Molecular Biological Studies on the Aflatoxin Biosynthetic Homologous Gene Cluster of Aspergillus oryzae

The biosynthetic gene cluster of fungal bioactive sesterterpenoids, variecolin (1) and variecolactone (2), was identified in Aspergillus aculeatus ATCC 16872. Heterologous production of 1 and 2 was achieved in Aspergillus oryzae by expressing the sesterterpene synthase VrcA and the cytochrome P450 VrcB. Intriguingly, the replacement of VrcB with homologous P450s from other fungal terpenoid pathways yielded three new variecolin analogues, one of which exhibited potent anticancer activity comparable to that of 1.

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Efficient Reconstitution of Basidiomycota Diterpene Erinacine Gene Cluster in Ascomycota Host Aspergillus oryzae Based on Genomic DNA Sequences

Journal of the American Chemical Society ◽

10.1021/jacs.9b08935 ◽

2019 ◽

Vol 141 (39) ◽

pp. 15519-15523 ◽

Cited By ~ 6

Author(s):

Chengwei Liu ◽

Atsushi Minami ◽

Taro Ozaki ◽

Jing Wu ◽

Hirokazu Kawagishi ◽

...

Keyword(s):

Gene Cluster ◽

Aspergillus Oryzae ◽

Dna Sequences ◽

Genomic Dna

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Identification of the Monooxygenase Gene Clusters Responsible for the Regioselective Oxidation of Phenol to Hydroquinone in Mycobacteria

Applied and Environmental Microbiology ◽

10.1128/aem.02316-10 ◽

2010 ◽

Vol 77 (4) ◽

pp. 1214-1220 ◽

Cited By ~ 21

Author(s):

Toshiki Furuya ◽

Satomi Hirose ◽

Hisashi Osanai ◽

Hisashi Semba ◽

Kuniki Kino

Keyword(s):

Gene Cluster ◽

Large Subunit ◽

Gene Clusters ◽

Small Subunit ◽

Homologous Gene ◽

Oxidation Activity ◽

Monooxygenase Gene ◽

Regioselective Oxidation ◽

Binuclear Iron ◽

Induced Proteins

ABSTRACTMycobacterium goodiistrain 12523 is an actinomycete that is able to oxidize phenol regioselectively at theparaposition to produce hydroquinone. In this study, we investigated the genes responsible for this unique regioselective oxidation. On the basis of the fact that the oxidation activity ofM. goodiistrain 12523 toward phenol is induced in the presence of acetone, we first identified acetone-induced proteins in this microorganism by two-dimensional electrophoretic analysis. The N-terminal amino acid sequence of one of these acetone-induced proteins shares 100% identity with that of the protein encoded by the open reading frame Msmeg_1971 inMycobacterium smegmatisstrain mc2155, whose genome sequence has been determined. Since Msmeg_1971, Msmeg_1972, Msmeg_1973, and Msmeg_1974 constitute a putative binuclear iron monooxygenase gene cluster, we cloned this gene cluster ofM. smegmatisstrain mc2155 and its homologous gene cluster found inM. goodiistrain 12523. Sequence analysis of these binuclear iron monooxygenase gene clusters revealed the presence of four genes designatedmimABCD, which encode an oxygenase large subunit, a reductase, an oxygenase small subunit, and a coupling protein, respectively. When themimAgene (Msmeg_1971) ofM. smegmatisstrain mc2155, which was also found to be able to oxidize phenol to hydroquinone, was deleted, this mutant lost the oxidation ability. This ability was restored by introduction of themimAgene ofM. smegmatisstrain mc2155 or ofM. goodiistrain 12523 into this mutant. Interestingly, we found that these gene clusters also play essential roles in propane and acetone metabolism in these mycobacteria.

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Two Different Lantibiotic-Like Peptides Originate from the Ericin Gene Cluster of Bacillus subtilis A1/3

Journal of Bacteriology ◽

10.1128/jb.184.6.1703-1711.2002 ◽

2002 ◽

Vol 184 (6) ◽

pp. 1703-1711 ◽

Cited By ~ 90

Author(s):

Torsten Stein ◽

Stefan Borchert ◽

Birgit Conrad ◽

Jörg Feesche ◽

Brigitte Hofemeister ◽

...

