scholarly journals Function of the cypX and moxY Genes in Aflatoxin Biosynthesis in Aspergillus parasiticus

2005 ◽  
Vol 71 (6) ◽  
pp. 3192-3198 ◽  
Author(s):  
Ying Wen ◽  
Hidemi Hatabayashi ◽  
Hatsue Arai ◽  
Hiroko K. Kitamoto ◽  
Kimiko Yabe
Keyword(s):  
Gene Cluster ◽  
Rapid Method ◽  
Aspergillus Parasiticus ◽  
Cell Disruption ◽  
Aflatoxin Biosynthesis ◽  
Feeding Experiments ◽  
Phenol Extraction ◽  
Versiconal Hemiacetal Acetate

ABSTRACT The pathway oxoaverantin (OAVN) → averufin (AVR) → hydroxyversicolorone (HVN) → versiconal hemiacetal acetate (VHA) is involved in aflatoxin biosynthesis, and the cypX and moxY genes, which are present in the aflatoxin gene cluster, have been previously suggested to be involved in this pathway. To clarify the function of these two genes in more detail, we disrupted the genes in aflatoxigenic Aspergillus parasiticus NRRL 2999. The cypX-deleted mutant lost aflatoxin productivity and accumulated AVR in the mycelia. Although this mutant converted HVN, versicolorone (VONE), VHA, and versiconol acetate (VOAc) to aflatoxins in feeding experiments, it could not produce aflatoxins from either OAVN or AVR. The moxY-deleted mutant also lost aflatoxin productivity, whereas it newly accumulated HVN and VONE. In feeding experiments, this mutant converted either VHA or VOAc to aflatoxins but did not convert OAVN, AVR, HVN, or VONE to aflatoxins. These results demonstrated that cypX encodes AVR monooxygenase, catalyzing the reaction from AVR to HVN, and moxY encodes HVN monooxygenase, catalyzing a Baeyer-Villiger reaction from HVN to VHA as well as from VONE to VOAc. In this work, we devised a simple and rapid method to extract DNA from many fungi for PCR analyses in which cell disruption with a shaker and phenol extraction were combined.

scholarly journals Enzymatic Conversion of Averufin to Hydroxyversicolorone and Elucidation of a Novel Metabolic Grid Involved in Aflatoxin Biosynthesis

2003 ◽  
Vol 69 (1) ◽  
pp. 66-73 ◽  
Author(s):  
Kimiko Yabe ◽  
Naomi Chihaya ◽  
Shioka Hamamatsu ◽  
Emi Sakuno ◽  
Takashi Hamasaki ◽  
...  
Keyword(s):  
Aspergillus Parasiticus ◽  
Cell Extract ◽  
Aflatoxin Biosynthesis ◽  
Cytosol Fraction ◽  
Feeding Experiments ◽  
A Cell ◽  
Molecular Masses ◽  
Microsome Fraction ◽  
Versicolorin A ◽  
Versiconal Hemiacetal Acetate

ABSTRACT The pathway from averufin (AVR) to versiconal hemiacetal acetate (VHA) in aflatoxin biosynthesis was investigated by using cell-free enzyme systems prepared from Aspergillus parasiticus. When (1′S,5′S)-AVR was incubated with a cell extract of this fungus in the presence of NADPH, versicolorin A and versicolorin B (VB), as well as other aflatoxin pathway intermediates, were formed. When the same substrate was incubated with the microsome fraction and NADPH, hydroxyversicolorone (HVN) and VHA were formed. However, (1′R,5′R)-AVR did not serve as the substrate. In cell-free experiments performed with the cytosol fraction and NADPH, VHA, versicolorone (VONE), and versiconol acetate (VOAc) were transiently produced from HVN in the early phase, and then VB and versiconol (VOH) accumulated later. Addition of dichlorvos (dimethyl 2,2-dichlorovinylphosphate) to the same reaction mixture caused transient formation of VHA and VONE, followed by accumulation of VOAc, but neither VB nor VOH was formed. When VONE was incubated with the cytosol fraction in the presence of NADPH, VOAc and VOH were newly formed, whereas the conversion of VOAc to VOH was inhibited by dichlorvos. The purified VHA reductase, which was previously reported to catalyze the reaction from VHA to VOAc, also catalyzed conversion of HVN to VONE. Separate feeding experiments performed with A. parasiticus NIAH-26 along with HVN, VONE, and versicolorol (VOROL) demonstrated that each of these substances could serve as a precursor of aflatoxins. Remarkably, we found that VONE and VOROL had ring-opened structures. Their molecular masses were 386 and 388 Da, respectively, which were 18 Da greater than the molecular masses previously reported. These data demonstrated that two kinds of reactions are involved in the pathway from AVR to VHA in aflatoxin biosynthesis: (i) a reaction from (1′S,5′S)-AVR to HVN, catalyzed by the microsomal enzyme, and (ii) a new metabolic grid, catalyzed by a new cytosol monooxygenase enzyme and the previously reported VHA reductase enzyme, composed of HVN, VONE, VOAc, and VHA. A novel hydrogenation-dehydrogenation reaction between VONE and VOROL was also discovered.

