scholarly journals RifZ (AMED_0655) Is a Pathway-Specific Regulator for Rifamycin Biosynthesis in Amycolatopsis mediterranei

2017 ◽  
Vol 83 (8) ◽  
Author(s):  
Chen Li ◽  
Xinqiang Liu ◽  
Chao Lei ◽  
Han Yan ◽  
Zhihui Shao ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Dna Sequences ◽  
Consensus Sequence ◽  
Biosynthetic Gene Cluster ◽  
High Yield ◽  
Biosynthetic Gene ◽  
Amycolatopsis Mediterranei ◽  
Promoter Regions ◽  
Mobility Shift ◽  
Content Type

ABSTRACT Rifamycin and its derivatives are particularly effective against the pathogenic mycobacteria Mycobacterium tuberculosis and Mycobacterium leprae. Although the biosynthetic pathway of rifamycin has been extensively studied in Amycolatopsis mediterranei, little is known about the regulation in rifamycin biosynthesis. Here, an in vivo transposon system was employed to identify genes involved in the regulation of rifamycin production in A. mediterranei U32. In total, nine rifamycin-deficient mutants were isolated, among which three mutants had the transposon inserted in AMED_0655 (rifZ, encoding a LuxR family regulator). The rifZ gene was further knocked out via homologous recombination, and the transcription of genes in the rifamycin biosynthetic gene cluster (rif cluster) was remarkably reduced in the rifZ null mutant. Based on the cotranscription assay results, genes within the rif cluster were grouped into 10 operons, sharing six promoter regions. By use of electrophoretic mobility shift assay and DNase I footprinting assay, RifZ was proved to specially bind to all six promoter regions, which was consistent with the fact that RifZ regulated the transcription of the whole rif cluster. The binding consensus sequence was further characterized through alignment using the RifZ-protected DNA sequences. By use of bionformatic analysis, another five promoters containing the RifZ box (CTACC-N8-GGATG) were identified, among which the binding of RifZ to the promoter regions of both rifK and orf18 (AMED_0645) was further verified. As RifZ directly regulates the transcription of all operons within the rif cluster, we propose that RifZ is a pathway-specific regulator for the rif cluster. IMPORTANCE To this day, rifamycin and its derivatives are still the first-line antituberculosis drugs. The biosynthesis of rifamycin has been extensively studied, and most biosynthetic processes have been characterized. However, little is known about the regulation of the transcription of the rifamycin biosynthetic gene cluster (rif cluster), and no regulator has been characterized. Through the employment of transposon screening, we here characterized a LuxR family regulator, RifZ, as a direct transcriptional activator for the rif cluster. As RifZ directly regulates the transcription of the entire rif cluster, it is considered a pathway-specific regulator for rifamycin biosynthesis. Therefore, as the first regulator characterized for direct regulation of rif cluster transcription, RifZ may provide a new clue for further engineering of high-yield industrial strains.

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 Identification of a Repressor for the Two iol Operons Required for Inositol Catabolism in Geobacillus Kaustophilus

2020 ◽  
Author(s):  
Ken-ichi Yoshida ◽  
Yusuke Shirae ◽  
Ryo Nishimura ◽  
Kaho Fukui ◽  
Shu Ishikawa
Keyword(s):  
Dna Binding ◽  
Gene Cluster ◽  
Binding Sites ◽  
Consensus Sequence ◽  
Binding Property ◽  
Promoter Regions ◽  
Mobility Shift ◽  
Geobacillus Kaustophilus ◽  
Inositol Dehydrogenase