Keyword(s):

Bacillus Subtilis ◽

Gene Cluster ◽

High Performance ◽

Genetic Locus ◽

Reversed Phase ◽

Homologous Gene ◽

Dependent Manner ◽

Maldi Tof Ms ◽

Maldi Tof ◽

Tof Ms

ABSTRACT A lantibiotic gene cluster was identified in Bacillus subtilis A1/3 showing a high degree of homology to the subtilin gene cluster and occupying the same genetic locus as the spa genes in B. subtilis ATCC 6633. The gene cluster exhibits diversity with respect to duplication of two subtilin-like genes which are separated by a sequence similar to a portion of a lanC gene. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) analyses of B. subtilis A1/3 culture extracts confirmed the presence of two lantibiotic-like peptides, ericin S (3,442 Da) and ericin A (2,986 Da). Disruption of the lanB-homologous gene eriB resulted in loss of production of both peptides, demonstrating that they are processed in an eriB-dependent manner. Although precursors of ericins S and A show only 75% of identity, the matured lantibiotic-like peptides reveal highly similar physical properties; separation was only achieved after multistep, reversed-phase high-performance liquid chromatography. Based on Edman and peptidase degradation in combination with MALDI-TOF MS, for ericin S a subtilin-like, lanthionine-bridging pattern is supposed. For ericin A two C-terminal rings are different from the lanthionine pattern of subtilin. Due to only four amino acid exchanges, ericin S and subtilin revealed similar antibiotic activities as well as similar properties in response to heat and protease treatment. For ericin A only minor antibiotic activity was found.

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Density-based binning of gene clusters to infer function or evolutionary history using GeneGrouper

10.1101/2021.05.27.446007 ◽

2021 ◽

Author(s):

Alexander G McFarland ◽

Nolan W Kennedy ◽

Carolyn E Mills ◽

Danielle Tullman-Ercek ◽

Curtis Huttenhower ◽

...

Keyword(s):

Gene Cluster ◽

Population Level ◽

Gene Clusters ◽

Homologous Gene ◽

Integrative And Conjugative Elements ◽

Density Based Clustering ◽

Command Line Tool ◽

Conserved Gene ◽

Similar Gene

Motivation: Identifying gene clusters of interest in phylogenetically proximate and distant taxa can help to infer phenotypes of interest. Conserved gene clusters may differ by only a few genes, which can be biologically meaningful, such as the formation of pseudogenes or insertions interrupting regulation. These qualities may allow for unsupervised clustering of similar gene clusters into bins that provide a population-level understanding of the genetic variation in similar gene clusters. Results: We developed GeneGrouper, a command-line tool that uses a density-based clustering method to group gene clusters into bins. GeneGrouper demonstrated high recall and precision in benchmarks for the detection of the 23-gene Salmonella enterica LT2 Pdu gene cluster and four-gene Pseudomonas aeruginosa PAO1 Mex gene cluster in 435 genomes containing mixed taxa. In a subsequent application investigating the diversity and impact of gene complete and incomplete LT2 Pdu gene clusters in 1130 S. enterica genomes, GeneGrouper identified a novel, frequently occurring pduN pseudogene. When replicated in vivo, disruption of pduN with a frameshift mutation negatively impacted microcompartment formation. We next demonstrated the versatility of GeneGrouper by clustering both distant homologous gene clusters and variable gene clusters found in integrative and conjugative elements.

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Identification of trans-AT polyketide clusters in two marine bacteria reveals cryptic similarities between distinct symbiosis factors

10.1101/2020.09.18.303172 ◽

2020 ◽

Author(s):

Dina Kačar ◽

Librada M Cañedo ◽

Pilar Rodríguez ◽

Elena Gonzalez ◽

Beatriz Galán ◽

...

