scholarly journals The fungal gene cluster for biosynthesis of the antibacterial agent viriditoxin

2019 ◽  
Author(s):  
Andrew S. Urquhart ◽  
Jinyu Hu ◽  
Yit-Heng Chooi ◽  
Alexander Idnurm
Keyword(s):  
Secondary Metabolites ◽  
Gene Cluster ◽  
Gene Disruption ◽  
Biosynthetic Pathway ◽  
Fluorescent Protein ◽  
Model Species ◽  
Paecilomyces Variotii ◽  
Bioinformatic Approaches ◽  
Green Fluorescent ◽  
Fungal Gene

AbstractBackgroundViriditoxin is one of the ‘classical’ secondary metabolites produced by fungi and that has antibacterial and other activities; however, the mechanism of its biosynthesis has remained unknown.ResultsHere, a gene cluster responsible for its synthesis was identified, using bioinformatic approaches from two species that produce viriditoxin and then through gene disruption and metabolite profiling. All eight genes in the cluster inPaecilomyces variotiiwere mutated, revealing their roles in the synthesis of this molecule and establishing its biosynthetic pathway which includes an interesting Baeyer-Villiger monooxygenase catalyzed reaction. Additionally, a candidate catalytically-inactive hydrolase was identified as being required for the stereoselective biosynthesis of (M)-viriditoxin. The localization of two proteins were assessed by fusing these proteins to green fluorescent protein, revealing that at least two intracellular structures are involved in the compartmentalization of the synthesis steps of this metabolite.ConclusionsThe full pathway for synthesis of viriditoxin was established by a combination of genomics, bioinformatics, gene disruption and chemical analysis processes. Hence, this work reveals the basis for the synthesis of an understudied class of fungal secondary metabolites and provides a new model species for understanding the synthesis of biaryl compounds with a chiral axis.

scholarly journals Biosynthesis of Fusarubins Accounts for Pigmentation of Fusarium fujikuroi Perithecia

2012 ◽  
Vol 78 (12) ◽  
pp. 4468-4480 ◽  
Author(s):  
Lena Studt ◽  
Philipp Wiemann ◽  
Karin Kleigrewe ◽  
Hans-Ulrich Humpf ◽  
Bettina Tudzynski
Keyword(s):  
Secondary Metabolites ◽  
Gene Cluster ◽  
Biosynthetic Pathway ◽  
Polyketide Synthase ◽  
Biological Function ◽  
Fusarium Fujikuroi ◽  
Diverse Group ◽  
Fruiting Bodies ◽  
Content Type ◽  
Encoding Gene

ABSTRACTFusarium fujikuroiproduces a variety of secondary metabolites, of which polyketides form the most diverse group. Among these are the highly pigmented naphthoquinones, which have been shown to possess different functional properties for the fungus. A group of naphthoquinones, polyketides related to fusarubin, were identified inFusariumspp. more than 60 years ago, but neither the genes responsible for their formation nor their biological function has been discovered to date. In addition, although it is known that the sexual fruiting bodies in which the progeny of the fungus develops are darkly colored by a polyketide synthase (PKS)-derived pigment, the structure of this pigment has never been elucidated. Here we present data that link the fusarubin-type polyketides to a defined gene cluster, which we designatefsr, and demonstrate that the fusarubins are the pigments responsible for the coloration of the perithecia. We studied their regulation and the function of the single genes within the cluster by a combination of gene replacements and overexpression of the PKS-encoding gene, and we present a model for the biosynthetic pathway of the fusarubins based on these data.

scholarly journals A new vector system for targeted integration and overexpression of genes in the crop pathogen Fusarium solani

2019 ◽  
Vol 6 (1) ◽  
Author(s):  
Mikkel Rank Nielsen ◽  
Anna Karolina Rilana Holzwarth ◽  
Emmett Brew ◽  
Natalia Chrapkova ◽  
Samba Evelyne Kabemba Kaniki ◽  
...  
Keyword(s):  
Secondary Metabolites ◽  
Gene Cluster ◽  
Fusarium Solani ◽  
Species Complex ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Vector System ◽  
Fungal Transformation ◽  
First Case