Abstract BackgroundGeobacillus kaustophilus HTA426, a thermophilic Gram-positive bacterium, grows on inositol as its sole carbon source, and an iol gene cluster required for inositol catabolism has been postulated with reference to the iol genes in Bacillus subtilis. The iol gene cluster consists of two tandem operons induced in the presence of inositol; however, the mechanism underlying the induction remains unclear. B. subtilis iolQ is known to be involved in the regulation of iolX encoding a scyllo-inositol dehydrogenase, and its homolog in HTA426 was found two genes upstream of the first gene (gk1899) of the iol gene cluster and termed as iolQ in G. kaustophilus.ResultsWhen iolQ was inactivated, not only the myo-inositol dehydrogenase activity in the cell due to the expression of gk1899 but also the transcription of the two iol operons became constitutive. IolQ was produced and purified as a C-terminal His-tag fusion in Escherichia coli and subjected to the in vitro gel mobility shift assay to examine its DNA binding property. It was observed that IolQ bound to the DNA fragments containing each of the two iol promoter regions, and its DNA binding was antagonized by myo-inositol. Moreover, DNase I footprint analyses were conducted to determine the two binding sites of IolQ within each of the iol promoter regions. By comparing the sequences of the binding sites, a consensus sequence for IolQ binding was deduced to be a palindrome of 5′-RGWAAGCGCTTSCY-3′ (where R = A or G, W = A or T, S = G or C, and Y = C or T).ConclusionIolQ functions as a transcriptional repressor regulating the induction of the two iol operons responding to myo-inositol.

scholarly journals Production of Diverse Beauveriolide Analogs in Closely Related Fungi: a Rare Case of Fungal Chemodiversity

mSphere ◽  
2020 ◽  
Vol 5 (5) ◽  
Author(s):  
Ying Yin ◽  
Bo Chen ◽  
Shuangxiu Song ◽  
Bing Li ◽  
Xiuqing Yang ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Growth Condition ◽  
Chain Length ◽  
Rare Case ◽  
Biosynthetic Gene Cluster ◽  
Biosynthetic Gene ◽  
Beauveria Brongniartii ◽  
Fungal Virulence ◽  
Content Type ◽  
A Chain

ABSTRACT Fungal chemodiversity is well known in part due to the production of diverse analogous compounds by a single biosynthetic gene cluster (BGC). Usually, similar or the same metabolites are produced by closely related fungal species under a given condition, the foundation of fungal chemotaxonomy. Here, we report a rare case of the production of the cyclodepsipeptide beauveriolides (BVDs) in three insect-pathogenic fungi. We found that the more closely related fungi Beauveria bassiana and Beauveria brongniartii produced structurally distinct analogs of BVDs, whereas the less-close relatives B. brongniartii and Cordyceps militaris biosynthesized structurally similar congeners under the same growth condition. It was verified that a conserved BGC containing four genes is responsible for BVD biosynthesis in three fungi, including a polyketide synthase (PKS) for the production of 3-hydroxy fatty acids (FAs) with chain length variations. In contrast to BVD production patterns, phylogenetic analysis of the BGC enzymes or enzyme domains largely resulted in the congruence relationship with fungal speciation. Feeding assays demonstrated that an FA with a chain length of eight carbon atoms was preferentially utilized, whereas an FA with a chain longer than 10 carbon atoms could not be used as a substrate for BVD biosynthesis. Insect survival assays suggested that the contribution of BVDs to fungal virulence might be associated with the susceptibility of insect species. The results of this study enrich the knowledge of fungal secondary metabolic diversity that can question the reliability of fungal chemotaxonomy. IMPORTANCE Fungal chemotaxonomy is an approach to classify fungi based on the fungal production profile of metabolites, especially the secondary metabolites. We found an atypical example that could question the reliability of fungal chemical classifications in this study, i.e., the more closely related entomopathogenic species Beauveria bassiana and Beauveria brongniartii produced structurally different congeners of the cyclodepsipeptide beauveriolides, whereas the rather divergent species B. brongniartii and Cordyceps militaris biosynthesized similar analogs under the same growth condition. The conserved biosynthetic gene cluster (BGC) containing four genes present in each species is responsible for beauveriolide production. In contrast to the compound formation profiles, the phylogenies of biosynthetic enzymes or enzymatic domains show associations with fungal speciation. Dependent on the insect species, production of beauveriolides may contribute to fungal virulence against the susceptible insect hosts. The findings in this study augment the diversity of fungal secondary metabolisms.