scholarly journals The Aspergillus parasiticus estA-Encoded Esterase Converts Versiconal Hemiacetal Acetate to Versiconal and Versiconol Acetate to Versiconol in Aflatoxin Biosynthesis

2004 ◽  
Vol 70 (6) ◽  
pp. 3593-3599 ◽  
Author(s):  
Perng-Kuang Chang ◽  
Kimiko Yabe ◽  
Jiujiang Yu
Keyword(s):  
Aspergillus Parasiticus ◽  
Enzyme Assays ◽  
Aflatoxin Biosynthesis ◽  
Cell Extracts ◽  
Esterase Gene ◽  
Versicolorin A ◽  
Versiconal Hemiacetal Acetate ◽  
Esterase Activities ◽  
Esterase Inhibitor

ABSTRACT In aflatoxin biosynthesis, the pathway for the conversion of 1-hydroxyversicolorone to versiconal hemiacetal acetate (VHA) to versiconal (VHOH) is part of a metabolic grid. In the grid, the steps from VHA to VHOH and from versiconol acetate (VOAc) to versiconol (VOH) may be catalyzed by the same esterase. Several esterase activities are associated with the conversion of VHA to VHOH, but only one esterase gene (estA) is present in the complete aflatoxin gene cluster of Aspergillus parasiticus. We deleted the estA gene from A. parasiticus SRRC 2043, an O-methylsterigmatocystin (OMST)-accumulating strain. The estA-deleted mutants were pigmented and accumulated mainly VHA and versicolorin A (VA). A small amount of VOAc and other downstream aflatoxin intermediates, including VHOH, versicolorin B, and OMST, also were accumulated. In contrast, a VA-accumulating mutant, NIAH-9, accumulated VA exclusively and neither VHA nor VOAc were produced. Addition of the esterase inhibitor dichlorvos (dimethyl 2,2-dichlorovinylphosphate) to the transformation recipient strain RHN1, an estA-deleted mutant, or NIAH-9 resulted in the accumulation of only VHA and VOAc. In in vitro enzyme assays, the levels of the esterase activities catalyzing the conversion of VHA to VHOH in the cell extracts of two estA-deleted mutants were decreased to approximately 10% of that seen with RHN1. Similar decreases in the esterase activities catalyzing the conversion of VOAc to VOH were also obtained. Thus, the estA-encoded esterase catalyzes the conversion of both VHA to VHOH and VOAc to VOH during aflatoxin biosynthesis.

Purification and characterization of the esterases involved in aflatoxin biosynthesis in Aspergillus parasiticus

1996 ◽  
Vol 42 (8) ◽  
pp. 804-810 ◽  
Author(s):  
Ken-Ichi Kusumoto ◽  
Dennis P. H. Hsieh
Keyword(s):  
Aspergillus Parasiticus ◽  
Specific Activity ◽  
Enzyme Stability ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Preparative Chromatography ◽  
Aflatoxin Biosynthesis ◽  
Anion Exchange Column ◽  
Versiconal Hemiacetal Acetate

The esterases from the cell-free extracts (CFEs) of Aspergillus parasiticus ATCC15517, an aflatoxin-producing strain, catalyzing the hydrolytic conversion of versiconal hemiacetal acetate (VHA) to versiconal was biochemically studied. The specific activity of the enzymes increased 2.5-fold during incubation of mycelia through 40–55 h. No metal ions were required for enzyme stability, but EDTA at 1 mM and dithiothreitol at 0.5–5 mM increased its stability. Three peaks of VHA esterase activity were resolved when the proteins in the CFEs prepared from the mycelia of different ages were separated by anion-exchange column chromatography, suggesting that at least three VHA esterases were present in the eluate of this purification step. One of these esterases extracted from the mycelia of a 55-h culture was partially purified in five steps by means of preparative chromatography and fast protein liquid chromatogaphy. The partially purified enzyme when reacted with [14C]diisopropylfluorophosphate followed by sodium dodecyl sulfate – polyacrylamide gel electrophoresis gave a single radiolabelled band, which corresponded to a protein of 32 kDa. The molecular mass of the partially purified VHA esterase determined with gel filtration was around 60 kDa. The results suggested that the enzyme consists of two isomeric subunits.Key words: aflatoxin biosynthesis, esterase, versiconal hemiacetal acetate, Aspergillus parasiticus.

