Cloning and expression of Penicillium minioluteum dextranase in Saccharomyces cerevisiae and its exploitation as a reporter in the detection of mycotoxins.
A dextranase gene from Penicillium minioluteum (strain IMI068219) has been cloned, sequenced and expressed in Saccharomyces cerevisiae via fusion of the DNA segment encoding the mature dextranase protein with alpha-factor signal sequence, and insertion into the GAL1-controlled expression vector pYES2/CT. Galactose-induced expression yielded extracellular dextranase activity of 0.63 units/ml and cell-associated dextranase activity of 0.48 units/ml, after 24 h incubation. The dextranase construct was introduced into a strain of S. cerevisiae expressing the human cytochrome P450 3A4 (CYP3A4) and the cognate reductase, which was then used to develop a microplate toxicity bioassay. Toxicity was signalled as inhibition of dextranase activity, assayed fluorimetrically. This novel bioassay was assessed using six economically significant mycotoxins.
Li
Xingmin
X
School of Science, University of Greenwich at Medway, Chatham Maritime, Kent, UK.
Millson
Stefan H
SH
Coker
Raymond D
RD
Evans
Ivor H
IH
D016604:Aflatoxin B1 / Q000378:metabolism; D001681:Biological Assay; D001709:Biotechnology / Q000295:instrumentation / Q000379:methods*; D003001:Cloning, Molecular; D003910:Dextranase / Q000235:genetics* / Q000378:metabolism*; D005690:Galactose / Q000378:metabolism; D005821:Genetic Techniques; D009183:Mycotoxins / Q000378:metabolism*; D010407:Penicillium / Q000201:enzymology*; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D013997:Time Factors
D009183:Mycotoxins; D016604:Aflatoxin B1; D003910:Dextranase; D005690:Galactose