scholarly journals Discovery of Gene Cluster for Mycosporine-Like Amino Acid Biosynthesis from Actinomycetales Microorganisms and Production of a Novel Mycosporine-Like Amino Acid by Heterologous Expression

2014 ◽  
Vol 80 (16) ◽  
pp. 5028-5036 ◽  
Author(s):  
Kiyoko T. Miyamoto ◽  
Mamoru Komatsu ◽  
Haruo Ikeda
Keyword(s):  
Amino Acid ◽  
Gene Cluster ◽  
Gene Clusters ◽  
Biosynthetic Gene Cluster ◽  
Dependent Manner ◽  
Biosynthetic Gene ◽  
Biosynthetic Gene Clusters ◽  
Gram Positive ◽  
Content Type ◽  
Gram Positive Bacteria

ABSTRACTMycosporines and mycosporine-like amino acids (MAAs), including shinorine (mycosporine-glycine-serine) and porphyra-334 (mycosporine-glycine-threonine), are UV-absorbing compounds produced by cyanobacteria, fungi, and marine micro- and macroalgae. These MAAs have the ability to protect these organisms from damage by environmental UV radiation. Although no reports have described the production of MAAs and the corresponding genes involved in MAA biosynthesis from Gram-positive bacteria to date, genome mining of the Gram-positive bacterial database revealed that two microorganisms belonging to the orderActinomycetales,Actinosynnema mirumDSM 43827 andPseudonocardiasp. strain P1, possess a gene cluster homologous to the biosynthetic gene clusters identified from cyanobacteria. When the two strains were grown in liquid culture,Pseudonocardiasp. accumulated a very small amount of MAA-like compound in a medium-dependent manner, whereasA. mirumdid not produce MAAs under any culture conditions, indicating that the biosynthetic gene cluster ofA. mirumwas in a cryptic state in this microorganism. In order to characterize these biosynthetic gene clusters, each biosynthetic gene cluster was heterologously expressed in an engineered host,Streptomyces avermitilisSUKA22. Since the resultant transformants carrying the entire biosynthetic gene cluster controlled by an alternative promoter produced mainly shinorine, this is the first confirmation of a biosynthetic gene cluster for MAA from Gram-positive bacteria. Furthermore,S. avermitilisSUKA22 transformants carrying the biosynthetic gene cluster for MAA ofA. mirumaccumulated not only shinorine and porphyra-334 but also a novel MAA. Structure elucidation revealed that the novel MAA is mycosporine-glycine-alanine, which substitutesl-alanine for thel-serine of shinorine.

scholarly journals Identification of the Herboxidiene Biosynthetic Gene Cluster in Streptomyces chromofuscus ATCC 49982

2012 ◽  
Vol 78 (6) ◽  
pp. 2034-2038 ◽  
Author(s):  
Lei Shao ◽  
Jiachen Zi ◽  
Jia Zeng ◽  
Jixun Zhan
Keyword(s):  
Gene Cluster ◽  
Genome Sequencing ◽  
Fermentation Broth ◽  
Biosynthetic Gene Cluster ◽  
The Novel ◽  
Biosynthetic Gene ◽  
Content Type ◽  
Minor Metabolite ◽  
Streptomyces Chromofuscus ◽  
A Minor

ABSTRACTThe 53-kb biosynthetic gene cluster for the novel anticholesterol natural product herboxidiene was identified inStreptomyces chromofuscusATCC 49982 by genome sequencing and gene inactivation. In addition to herboxidiene, a biosynthetic intermediate, 18-deoxy-herboxidiene, was also isolated from the fermentation broth ofS. chromofuscusATCC 49982 as a minor metabolite.

scholarly journals Aspergillus fumigatus SidJ Mediates Intracellular Siderophore Hydrolysis