Genomic deletions in Rhodococcus based on transformation of linear heterologous DNA

Microbiology ◽  
2021 ◽  
Author(s):  
Divya Singhi ◽  
Shabnam Parwin ◽  
Preeti Srivastava
Keyword(s):  
Rapid Method ◽  
Genome Engineering ◽  
Cost Effective ◽  
Cell Disruption ◽  
Content Type ◽  
First Report ◽  
Genomic Deletions ◽  
Linear Dna ◽  
Link Type ◽  
Successful Expression

Several genome engineering methods have been developed for Rhodococcus . However, they suffer from limitations such as extensive cloning, multiple steps, successful expression of heterologous genes via plasmid etc. Here, we report a rapid method for performing genomic deletions/disruptions in Rhodococcus spp. using heterologous linear DNA. The method is cost effective and less labour intensive. The applicability of the method was demonstrated by successful disruption of rodA and orphan parA. None of the disrupted genes were found to be essential for the viability of the cell. Disruption of orphan parA and rodA resulted in elongated cells and short rods, respectively. This is the first report demonstrating disruption of rodA and orphan parA genes by electroporation of heterologous linear DNA in Rhodococcus spp.

scholarly journals The effect of Propolis on inhibition of Aspergillus parasiticus growth, aflatoxin production and expression of aflatoxin biosynthesis pathway genes

2020 ◽  
Vol 18 (1) ◽  
pp. 297-302 ◽  
Author(s):  
Hamideh Mahmoodzadeh Hosseini ◽  
Siavash Hamzeh Pour ◽  
Jafar Amani ◽  
Sima Jabbarzadeh ◽  
Mostafa Hosseinabadi ◽  
...  
Keyword(s):  
Aspergillus Parasiticus ◽  
Aflatoxin Production ◽  
Biosynthesis Pathway ◽  
Aflatoxin Biosynthesis

scholarly journals Aspergillus Volatiles Regulate Aflatoxin Synthesis and Asexual Sporulation in Aspergillus parasiticus

2007 ◽  
Vol 73 (22) ◽  
pp. 7268-7276 ◽  
Author(s):  
Ludmila V. Roze ◽  
Randolph M. Beaudry ◽  
Anna E. Arthur ◽  
Ana M. Calvo ◽  
John E. Linz
Keyword(s):  
Aspergillus Parasiticus ◽  
Primary Source ◽  
Aflatoxin Contamination ◽  
Aflatoxin Biosynthesis ◽  
Transcript Accumulation ◽  
Potential Health ◽  
Asexual Sporulation ◽  
Norsolorinic Acid ◽  
Aflatoxin Accumulation ◽  
Regulatory Effects

ABSTRACT Aspergillus parasiticus is one primary source of aflatoxin contamination in economically important crops. To prevent the potential health and economic impacts of aflatoxin contamination, our goal is to develop practical strategies to reduce aflatoxin synthesis on susceptible crops. One focus is to identify biological and environmental factors that regulate aflatoxin synthesis and to manipulate these factors to control aflatoxin biosynthesis in the field or during crop storage. In the current study, we analyzed the effects of aspergillus volatiles on growth, development, aflatoxin biosynthesis, and promoter activity in the filamentous fungus A. parasiticus. When colonies of Aspergillus nidulans and A. parasiticus were incubated in the same growth chamber, we observed a significant reduction in aflatoxin synthesis and asexual sporulation by A. parasiticus. Analysis of the headspace gases demonstrated that A. nidulans produced much larger quantities of 2-buten-1-ol (CA) and 2-ethyl-1-hexanol (EH) than A. parasiticus. In its pure form, EH inhibited growth and increased aflatoxin accumulation in A. parasiticus at all doses tested; EH also stimulated aflatoxin transcript accumulation. In contrast, CA exerted dose-dependent up-regulatory or down-regulatory effects on aflatoxin accumulation, conidiation, and aflatoxin transcript accumulation. Experiments with reporter strains carrying nor-1 promoter deletions and mutations suggested that the differential effects of CA were mediated through separate regulatory regions in the nor-1 promoter. The potential efficacy of CA as a tool for analysis of transcriptional regulation of aflatoxin biosynthesis is discussed. We also identify a novel, rapid, and reliable method to assess norsolorinic acid accumulation in solid culture using a Chroma Meter CR-300 apparatus.