Keyword(s):

Gene Cluster ◽

Marine Bacteria ◽

Genome Mining ◽

Evolutionary Relationship ◽

Gene Clusters ◽

Comparative Genomic ◽

Homologous Gene ◽

Modular Architecture ◽

Orphan Gene ◽

Strong Antitumor Activity

AbstractGlutaramide-containing polyketides are known as potent antitumoral and antimetastatic agents. However, the associated gene clusters have only been identified and studied in a few Streptomyces producers and sole Burkholderia gladioli symbiont. The new glutaramide-family polyketides, denominated sesbanimides D, E and F along with the previously known sesbanimide A and C, were isolated from two marine alphaproteobacteria Stappia indica PHM037 and Labrenzia aggregata PHM038. Structures of the isolated compounds were elucidated based on 1D and 2D homo and heteronuclear NMR analyses and ESI-MS spectrometry. All compounds exhibited strong antitumor activity in lung, breast and colorectal cancer cell lines. Subsequent whole genome sequencing and genome mining revealed the presence of the trans-AT PKS gene cluster responsible for the sesbanimide biosynthesis, described as sbn cluster, and the sesbanimide modular assembly is proposed. Interestingly, numerous homologous orphan gene clusters were localized in distantly related bacteria and used as comparative genomic assets for a more global characterization of sbn like-clusters. Strikingly, the modular architecture of downstream mixed type PKS/NRPS, SbnQ, revealed high similarity to PedH in pederin and Lab13 in labrenzin gene clusters, although those clusters are responsible for the production of structurally completely different molecules. The unexpected presence of SbnQ homologs in unrelated polyketide gene clusters across phylogenetically distant bacteria, raises intriguing questions about the evolutionary relationship between glutaramide-like and pederin-like pathways, as well as the functionality of their synthetic products.SignificanceGlutaramide-containing polyketides are still a largely understudied group of polyketides, produced mainly by the genera Streptomyces, with a great potential for antitumor drug production. Here, we describe genomes of two cultivable marine bacteria, Stappia indica PHM037 and Labrenzia aggregata PHM038, producers of the cytotoxic glutaramide-family polyketides sesbanimide A and C with chemical elucidation of newly identified analogs D, E and F. Genome mining revealed trans-AT PKS gene cluster responsible for sesbanimide biosynthesis. Although there are numerous homologous gene clusters present in remarkably different bacteria, this is the first time that the biosynthesis product has been reported. The comparative genome analysis reveals stunning, cryptic evolutionary relationship between sesbanimides, glutaramides from Streptomyces spp. and the pederin-family gene clusters.

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Global distribution of anaerobic dichloromethane degradation potential

10.1101/2021.08.30.458270 ◽

2021 ◽

Author(s):

Robert W. Murdoch ◽

Gao Chen ◽

Fadime Kara Murdoch ◽

E. Erin Mack ◽

Manuel I. Villalobos Solis ◽

...

Keyword(s):

Gene Cluster ◽

Methylene Chloride ◽

Control Function ◽

Stratospheric Ozone ◽

Bacterial Species ◽

Gene Clusters ◽

Gene Cassette ◽

Anaerobic Growth ◽

Diagnostic Tools ◽

Homologous Gene

AbstractAnthropogenic activities and natural processes release dichloromethane (DCM), a toxic chemical with substantial ozone-depleting capacity. Specialized anaerobic bacteria metabolize DCM; however, the genetic basis for this process has remained elusive. Comparative genomics of the three known anaerobic DCM-degrading bacterial species revealed a homologous gene cluster, designated the methylene chloride catabolism (mec) gene cassette, comprising eight to ten genes with predicted 79.6 – 99.7% amino acid identity. Functional annotation identified genes encoding a corrinoid-dependent methyltransferase system, and shotgun proteomics applied to two DCM-catabolizing cultures revealed high expression of proteins encoded on the mec gene cluster during anaerobic growth with DCM. In a DCM-contaminated groundwater plume, the abundance of mec genes strongly correlated with DCM concentrations (R2 = 0.71 – 0.85) indicating their value as process-specific bioremediation biomarkers. mec gene clusters were identified in metagenomes representing peat bogs, the deep subsurface, and marine ecosystems including oxygen minimum zones (OMZs), suggesting DCM turnover in diverse habitats. The broad distribution of anaerobic DCM catabolic potential suggests a relevant control function for emissions to the atmosphere, and a role for DCM as a microbial energy source in critical zone environments. The findings imply that the global DCM flux might be far greater than emission measurements suggest.ImportanceDichloromethane (DCM) is an increasing threat to stratospheric ozone with both anthropogenic and natural emission sources. Anaerobic bacterial metabolism of DCM has not yet been taken into consideration as a factor in the global DCM cycle. The discovery of the mec gene cassette associated with anaerobic bacterial DCM metabolism and its widespread distribution in environmental systems highlight a strong attenuation potential for DCM. Knowledge of the mec cassette offers new opportunities to delineate DCM sources, enables more robust estimates of DCM fluxes, supports refined DCM emission modeling and simulation of the stratospheric ozone layer, reveals a novel, ubiquitous C1 carbon metabolic system, and provides prognostic and diagnostic tools supporting bioremediation of groundwater aquifers impacted by DCM.

