Lactobacillus plantarum and Streptococcus thermophilus as starter cultures for a donkey milk fermented beverage.

Donkey milk is recently gaining attention due to its nutraceutical properties. Its low casein content does not allow caseification, so the production of a fermented milk would represent an alternative way to increase donkey milk shelf life. The aim of this study was to investigate the possibility of employing selected Streptococcus thermophilus and Lactobacillus plantarum isolates for the production of a novel donkey milk fermented beverage.

Tight control of mycotoxin biosynthesis gene expression in Aspergillus flavus by temperature as revealed by RNA-Seq.

To better understand the effect of temperature on mycotoxin biosynthesis, RNA-Seq technology was used to profile the Aspergillus flavus transcriptome under different temperature conditions. This approach allowed us to quantify transcript abundance for over 80% of fungal genes including 1153 genes that were differentially expressed at 30 and 37 °C. Eleven of the 55 secondary metabolite clusters were upregulated at the lower temperature, including aflatoxin biosynthesis genes, which were among the most highly upexpressed genes.

Are the genes nadA and norB involved in formation of aflatoxin G(1)?

Aflatoxins, the most toxic and carcinogenic family of fungal secondary metabolites, are frequent contaminants of foods intended for human consumption. Previous studies showed that formation of G-group aflatoxins (AFs) from O-methylsterigmatocystin (OMST) by certain Aspergillus species involves oxidation by the cytochrome P450 monooxygenases, OrdA (AflQ) and CypA (AflU). However, some of the steps in the conversion have not yet been fully defined.

Analysis of single nucleotide polymorphisms in three genes shows evidence for genetic isolation of certain Aspergillus flavus vegetative compatibility groups.

Genetic exchange by asexual filamentous fungi is presumed to be limited to isolates in the same vegetative compatibility group (VCG). To evaluate genetic isolation of Aspergillus flavus due to vegetative incompatibility, three gene regions were chosen that contained closely spaced nucleotides that were polymorphic among some of the six VCGs examined. A member of each VCG was collected from five regions across the southern United States.

Aflatoxin-producing Aspergillus species from Thailand.

Aflatoxin-producing Aspergillus species were isolated from soil samples from ten different regions within Thailand. Aspergillus flavus was present in all of the soil samples. Unlike previous studies, we found no A.

The aflatoxin biosynthesis cluster gene, aflX, encodes an oxidoreductase involved in conversion of versicolorin A to demethylsterigmatocystin.

Biosynthesis of the toxic and carcinogenic aflatoxins by the fungus Aspergillus flavus is a complicated process involving more that 27 enzymes and regulatory factors encoded by a clustered group of genes. Previous studies found that three enzymes, encoded by verA, ver-1, and aflY, are required for conversion of versicolorin A (VA), to demethylsterigmatocystin. We now show that a fourth enzyme, encoded by the previously uncharacterized gene, aflX (ordB), is also required for this conversion.

An aflatoxin biosynthesis cluster gene encodes a novel oxidase required for conversion of versicolorin a to sterigmatocystin.

Disruption of the aflatoxin biosynthesis cluster gene aflY (hypA) gave Aspergillus parasiticus transformants that accumulated versicolorin A. This gene is predicted to encode the Baeyer-Villiger oxidase necessary for formation of the xanthone ring of the aflatoxin precursor demethylsterigmatocystin.

Divergent regulation of aflatoxin production at acidic pH by two Aspergillus strains.

Production of aflatoxins (AF) by Aspergillus flavus and A. parasiticus is known to occur only at acidic pH. Although typical A.

Sequence comparison of aflR from different Aspergillus species provides evidence for variability in regulation of aflatoxin production.

Aflatoxin contamination of foods and feeds is a world-wide agricultural problem. Aflatoxin production requires expression of the biosynthetic pathway regulatory gene, aflR, which encodes a Cys6Zn2-type DNA-binding protein. Homologs of aflR from Aspergillus nomius, bombycis, parasiticus, flavus, and pseudotamarii were compared to investigate the molecular basis for variation among aflatoxin-producing taxa in the regulation of aflatoxin production.

Promoter elements in the aflatoxin pathway polyketide synthase gene.

PksA catalyzes the formation of the polyketide backbone necessary for aflatoxin biosynthesis. Based on reporter assays and sequence comparisons of the nor1-pksA intergenic region in different aflatoxin-producing Aspergillus species, cis-acting elements for the aflatoxin pathway-specific regulatory protein, AflR, and the global-acting regulatory proteins BrlA and PacC are involved in pksA promoter activity.

adhA in Aspergillus parasiticus is involved in conversion of 5'-hydroxyaverantin to averufin.