Abstract Background Besides their ability to produce several interesting bioactive secondary metabolites, members of the Fusarium solani species complex comprise important pathogens of plants and humans. One of the major obstacles in understanding the biology of this species complex is the lack of efficient molecular tools for genetic manipulation. Results To remove this obstacle we here report the development of a reliable system where the vectors are generated through yeast recombinational cloning and inserted into a specific site in F. solani through Agrobacterium tumefaciens-mediated transformation. As proof-of-concept, the enhanced yellow fluorescent protein (eYFP) was inserted in a non-coding genomic position of F. solani and subsequent analyses showed that the resulting transformants were fluorescent on all tested media. In addition, we cloned and overexpressed the Zn(II)2Cys6 transcriptional factor fsr6 controlling mycelial pigmentation. A transformant displayed deep red/purple pigmentation stemming from bostrycoidin and javanicin. Conclusion By creating streamlined plasmid construction and fungal transformation systems, we are now able to express genes in the crop pathogen F. solani in a reliable and fast manner. As a case study, we targeted and activated the fusarubin (PKS3: fsr) gene cluster, which is the first case study of secondary metabolites being directly associated with the responsible gene cluster in F. solani via targeted activation. The system provides an approach that in the future can be used by the community to understand the biochemistry and genetics of the Fusarium solani species complex, and is obtainable from Addgene catalog #133094. Graphic abstract

scholarly journals Comparison of CRISPR–Cas9 Tools for Transcriptional Repression and Gene Disruption in the BEVS

Viruses ◽  
2021 ◽  
Vol 13 (10) ◽  
pp. 1925
Author(s):  
Mark R. Bruder ◽  
Sadru-Dean Walji ◽  
Marc G. Aucoin
Keyword(s):  
Transcriptional Repression ◽  
Gene Disruption ◽  
Protein Gene ◽  
Fluorescent Protein ◽  
Green Fluorescent Protein Gene ◽  
Proof Of Concept ◽  
Knock Out ◽  
Targeted Gene Disruption ◽  
Green Fluorescent ◽  
Affect Gene Expression

The generation of knock-out viruses using recombineering of bacmids has greatly accelerated scrutiny of baculovirus genes for a variety of applications. However, the CRISPR–Cas9 system is a powerful tool that simplifies sequence-specific genome editing and effective transcriptional regulation of genes compared to traditional recombineering and RNAi approaches. Here, the effectiveness of the CRISPR–Cas9 system for gene disruption and transcriptional repression in the BEVS was compared. Cell lines constitutively expressing the cas9 or dcas9 gene were developed, and recombinant baculoviruses delivering the sgRNA were evaluated for disruption or repression of a reporter green fluorescent protein gene. Finally, endogenous AcMNPV genes were targeted for disruption or downregulation to affect gene expression and baculovirus replication. This study provides a proof-of-concept that CRISPR–Cas9 technology may be an effective tool for efficient scrutiny of baculovirus genes through targeted gene disruption and transcriptional repression.

scholarly journals PRELI (protein of relevant evolutionary and lymphoid interest) is located within an evolutionarily conserved gene cluster on chromosome 5q34-q35 and encodes a novel mitochondrial protein

2004 ◽  
Vol 378 (3) ◽  
pp. 817-825 ◽  
Author(s):  
Elizabeth J. FOX ◽  
Sally A. STUBBS ◽  
Jimmy KYAW TUN ◽  
Jack P. LEEK ◽  
Alexander F. MARKHAM ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Fluorescent Protein ◽  
Mitochondrial Protein ◽  
Subcellular Location ◽  
Protein Fusion ◽  
Evolutionarily Conserved ◽  
N Terminus ◽  
Targeting Signal ◽  
Conserved Gene ◽  
Green Fluorescent

The characterization of mitochondrial proteins is important for the understanding of both normal cellular function and mitochondrial disease. In the present study we identify a novel mitochondrial protein, PRELI (protein of relevant evolutionary and lymphoid interest), that is encoded within the evolutionarily conserved MAD3/PRELI/RAB24 gene cluster located at chromosome 5q34–q35. Mouse Preli is expressed at high levels in all settings analysed; it is co-expressed with Rab24 from a strong bi-directional promoter, and is regulated independently from the S-phase-specific Mad3 gene located at its 3´ end. PRELI contains a stand-alone 170 amino acid PRELI/MSF1p´ motif at its N-terminus. This domain is found in a variety of proteins from diverse eukaryotes including yeast, Drosophila and mammals, but its function is unknown, and the subcellular location of higher eukaryotic PRELI/MSF1P´ proteins has not been determined previously. We show here that PRELI is located in the mitochondria, and by using green-fluorescent-protein fusion proteins we identify a mitochondrial targeting signal at its N-terminus.

scholarly journals Secondary Metabolites Produced by the Marine Bacterium Halobacillus salinus That Inhibit Quorum Sensing-Controlled Phenotypes in Gram-Negative Bacteria