2013 ◽  
Vol 79 (23) ◽  
pp. 7534-7536 ◽  
Author(s):  
Mario Gründlinger ◽  
Fabio Gsaller ◽  
Markus Schrettl ◽  
Herbert Lindner ◽  
Hubertus Haas
Keyword(s):  
Aspergillus Fumigatus ◽  
Gene Cluster ◽  
Biosynthetic Gene Cluster ◽  
Protein Family ◽  
Biosynthetic Gene ◽  
Intracellular Accumulation ◽  
Iron Starvation ◽  
Content Type ◽  
Hydrolysis Products ◽  
Encoding Gene

ABSTRACTSiderophore-mediated iron handling is crucial for the virulence ofAspergillus fumigatus. Here we identified a new component of its siderophore metabolism, termed SidJ, which is encoded by AFUA_3G03390. The encoding gene is localized in a siderophore biosynthetic gene cluster that is conserved in a variety of fungi. During iron starvation, SidJ deficiency resulted in decreased growth and increased intracellular accumulation of hydrolysis products of the siderophore fusarinine C. The implied role in siderophore hydrolysis is consistent with a putative esterase domain in SidJ, which now represents the first functionally characterized member of the DUF1749 (domain ofunknownfunction) protein family, with members found exclusively in fungi and plants.

scholarly journals Alternative Biosynthetic Starter Units Enhance the Structural Diversity of Cyanobacterial Lipopeptides

2018 ◽  
Vol 85 (4) ◽  
Author(s):  
Jan Mareš ◽  
Jan Hájek ◽  
Petra Urajová ◽  
Andreja Kust ◽  
Jouni Jokela ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Gene Cluster ◽  
Plasma Membranes ◽  
Gene Clusters ◽  
Biosynthetic Gene Cluster ◽  
Published Data ◽  
Biosynthetic Gene ◽  
Structural Variants ◽  
Biosynthetic Gene Clusters ◽  
Content Type

ABSTRACT Puwainaphycins (PUWs) and minutissamides (MINs) are structurally analogous cyclic lipopeptides possessing cytotoxic activity. Both types of compound exhibit high structural variability, particularly in the fatty acid (FA) moiety. Although a biosynthetic gene cluster responsible for synthesis of several PUW variants has been proposed in a cyanobacterial strain, the genetic background for MINs remains unexplored. Herein, we report PUW/MIN biosynthetic gene clusters and structural variants from six cyanobacterial strains. Comparison of biosynthetic gene clusters indicates a common origin of the PUW/MIN hybrid nonribosomal peptide synthetase and polyketide synthase. Surprisingly, the biosynthetic gene clusters encode two alternative biosynthetic starter modules, and analysis of structural variants suggests that initiation by each of the starter modules results in lipopeptides of differing lengths and FA substitutions. Among additional modifications of the FA chain, chlorination of minutissamide D was explained by the presence of a putative halogenase gene in the PUW/MIN gene cluster of Anabaena minutissima strain UTEX B 1613. We detected PUW variants bearing an acetyl substitution in Symplocastrum muelleri strain NIVA-CYA 644, consistent with an O-acetyltransferase gene in its biosynthetic gene cluster. The major lipopeptide variants did not exhibit any significant antibacterial activity, and only the PUW F variant was moderately active against yeast, consistent with previously published data suggesting that PUWs/MINs interact preferentially with eukaryotic plasma membranes. IMPORTANCE Herein, we deciphered the most important biosynthetic traits of a prominent group of bioactive lipopeptides. We reveal evidence for initiation of biosynthesis by two alternative starter units hardwired directly in the same gene cluster, eventually resulting in the production of a remarkable range of lipopeptide variants. We identified several unusual tailoring genes potentially involved in modifying the fatty acid chain. Careful characterization of these biosynthetic gene clusters and their diverse products could provide important insight into lipopeptide biosynthesis in prokaryotes. Some of the variants identified exhibit cytotoxic and antifungal properties, and some are associated with a toxigenic biofilm-forming strain. The findings may prove valuable to researchers in the fields of natural product discovery and toxicology.

