scholarly journals Biosynthetic Genes for the Tetrodecamycin Antibiotics

2016 ◽  
Vol 198 (14) ◽  
pp. 1965-1973 ◽  
Author(s):  
Tomas Gverzdys ◽  
Justin R. Nodwell
Keyword(s):  
Biosynthetic Pathway ◽  
Genetic Manipulation ◽  
Biosynthetic Gene Cluster ◽  
Mechanisms Of Action ◽  
Covalent Modification ◽  
Biosynthetic Gene ◽  
Producer Strain ◽  
Biosynthetic Genes ◽  
Content Type ◽  
Distinct Subgroup

ABSTRACTWe recently described 13-deoxytetrodecamycin, a new member of the tetrodecamycin family of antibiotics. A defining feature of these molecules is the presence of a five-membered lactone called a tetronate ring. By sequencing the genome of a producer strain,Streptomycessp. strain WAC04657, and searching for a gene previously implicated in tetronate ring formation, we identified the biosynthetic genes responsible for producing 13-deoxytetrodecamycin (thetedgenes). Using thetedcluster in WAC04657 as a reference, we found related clusters in three other organisms:Streptomyces atroolivaceusATCC 19725,Streptomyces globisporusNRRL B-2293, andStreptomycessp. strain LaPpAH-202. Comparing the four clusters allowed us to identify the cluster boundaries. Genetic manipulation of the cluster confirmed the involvement of thetedgenes in 13-deoxytetrodecamycin biosynthesis and revealed several additional molecules produced through thetedbiosynthetic pathway, including tetrodecamycin, dihydrotetrodecamycin, and another, W5.9, a novel molecule. Comparison of the bioactivities of these four molecules suggests that they may act through the covalent modification of their target(s).IMPORTANCEThe tetrodecamycins are a distinct subgroup of the tetronate family of secondary metabolites. Little is known about their biosynthesis or mechanisms of action, making them an attractive subject for investigation. In this paper we present the biosynthetic gene cluster for 13-deoxytetrodecamycin inStreptomycessp. strain WAC04657. We identify related clusters in several other organisms and show that they produce related molecules.

scholarly journals Streptomyces lividans Blasticidin S Deaminase and Its Application in Engineering a Blasticidin S-Producing Strain for Ease of Genetic Manipulation

2013 ◽  
Vol 79 (7) ◽  
pp. 2349-2357 ◽  
Author(s):  
Li Li ◽  
Jun Wu ◽  
Zixin Deng ◽  
T. Mark Zabriskie ◽  
Xinyi He
Keyword(s):  
Gene Cluster ◽  
Genetic Manipulation ◽  
Biosynthetic Gene Cluster ◽  
Streptomyces Lividans ◽  
Dna Uptake ◽  
Biosynthetic Gene ◽  
Content Type ◽  
Blasticidin S

ABSTRACTBlasticidin S is a peptidyl nucleoside antibiotic produced byStreptomyces griseochromogenesthat exhibits strong fungicidal activity. To circumvent an effective DNA uptake barrier system in the native producer and investigate its biosynthesisin vivo, the blasticidin S biosynthetic gene cluster (bls) was engrafted to the chromosome ofStreptomyces lividans. However, the resulting mutant, LL2, produced the inactive deaminohydroxyblasticidin S instead of blasticidin S. Subsequently, a blasticidin S deaminase (SLBSD, forS. lividansblasticidin S deaminase) was identified inS. lividansand shown to govern thisin vivoconversion. Purified SLBSD was found to be capable of transforming blasticidin S to deaminohydroxyblasticidin Sin vitro. It also catalyzed deamination of the cytosine moiety of cytosylglucuronic acid, an intermediate in blasticidin S biosynthesis. Disruption of the SLBSD gene inS. lividansLL2 led to successful production of active blasticidin S in the resultant mutant,S. lividansWJ2. To demonstrate the easy manipulation of the blasticidin S biosynthetic gene cluster,blsE,blsF, andblsL, encoding a predicted radicalS-adenosylmethionine (SAM) protein, an unknown protein, and a guanidino methyltransferase, were individually inactivated to access their role in blasticidin S biosynthesis.

scholarly journals Refactoring the formicamycin biosynthetic gene cluster to make high-level producing strains and new molecules