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The ADEP Biosynthetic Gene Cluster in Streptomyces hawaiiensis NRRL 15010 Reveals an Accessory clpP Gene as a Novel Antibiotic Resistance Factor

Applied and Environmental Microbiology ◽

10.1128/aem.01292-19 ◽

2019 ◽

Vol 85 (20) ◽

Cited By ~ 6

Author(s):

Dhana Thomy ◽

Elizabeth Culp ◽

Martina Adamek ◽

Eric Y. Cheng ◽

Nadine Ziemert ◽

...

Keyword(s):

Antibiotic Resistance ◽

Gene Cluster ◽

Natural Compound ◽

Biosynthetic Gene Cluster ◽

Resistance Factor ◽

Homologous Gene ◽

Biosynthetic Gene ◽

Content Type ◽

New Class ◽

Caseinolytic Protease

ABSTRACT The increasing threat posed by multiresistant bacterial pathogens necessitates the discovery of novel antibacterials with unprecedented modes of action. ADEP1, a natural compound produced by Streptomyces hawaiiensis NRRL 15010, is the prototype for a new class of acyldepsipeptide (ADEP) antibiotics. ADEP antibiotics deregulate the proteolytic core ClpP of the bacterial caseinolytic protease, thereby exhibiting potent antibacterial activity against Gram-positive bacteria, including multiresistant pathogens. ADEP1 and derivatives, here collectively called ADEP, have been previously investigated for their antibiotic potency against different species, structure-activity relationship, and mechanism of action; however, knowledge on the biosynthesis of the natural compound and producer self-resistance have remained elusive. In this study, we identified and analyzed the ADEP biosynthetic gene cluster in S. hawaiiensis NRRL 15010, which comprises two NRPSs, genes necessary for the biosynthesis of (4S,2R)-4-methylproline, and a type II polyketide synthase (PKS) for the assembly of highly reduced polyenes. While no resistance factor could be identified within the gene cluster itself, we discovered an additional clpP homologous gene (named clpPADEP) located further downstream of the biosynthetic genes, separated from the biosynthetic gene cluster by several transposable elements. Heterologous expression of ClpPADEP in three ADEP-sensitive Streptomyces species proved its role in conferring ADEP resistance, thereby revealing a novel type of antibiotic resistance determinant. IMPORTANCE Antibiotic acyldepsipeptides (ADEPs) represent a promising new class of potent antibiotics and, at the same time, are valuable tools to study the molecular functioning of their target, ClpP, the proteolytic core of the bacterial caseinolytic protease. Here, we present a straightforward purification procedure for ADEP1 that yields substantial amounts of the pure compound in a time- and cost-efficient manner, which is a prerequisite to conveniently study the antimicrobial effects of ADEP and the operating mode of bacterial ClpP machineries in diverse bacteria. Identification and characterization of the ADEP biosynthetic gene cluster in Streptomyces hawaiiensis NRRL 15010 enables future bioinformatics screenings for similar gene clusters and/or subclusters to find novel natural compounds with specific substructures. Most strikingly, we identified a cluster-associated clpP homolog (named clpPADEP) as an ADEP resistance gene. ClpPADEP constitutes a novel bacterial resistance factor that alone is necessary and sufficient to confer high-level ADEP resistance to Streptomyces across species.

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