddbv37) and of six operons (dbv2-dbv1,dbv14-dbv8,dbv17-dbv15,dbv21-dbv20,dbv24-dbv28, anddbv30-dbv35). We propose a complex and coordinated model of regulation in which Dbv3 directly or indirectly activates transcription ofdbv4and controls biosynthesis of 4-hydroxyphenylglycine and the heptapeptide backbone, A40926 export, and some tailoring reactions (mannosylation and hexose oxidation), while Dbv4 directly regulates biosynthesis of 3,5-dihydroxyphenylglycine and other tailoring reactions, including the four cross-links, halogenation, glycosylation, and acylation.IMPORTANCEThis report expands knowledge of the regulatory mechanisms used to control the biosynthesis of the glycopeptide antibiotic A40926 in the actinomyceteNonomuraeasp. strain ATCC 39727. A40926 is the precursor of dalbavancin, approved for treatment of skin infections by Gram-positive bacteria. Therefore, understanding the regulation of its biosynthesis is also of industrial importance. So far, the regulatory mechanisms used to control two other similar glycopeptides (balhimycin and teicoplanin) have been elucidated, and beyond a common step, different clusters seem to have devised different strategies to control glycopeptide production. Thus, our work provides one more example of the pitfalls of deducing regulatory roles from bioinformatic analyses only, even when analyzing gene clusters directing the synthesis of structurally related compounds.

scholarly journals Hierarchical control of virginiamycin production in Streptomyces virginiae by three pathway-specific regulators: VmsS, VmsT and VmsR

Microbiology ◽  
2009 ◽  
Vol 155 (4) ◽  
pp. 1250-1259 ◽  
Author(s):  
Nattika Pulsawat ◽  
Shigeru Kitani ◽  
Eriko Fukushima ◽  
Takuya Nihira
Keyword(s):  
Gene Cluster ◽  
Response Regulator ◽  
Expression Profiles ◽  
Regulatory Protein ◽  
Regulatory Region ◽  
Biosynthetic Gene Cluster ◽  
Transcriptional Analysis ◽  
Biosynthetic Gene ◽  
Biosynthetic Genes ◽  
Streptomyces Virginiae

Two regulatory genes encoding a Streptomyces antibiotic regulatory protein (vmsS) and a response regulator (vmsT) of a bacterial two-component signal transduction system are present in the left-hand region of the biosynthetic gene cluster of the antibiotic virginiamycin, which is composed of virginiamycin M (VM) and virginiamycin S (VS), in Streptomyces virginiae. Disruption of vmsS abolished both VM and VS biosynthesis, with drastic alteration of the transcriptional profile for virginiamycin biosynthetic genes, whereas disruption of vmsT resulted in only a loss of VM biosynthesis, suggesting that vmsS is a pathway-specific regulator for both VM and VS biosynthesis, and that vmsT is a pathway-specific regulator for VM biosynthesis alone. Gene expression profiles determined by semiquantitative RT-PCR on the virginiamycin biosynthetic gene cluster demonstrated that vmsS controls the biosynthetic genes for VM and VS, and vmsT controls unidentified gene(s) of VM biosynthesis located outside the biosynthetic gene cluster. In addition, transcriptional analysis of a deletion mutant of vmsR located in the clustered regulatory region in the virginiamycin cluster (and which also acts as a SARP-family activator for both VM and VS biosynthesis) indicated that the expression of vmsS and vmsT is under the control of vmsR, and vmsR also contributes to the expression of VM and VS biosynthetic genes, independent of vmsS and vmsT. Therefore, coordinated virginiamycin biosynthesis is controlled by three pathway-specific regulators which hierarchically control the expression of the biosynthetic gene cluster.

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