2020 ◽  
Author(s):  
Rebecca Devine ◽  
Hannah McDonald ◽  
Zhiwei Qin ◽  
Corinne Arnold ◽  
Katie Noble ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Large Scale ◽  
Liquid Culture ◽  
Gene Clusters ◽  
Biosynthetic Gene Cluster ◽  
Resistant Bacteria ◽  
Drug Resistant ◽  
Biosynthetic Gene ◽  
Biosynthetic Genes

AbstractThe formicamycins are promising antibiotics with potent activity against Gram-positive pathogens including VRE and MRSA and display a high barrier to selection of resistant isolates. They were first identified in Streptomyces formicae KY5, which produces the formicamycins at low levels on solid agar but not in liquid culture, thus hindering further investigation of these promising antibacterial compounds. We hypothesised that by understanding the organisation and regulation of the for biosynthetic gene cluster, we could rationally refactor the cluster to increase production levels. Here we report that the for biosynthetic gene cluster consists of 24 genes expressed on nine transcripts. Seven of these transcripts, including those containing all the major biosynthetic genes, are repressed by the MarR-regulator ForJ which also controls the expression of the ForGF two-component system that initiates biosynthesis. A third cluster-situated regulator, ForZ, autoregulates and controls production of the putative MFS transporter ForAA. Consistent with these findings, deletion of forJ increased formicamycin biosynthesis 5-fold, while over-expression of forGF in the ΔforJ background increased production 10-fold compared to the wild-type. De-repression by deleting forJ also switched on biosynthesis in liquid-culture and induced the production of two novel formicamycin congeners. By combining mutations in regulatory and biosynthetic genes, six new biosynthetic precursors with antibacterial activity were also isolated. This work demonstrates the power of synthetic biology for the rational redesign of antibiotic biosynthetic gene clusters both to engineer strains suitable for fermentation in large scale bioreactors and to generate new molecules.ImportanceAntimicrobial resistance is a growing threat as existing antibiotics become increasingly ineffective against drug resistant pathogens. Here we determine the transcriptional organisation and regulation of the gene cluster encoding biosynthesis of the formicamycins, promising new antibiotics with activity against drug resistant bacteria. By exploiting this knowledge, we construct stable mutant strains which over-produce these molecules in both liquid and solid culture whilst also making some new compound variants. This will facilitate large scale purification of these molecules for further study including in vivo experiments and the elucidation of their mechanism of action. Our work demonstrates that understanding the regulation of natural product biosynthetic pathways can enable rational improvement of the producing strains.

scholarly journals Composite Long- and Short-Read Sequencing Delivers a Complete Genome Sequence of B04Sm5, a Reutericyclin- and Mutanocyclin-Producing Strain of Streptococcus mutans

2020 ◽  
Vol 9 (47) ◽  
Author(s):  
Jonathon L. Baker ◽  
Anna Edlund
Keyword(s):  
Streptococcus Mutans ◽  
Genome Sequence ◽  
Complete Genome Sequence ◽  
Complete Genome ◽  
Biosynthetic Gene Cluster ◽  
Commensal Bacteria ◽  
Biosynthetic Gene ◽  
Tetramic Acid ◽  
Content Type ◽  
Short Read Sequencing

ABSTRACT Streptococcus mutans strain B04Sm5 was recently shown to inhibit the growth of neighboring commensal bacteria using reutericyclin, an acylated tetramic acid produced by the muc biosynthetic gene cluster. Here, a complete genome sequence of B04Sm5 is reported.

Biosynthesis of the uridine-derived nucleoside antibiotic A-94964: identification and characterization of the biosynthetic gene cluster provide insight into the biosynthetic pathway

2019 ◽  
Vol 17 (3) ◽  
pp. 461-466 ◽  
Author(s):  
Taro Shiraishi ◽  
Makoto Nishiyama ◽  
Tomohisa Kuzuyama
Keyword(s):  
Gene Cluster ◽  
In Silico ◽  
Biosynthetic Pathway ◽  
Gene Deletion ◽  
Biosynthetic Gene Cluster ◽  
Biosynthetic Gene ◽  
Identification And Characterization ◽  
Insight Into

The biosynthetic pathway of the uridine-derived nucleoside antibiotic A-94964 was proposed via in silico analysis coupled with gene deletion experiments.

scholarly journals Tracking Down Biotransformation to the Genetic Level: Identification of a Highly Flexible Glycosyltransferase from Saccharothrix espanaensis