Two routes for the conversion of 5'-hydroxyaverantin (HAVN) to averufin (AVF) in the synthesis of aflatoxin have been proposed. One involves the dehydration of HAVN to the lactone averufanin (AVNN), which is then oxidized to AVF. Another requires dehydrogenation of HAVN to 5'-ketoaverantin, the open-chain form of AVF, which then cyclizes spontaneously to AVF.

Promoter elements involved in the expression of the Aspergillus parasiticus aflatoxin biosynthesis pathway gene avnA.

One of the early genes in aflatoxin biosynthesis, avnA, encodes a pathway-specific cytochrome P-450 monooxygenase that catalyzes the hydroxylation of the polyketide anthraquinone, averantin. Based on beta-glucuronidase (GUS) reporter and electrophoretic mobility shift assays, promoter sites upstream of -118 bp in the 367-bp verB-avnA intergenic region are not required for avnA gene activity. Therefore, only the -100 to -110 site of the four putative binding sites for AFLR, the aflatoxin biosynthetic pathway transcription regulatory protein (consensus binding sequence: 5'-TCGN(5)CGR-3') was required for elevated avnA expression.

Binding of the C6-zinc cluster protein, AFLR, to the promoters of aflatoxin pathway biosynthesis genes in Aspergillus parasiticus.

AFLR is a Zn2Cys6-type sequence-specific DNA-binding protein that is thought to be necessary for expression of most of the genes in the aflatoxin pathway gene cluster in Aspergillus parasiticus and A. flavus, and the sterigmatocystin gene cluster in A. nidulans.

Characterization of the promoter for the gene encoding the aflatoxin biosynthetic pathway regulatory protein AFLR.

Most genes in the aflatoxin biosynthetic pathway in Aspergillus parasiticus are regulated by the binuclear zinc cluster DNA-binding protein AFLR. The aflR promoter was analyzed in beta-glucuronidase reporter assays to elucidate some of the elements involved in the gene's transcription control. Truncation at 118 bp upstream of the translational start site increased promoter activity 5-fold, while truncation at -100 reduced activity about 20-fold.

Characterization of the critical amino acids of an Aspergillus parasiticus cytochrome P-450 monooxygenase encoded by ordA that is involved in the biosynthesis of aflatoxins B1, G1, B2, and G2.

The conversion of O-methylsterigmatocystin (OMST) and dihydro-O-methylsterigmatocystin to aflatoxins B1, G1, B2, and G2 requires a cytochrome P-450 type of oxidoreductase activity. ordA, a gene adjacent to the omtA gene, was identified in the aflatoxin-biosynthetic pathway gene cluster by chromosomal walking in Aspergillus parasiticus. The ordA gene was a homolog of the Aspergillus flavus ord1 gene, which is involved in the conversion of OMST to aflatoxin B1.

Alteration of different domains in AFLR affects aflatoxin pathway metabolism in Aspergillus parasiticus transformants.

AFLR, a zinc binuclear cluster DNA-binding protein, is required for activation of genes comprising the aflatoxin biosynthetic pathway in Aspergillus spp. Transformation of Aspergillus parasiticus with plasmids containing the intact aflR gene gave clones that produced fivefold more aflatoxin pathway metabolites than did the untransformed strain. When a 13-bp region in the aflR promoter (position -102 to -115 with respect to the ATG) was deleted, including a portion of a palindromic site previously shown to bind recombinant AFLR, metabolite production was 40% that of transformants with intact aflR.

An acid phosphatase from Aspergillus ficuum has homology to Penicillium chrysogenum PhoA.

Three secreted acid phosphatases had previously been characterized from Aspergillus ficuum grown under conditions of limited phosphate. One of these could not be readily separated from AFPhyB, a pH 2.5 optimum acid phosphatase with phytase activity.

Identification and cloning of a second phytase gene (phyB) from Aspergillus niger (ficuum).

An Aspergillus niger (ficuum) genomic DNA lambda EMBL3 library was probed with a 354-bp DNA fragment obtained by polymerase chain reaction of A. niger DNA with oligonucleotides based on partial amino acid sequence of a pH 2.5 optimum acid phosphatase.

An ethidium bromide-agarose plate assay for the nonradioactive detection of cDNA synthesis.

Ethidium bromide was used to determine the success of cDNA synthesis reactions. Since ethidium bromide in agarose can be used to quantitate RNA and DNA, conditions under which the greater fluorescence of double-stranded DNA (dsDNA) is utilized were devised to assay dsDNA synthesis from mRNA. Ethidium bromide at 5 micrograms/ml in agarose allowed quantitative detection of cDNA in the range of 0.