A sensitive bioassay for the mycotoxin aflatoxin B(1), which also responds to the mycotoxins aflatoxin G(1) 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.
Li
Xingmin
X
School of Science, University of Greenwich at Medway, Central Avenue, Chatham Maritime, Kent ME44TB, United Kingdom.
Millson
Stefan
S
Coker
Raymond
R
Evans
Ivor
I
D000348:Aflatoxins / Q000494:pharmacology*; D001681:Biological Assay / Q000379:methods*; D051544:Cytochrome P-450 CYP3A / Q000235:genetics / Q000378:metabolism; D003910:Dextranase / Q000235:genetics / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D005818:Genetic Engineering; D006801:Humans; D055160:Lipomyces / Q000201:enzymology; D012441:Saccharomyces cerevisiae / Q000187:drug effects* / Q000235:genetics* / Q000378:metabolism; D000516:alpha-Amylases / Q000235:genetics / Q000378:metabolism
D000348:Aflatoxins; D005656:Fungal Proteins; D051544:Cytochrome P-450 CYP3A; D000516:alpha-Amylases; D003910:Dextranase