2008 ◽  
Vol 75 (3) ◽  
pp. 567-572 ◽  
Author(s):  
Margaret E. Teasdale ◽  
Jiayuan Liu ◽  
Joselynn Wallace ◽  
Fatemeh Akhlaghi ◽  
David C. Rowley
Keyword(s):  
Quorum Sensing ◽  
Secondary Metabolites ◽  
Fluorescent Protein ◽  
Phenotypic Expression ◽  
Antibiotic Biosynthesis ◽  
Gram Negative ◽  
Bacterial Quorum Sensing ◽  
Bioassay Guided Fractionation ◽  
Bacterial Quorum ◽  
Green Fluorescent

ABSTRACT Certain bacteria use cell-to-cell chemical communication to coordinate community-wide phenotypic expression, including swarming motility, antibiotic biosynthesis, and biofilm production. Here we present a marine gram-positive bacterium that secretes secondary metabolites capable of quenching quorum sensing-controlled behaviors in several gram-negative reporter strains. Isolate C42, a Halobacillus salinus strain obtained from a sea grass sample, inhibits bioluminescence production by Vibrio harveyi in cocultivation experiments. With the use of bioassay-guided fractionation, two phenethylamide metabolites were identified as the active agents. The compounds additionally inhibit quorum sensing-regulated violacein biosynthesis by Chromobacterium violaceum CV026 and green fluorescent protein production by Escherichia coli JB525. Bacterial growth was unaffected at concentrations below 200 μg/ml. Evidence is presented that these nontoxic metabolites may act as antagonists of bacterial quorum sensing by competing with N-acyl homoserine lactones for receptor binding.

scholarly journals Improved pyrrolysine biosynthesis through continuous directed evolution of the complete pathway

2020 ◽  
Author(s):  
Joanne Ho ◽  
Corwin Miller ◽  
Jacob Mattia ◽  
Matthew Bennett
Keyword(s):  
Escherichia Coli ◽  
Green Fluorescent Protein ◽  
Directed Evolution ◽  
Biosynthetic Pathway ◽  
Fluorescent Protein ◽  
Proteinogenic Amino Acid ◽  
E Coli ◽  
Rational Engineering ◽  
Fundamental Building Block ◽  
Green Fluorescent

Abstract Pyrrolysine (Pyl, O) exists in nature as the 22nd proteinogenic amino acid. Despite being a fundamental building block of proteins, studies of Pyl have been hindered by the difficulty and inefficiency of both its chemical and biological syntheses. Here, we improved Pyl biosynthesis via rational engineering and directed evolution of the entire biosynthetic pathway. To accommodate toxicity of Pyl biosynthetic genes in Escherichia coli, we devised an approach termed Alternating Phage Assisted Non-Continuous Evolution (Alt-PANCE) that alternates mutagenic and selective phage growths. The evolved pathway exhibited a 32-fold improved yield of Pyl-containing super- folder green fluorescent protein (sfGFP) compared to the rationally engineered ancestor, whereas the WT pathway produced no detectable quantities of Pyl-containing sfGFP. This study demonstrates that Alt-PANCE provides a general approach for evolving proteins exhibiting toxic side effects, and further provides an improved pathway capable of producing substantially greater quantities of Pyl- proteins in E. coli.

scholarly journals The Yersiniabactin Biosynthetic Gene Cluster of Yersinia enterocolitica: Organization and Siderophore-Dependent Regulation

1998 ◽  
Vol 180 (3) ◽  
pp. 538-546 ◽  
Author(s):  
C. Pelludat ◽  
A. Rakin ◽  
C. A. Jacobi ◽  
S. Schubert ◽  
J. Heesemann
Keyword(s):  
Gene Cluster ◽  
Polyketide Synthase ◽  
Fluorescent Protein ◽  
Vibrio Anguillarum ◽  
High Specificity ◽  
Biosynthetic Gene Cluster ◽  
Cross Reactivity ◽  
Biosynthetic Gene ◽  
Chromosomal Dna ◽  
Green Fluorescent