scholarly journals In Vivo Analysis of the Regulatory Genes in the Nystatin Biosynthetic Gene Cluster of Streptomyces noursei ATCC 11455 Reveals Their Differential Control Over Antibiotic Biosynthesis

2004 ◽  
Vol 186 (5) ◽  
pp. 1345-1354 ◽  
Author(s):  
Olga N. Sekurova ◽  
Trygve Brautaset ◽  
Håvard Sletta ◽  
Sven E. F. Borgos ◽  
Øyvind M. Jakobsen ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Biosynthetic Gene Cluster ◽  
Regulatory Genes ◽  
Antibiotic Biosynthesis ◽  
Biosynthetic Gene ◽  
Promoter Regions ◽  
Streptomyces Noursei ◽  
In Vivo Analysis ◽  
Differential Control

ABSTRACT Six putative regulatory genes are located at the flank of the nystatin biosynthetic gene cluster in Streptomyces noursei ATCC 11455. Gene inactivation and complementation experiments revealed that nysRI, nysRII, nysRIII, and nysRIV are necessary for efficient nystatin production, whereas no significant roles could be demonstrated for the other two regulatory genes. To determine the in vivo targets for the NysR regulators, chromosomal integration vectors with the xylE reporter gene under the control of seven putative promoter regions upstream of the nystatin structural and regulatory genes were constructed. Expression analyses of the resulting vectors in the S. noursei wild-type strain and regulatory mutants revealed that the four regulators differentially affect certain promoters. According to these analyses, genes responsible for initiation of nystatin biosynthesis and antibiotic transport were the major targets for regulation. Data from cross-complementation experiments showed that nysR genes could in some cases substitute for each other, suggesting a functional hierarchy of the regulators and implying a cascade-like mechanism of regulation of nystatin biosynthesis.

scholarly journals Improved molecular characterization of the Klebsiella oxytoca complex reveals the prevalence of the kleboxymycin biosynthetic gene cluster

2021 ◽  
Vol 7 (6) ◽  
Author(s):  
Preetha Shibu ◽  
Frazer McCuaig ◽  
Anne L. McCartney ◽  
Magdalena Kujawska ◽  
Lindsay J. Hall ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Type Species ◽  
Phylogenetic Analyses ◽  
Klebsiella Oxytoca ◽  
Biosynthetic Gene Cluster ◽  
Biosynthetic Gene ◽  
Genome Sequences ◽  
Content Type ◽  
Link Type

As part of the ongoing studies with clinically relevant Klebsiella spp., we characterized the genomes of three clinical GES-5-positive ST138 strains originally identified as Klebsiella oxytoca. bla OXY gene, average nucleotide identity and phylogenetic analyses showed the strains to be Klebsiella michiganensis . Affiliation of the strains to ST138 led us to demonstrate that the current multi-locus sequence typing scheme for K. oxytoca can be used to distinguish members of this genetically diverse complex of bacteria. The strains encoded the kleboxymycin biosynthetic gene cluster (BGC), previously only found in K. oxytoca strains and one strain of Klebsiella grimontii . The finding of this BGC, associated with antibiotic-associated haemorrhagic colitis, in K. michiganensis led us to carry out a wide-ranging study to determine the prevalence of this BGC in Klebsiella spp. Of 7170 publicly available Klebsiella genome sequences screened, 88 encoded the kleboxymycin BGC. All BGC-positive strains belonged to the K. oxytoca complex, with strains of four ( K. oxytoca , K. pasteurii , K. grimontii , K. michiganensis ) of the six species of complex found to encode the complete BGC. In addition to being found in K. grimontii strains isolated from preterm infants, the BGC was found in K. oxytoca and K. michiganensis metagenome-assembled genomes recovered from neonates. Detection of the kleboxymycin BGC across the K. oxytoca complex may be of clinical relevance and this cluster should be included in databases characterizing virulence factors, in addition to those characterizing BGCs.

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