A sensitive bioassay for the mycotoxin aflatoxin B1, which also responds to the mycotoxins aflatoxin G1 and T-2 toxin, using engineered baker's yeast

A novel aflatoxin B(1) bioassay was created by introducing a Lipomyces kononenkoae alpha-amylase gene into a strain of S. cerevisiae capable of expressing the human cytochrome P450 3A4 (CYP3A4), and the cognate human CYP450 reductase. This strain and a dextranase-expressing strain were used in the development of a microtitre plate mycotoxin bioassay, which employed methanol as the solvent and polymyxin B nonapeptide as a permeation enhancer. Stable co-expression of the CYP3A4 gene system and of the dextranase and amylase genes in the two bioassay strains was demonstrated. The bioassay signalled toxicity as inhibition of secreted carbohydrase activity, using sensitive fluorimetric assays. The amylase-expressing strain could detect aflatoxin B(1) at 2 ng/ml, and was more sensitive than the dextranase-expressing strain. Aflatoxin G(1) could be detected at 2 microg/ml, and the trichothecene mycotoxin T-2 toxin was detectable at 100 ng/ml.

Multiple dextranases from the yeast Lipomyces starkeyi

The soil yeast Lipomyces starkeyi (NCYC 1436) secretes dextranase activity into the growth medium. Resolution of a dextranase-active protein fraction by SDS-PAGE produced three protein bands, of 66 kDa, 68 kDa and 78 kDa, and isoelectric focusing of the same fraction resulted in seven protein bands, of pIs 3.50, 3.85, 4.20, 4.80, 4.85, 5.00 and 5.30. Dextranase activity was demonstrated for all the isoelectric forms, and for the 78 kDa species in the presence of SDS. Amino acid compositions of the 66 kDa, 68 kDa and 78 kDa protein bands were determined, and the N-termini of the 66 kDa and 78 kDa protein bands were sequenced: the first two amino acids at the N-terminus of each protein were alanine and valine, respectively; an alanine-valine pair is seen early in the N-terminal coding sequences of the dextranases and the isopullulanase produced by the phylogenetically disparate organisms contributing to glycosyl hydrolase family 49.