2013 ◽  
Vol 79 (17) ◽  
pp. 5224-5232 ◽  
Author(s):  
Tina Strobel ◽  
Yvonne Schmidt ◽  
Anton Linnenbrink ◽  
Andriy Luzhetskyy ◽  
Marta Luzhetska ◽  
...  
Keyword(s):  
Natural Products ◽  
Phenolic Compounds ◽  
Natural Product ◽  
Defense Mechanism ◽  
Biosynthetic Gene Cluster ◽  
Biosynthetic Gene ◽  
Gene Encoding ◽  
Content Type ◽  
Genetic Level ◽  
Level Identification

ABSTRACTSaccharothrix espanaensisis a member of the orderActinomycetales. The genome of the strain has been sequenced recently, revealing 106 glycosyltransferase genes. In this paper, we report the detection of a glycosyltransferase fromSaccharothrix espanaensiswhich is able to rhamnosylate different phenolic compounds targeting different positions of the molecules. The gene encoding the flexible glycosyltransferase is not located close to a natural product biosynthetic gene cluster. Therefore, the native function of this enzyme might be not the biosynthesis of a secondary metabolite but the glycosylation of internal and external natural products as part of a defense mechanism.

scholarly journals Regiospecificities and Prenylation Mode Specificities of the Fungal Indole Diterpene Prenyltransferases AtmD and PaxD

2013 ◽  
Vol 79 (23) ◽  
pp. 7298-7304 ◽  
Author(s):  
Chengwei Liu ◽  
Atsushi Minami ◽  
Motoyoshi Noike ◽  
Hiroaki Toshima ◽  
Hideaki Oikawa ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Biosynthetic Gene Cluster ◽  
Amino Acid Identity ◽  
Biosynthetic Gene ◽  
Content Type ◽  
Acid Identity ◽  
Dimethylallyl Diphosphate ◽  
Regular Type ◽  
Penicillium Paxilli ◽  
Related Compounds

ABSTRACTWe recently reported the function ofpaxD, which is involved in the paxilline (compound 1) biosynthetic gene cluster inPenicillium paxilli. Recombinant PaxD catalyzed a stepwise regular-type diprenylation at the 21 and 22 positions of compound 1 with dimethylallyl diphosphate (DMAPP) as the prenyl donor. In this study,atmD, which is located in the aflatrem (compound 2) biosynthetic gene cluster inAspergillus flavusand encodes an enzyme with 32% amino acid identity to PaxD, was characterized using recombinant enzyme. When compound 1 and DMAPP were used as substrates, two major products and a trace of minor product were formed. The structures of the two major products were determined to be reversely monoprenylated compound 1 at either the 20 or 21 position. Because compound 2 and β-aflatrem (compound 3), both of which are compound 1-related compounds produced byA. flavus, have the same prenyl moiety at the 20 and 21 position, respectively, AtmD should catalyze the prenylation in compound 2 and 3 biosynthesis. More importantly and surprisingly, AtmD accepted paspaline (compound 4), which is an intermediate of compound 1 biosynthesis that has a structure similar to that of compound 1, and catalyzed a regular monoprenylation of compound 4 at either the 21 or 22 position, though the reverse prenylation was observed with compound 1. This suggests that fungal indole diterpene prenyltransferases have the potential to alter their position and regular/reverse specificities for prenylation and could be applicable for the synthesis of industrially useful compounds.

scholarly journals Cloning and Characterization of the Biosynthetic Gene Cluster for Tomaymycin, an SJG-136 Monomeric Analog

2009 ◽  
Vol 75 (9) ◽  
pp. 2958-2963 ◽  
Author(s):  
Wei Li ◽  
ShenChieh Chou ◽  
Ankush Khullar ◽  
Barbara Gerratana
Keyword(s):  
Cluster Analysis ◽  
Gene Cluster ◽  
Biosynthetic Pathway ◽  
Biosynthetic Gene Cluster ◽  
Gene Replacement ◽  
Biosynthetic Gene

ABSTRACT Tomaymycin produced by Streptomyces achromogenes is a naturally produced pyrrolobenzodiazepine (PBD). The biosynthetic gene cluster for tomaymycin was identified and sequenced. The gene cluster analysis reveals a novel biosynthetic pathway for the anthranilate moiety of PBDs. Gene replacement and chemical complementation studies were used to confirm the proposed biosynthetic pathway.