scholarly journals Involvement of Two Cytosolic Enzymes and a Novel Intermediate, 5′-Oxoaverantin, in the Pathway from 5′-Hydroxyaverantin to Averufin in Aflatoxin Biosynthesis

2003 ◽  
Vol 69 (11) ◽  
pp. 6418-6426 ◽  
Author(s):  
Emi Sakuno ◽  
Kimiko Yabe ◽  
Hiromitsu Nakajima
Keyword(s):  
Aspergillus Parasiticus ◽  
Laser Desorption ◽  
Mass Spectrometry Analysis ◽  
Laser Desorption Ionization ◽  
Aflatoxin Biosynthesis ◽  
Cytosol Fraction ◽  
Ionization Time ◽  
Spectrometry Analysis ◽  
Flight Mass Spectrometry ◽  
Physicochemical Methods

ABSTRACT During aflatoxin biosynthesis, 5′-hydroxyaverantin (HAVN) is converted to averufin (AVR). Although we had previously suggested that this occurs in one enzymatic step, we demonstrate here that this conversion is composed of two enzymatic steps by showing that the two enzyme activities in the cytosol fraction of Aspergillus parasiticus were clearly separated by Mono Q column chromatography. An enzyme, HAVN dehydrogenase, catalyzes the first reaction from HAVN to a novel intermediate, another new enzyme catalyzes the next reaction from the intermediate to AVR, and the intermediate is a novel substance, 5′-oxoaverantin (OAVN), which was determined by physicochemical methods. We also purified both of the enzymes, HAVN dehydrogenase and OAVN cyclase, from the cytosol fraction of A. parasiticus by using ammonium sulfate fractionation and successive chromatographic steps. The HAVN dehydrogenase is a homodimer composed of 28-kDa subunits, and it requires NAD, but not NADP, as a cofactor for its activity. Matrix-assisted laser desorption ionization-time of flight mass spectrometry analysis of tryptic peptides of the purified HAVN dehydrogenase revealed that this enzyme coincides with a protein deduced from the adhA gene in the aflatoxin gene cluster of A. parasiticus. Also, the OAVN cyclase enzyme is a homodimer composed of 79-kDa subunits which does not require any cofactor for its activity. Further characterizations of both enzymes were performed.

Evidence for de novo synthesis of an aflatoxin pathway methyltransferase near the cessation of active growth and the onset of aflatoxin biosynthesis in Aspergillus parasiticus mycelia

1990 ◽  
Vol 36 (1) ◽  
pp. 1-5 ◽  
Author(s):  
T. E. Cleveland ◽  
D. Bhatnagar
Keyword(s):  
Protein Synthesis ◽  
Soluble Protein ◽  
Aspergillus Parasiticus ◽  
De Novo ◽  
Protein Fractions ◽  
Time Interval ◽  
Aflatoxin Biosynthesis ◽  
Active Growth ◽  
Mtase Activity ◽  
De Novo Protein Synthesis

The accumulation of both activity and protein of a methyltransferase (MTase) from Aspergillus parasiticus, which catalyzes conversion of sterigmatocystin to O-methylsterigmatocystin in the aflatoxin pathway, was detected in fungal mycelia slightly before the onset of aflatoxin biosynthesis in the same cultures. MTase protein was identified in mycelial postmicrosomal (soluble protein) fractions by electrophoresis and subsequent immunoblotting using antiserum raised against purified MTase protein; MTase activity was determined by measuring the rate of conversion of sterigmatocystin to O-methylsterigmatocystin in the presence of soluble protein fractions. Using the above technique, it was determined that MTase protein as well as MTase activity increased sharply in mycelia 30 to 45 h after inoculation, shortly after which, mycelial growth rate began to decline. During the subsequent time interval (45 to 70 h after inoculation), a sharp increase in aflatoxin levels was detected in the culture medium. Results obtained from an experiment in which cycloheximide was added to cultures at various times to inhibit protein synthesis and from an experiment in which mycelial proteins were radiolabelled to identify newly synthesized proteins indicated that accumulation of MTase activity and protein in late growth phase mycelia is due to de novo protein synthesis. Key words: aflatoxin, methyltransferase, biosynthetic pathway.

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