ABSTRACT The ability to synthesize and uptake the Yersiniasiderophore yersiniabactin is a hallmark of the highly pathogenic, mouse-lethal species Yersinia pestis, Y. pseudotuberculosis, and Y. enterocolitica 1B. We have identified four genes, irp1, irp3,irp4, and irp5, on a 13-kb chromosomal DNA fragment of Y. enterocolitica O8, WA-314. These genes constitute the yersiniabactin biosynthetic gene cluster together with the previously defined irp2. The irp1 gene consists of 9,486 bp capable of encoding a 3,161-amino-acid high-molecular-weight protein 1 (HMWP1) polypeptide with a predicted mass of 384.6 kDa. The first 3,000 bp of irp1 show similarity to the corresponding regions of the polyketide synthase genes of Bacillus subtilis and Streptomyces antibioticus. The remaining part of irp1 is most similar to irp2, encoding HMWP2, which might be the reason for immunological cross-reactivity of the two polypeptides. Irp4 was found to have 41.7% similarity to thioesterase-like protein of the anguibactin biosynthetic genes of Vibrio anguillarum. Irp5 shows 41% similarity to EntE, the 2,3-dihydroxybenzoic acid-activating enzyme utilized in enterobactin synthesis of Escherichia coli. Irp4 and Irp5 are nearly identical to YbtT and YbtE, recently identified in Y. pestis. irp3 has no similarity to any known gene. Inactivation of either irp1 orirp2 abrogates yersiniabactin synthesis. Mutations inirp1 or fyuA (encoding yersiniabactin/pesticin receptor) result in downregulation of irp2 that can be upregulated by the addition of yersiniabactin. A FyuA-green fluorescent protein translational fusion was downregulated in an irp1mutant. Upregulation was achieved by addition of yersiniabactin but not desferal, pesticin, or pyochelin, which indicates high specificity of the FyuA receptor and autoregulation of genes involved in synthesis and uptake of yersiniabactin.

scholarly journals Targeted gene disruption reveals a role for vacuolin B in the late endocytic pathway and exocytosis

1998 ◽  
Vol 111 (1) ◽  
pp. 61-70 ◽  
Author(s):  
N. Jenne ◽  
R. Rauchenberger ◽  
U. Hacker ◽  
T. Kast ◽  
M. Maniak
Keyword(s):  
Gene Disruption ◽  
Fluorescent Protein ◽  
Fluid Phase ◽  
Endocytic Pathway ◽  
Knock Out ◽  
Targeted Gene Disruption ◽  
Green Fluorescent ◽  
Cytoplasmic Side ◽  
Dependent Mechanism

Cells of Dictyostelium discoideum take up fluid by macropinocytosis. The contents of macropinosomes are acidified and digested by lysosomal enzymes. Thereafter, an endocytic marker progresses in an F-actin dependent mechanism from the acidic lysosomal phase to a neutral post-lysosomal phase. From the post-lysosomal compartment indigestible remnants are released by exocytosis. This compartment is characterised by two isoforms of vacuolin, A and B, which are encoded by different genes. Fusions of the vacuolin isoforms to the green fluorescent protein associate with the cytoplasmic side of post-lysosomal vacuoles in vivo. Vacuolin isoforms also localise to patches at the plasma membrane. Since vacuolins have no homologies to known proteins and do not contain domains of obvious function, we investigated their role by knocking out the genes separately. Although the sequences of vacuolins A and B are about 80% identical, only deletion of the vacuolin B gene results in a defect in the endocytic pathway; the vacuolin A knock-out appeared to be phenotypically normal. In vacuolin B- mutants endocytosis is normal, but the progression of fluid-phase marker from acidic to neutral pH is impaired. Furthermore, in the mutants post-lysosomal vacuoles are dramatically increased in size and accumulate endocytic marker, suggesting a role for vacuolin B in targeting the vacuole for exocytosis.

scholarly journals Detection of a Gene Cluster That Is Dispensable for Human Herpesvirus 6 Replication and Latency

2003 ◽  
Vol 77 (19) ◽  
pp. 10719-10724 ◽  
Author(s):  
Kazuhiro Kondo ◽  
Hideo Nozaki ◽  
Kazuya Shimada ◽  
Koichi Yamanishi
Keyword(s):  
Gene Cluster ◽  
Fluorescent Protein ◽  
Human Herpesvirus ◽  
Gene Cassette ◽  
Human Herpesvirus 6 ◽  
Transcription Start ◽  
Transcription Start Sites ◽  
Herpesvirus 6 ◽  
Green Fluorescent ◽  
Major Immediate Early Promoter

ABSTRACT The U3-U7 gene cluster of human herpesvirus 6 (HHV-6) was replaced with an enhanced green fluorescent protein-puromycin gene cassette containing the cytomegalovirus major immediate-early promoter. Neither viral replication in T cells nor latency and reactivation in macrophages was impaired. During HHV-6 latency, the cytomegalovirus promoter used the transcription start sites employed in cytomegalovirus latency.

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