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.

scholarly journals Pathway for the Biosynthesis of the Pigment Chrysogine by Penicillium chrysogenum

2017 ◽  
Vol 84 (4) ◽  
Author(s):  
Annarita Viggiano ◽  
Oleksandr Salo ◽  
Hazrat Ali ◽  
Wiktor Szymanski ◽  
Peter P. Lankhorst ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Filamentous Fungi ◽  
Penicillium Chrysogenum ◽  
Biosynthetic Pathway ◽  
Biosynthetic Gene Cluster ◽  
Culture Broth ◽  
Yellow Pigment ◽  
Metabolic Potential ◽  
Content Type ◽  
Related Compounds

ABSTRACT Chrysogine is a yellow pigment produced by Penicillium chrysogenum and other filamentous fungi. Although the pigment was first isolated in 1973, its biosynthetic pathway has so far not been resolved. Here, we show that deletion of the highly expressed nonribosomal peptide synthetase (NRPS) gene Pc21g12630 ( chyA ) resulted in a decrease in the production of chrysogine and 13 related compounds in the culture broth of P. chrysogenum . Each of the genes of the chyA -containing gene cluster was individually deleted, and corresponding mutants were examined by metabolic profiling in order to elucidate their function. The data suggest that the NRPS ChyA mediates the condensation of anthranilic acid and alanine into the intermediate 2-(2-aminopropanamido)benzoic acid, which was verified by feeding experiments of a ΔchyA strain with the chemically synthesized product. The remainder of the pathway is highly branched, yielding at least 13 chrysogine-related compounds. IMPORTANCE Penicillium chrysogenum is used in industry for the production of β-lactams, but also produces several other secondary metabolites. The yellow pigment chrysogine is one of the most abundant metabolites in the culture broth, next to β-lactams. Here, we have characterized the biosynthetic gene cluster involved in chrysogine production and elucidated a complex and highly branched biosynthetic pathway, assigning each of the chrysogine cluster genes to biosynthetic steps and metabolic intermediates. The work further unlocks the metabolic potential of filamentous fungi and the complexity of secondary metabolite pathways.

scholarly journals Heme Protein and Hydroxyarginase Necessary for Biosynthesis of d-Cycloserine

2012 ◽  
Vol 56 (7) ◽  
pp. 3682-3689 ◽  
Author(s):  
Takanori Kumagai ◽  
Kisho Takagi ◽  
Yusuke Koyama ◽  
Yasuyuki Matoba ◽  
Kosuke Oda ◽  
...  
Keyword(s):  
Biosynthetic Pathway ◽  
Culture Medium ◽  
Biosynthetic Gene Cluster ◽  
Heme Protein ◽  
Content Type ◽  
Streptomyces Lavendulae ◽  
Spectroscopic Analyses ◽  
Encoding Gene ◽  
Oxide Synthase ◽  
Hydrolysis Of

ABSTRACTWe have recently cloned ad-cycloserine (DCS) biosynthetic gene cluster that consists of 10 genes, designateddcsA∼dcsJ, fromStreptomyces lavendulaeATCC 11924 (16). In the predicted pathway of hydroxyurea (HU) formation in DCS biosynthesis,l-arginine (L-Arg) must first be hydroxylated, prior to the hydrolysis ofNω-hydroxy-l-arginine (NHA) by DcsB, an arginase homolog. The hydroxylation of L-Arg is known to be catalyzed by nitric oxide synthase (NOS). In this study, to verify the supply route of HU, we created adcsB-disrupted mutant, ΔdcsB. While the mutant lost DCS productivity, its productivity was restored by complementation ofdcsB, and also by the addition of HU but not NHA, suggesting that HU is supplied by DcsB. A NOS-encoding gene,nos, fromS. lavendulaechromosome was cloned, to create anos-disrupted mutant. However, the mutant maintained the DCS productivity, suggesting that NOS is not necessary for DCS biosynthesis. To clarify the identity of an enzyme necessary for NHA formation, adcsA-disrupted mutant, designated ΔdcsA, was also created. The mutant lost DCS productivity, whereas the DCS productivity was restored by complementation ofdcsA. The addition of NHA to the culture medium of ΔdcsAmutant was also effective to restore DCS production. These results indicate that thedcsAgene product, DcsA, is an enzyme essential to generate NHA as a precursor in the DCS biosynthetic pathway. Spectroscopic analyses of the recombinant DcsA revealed that it is a heme protein, supporting an idea that DcsA is an enzyme catalyzing hydroxylation.

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