Functional expression of bacterial beta-glucuronidase and its use as a reporter system in the yeast Yarrowia lipolytica.
The use of beta-glucuronidase (beta-GUS) as a reporter and sensitive detection system for Yarrowia lipolytica was studied. The Escherichia coli gusA gene was expressed under control of the homologous LEU2 promoter in a transcriptional fusion. An NcoI restriction site was introduced at the translational start-ATG, conserving the most favorable context for initiation of translation. The chimeric LEU2'-gusA gene was integrated into the LEU2 locus by homologous recombination. The beta-GUS assay was very sensitive and highly reproducible, using the cytosolic fraction or a total cell extract as the source of enzyme. In a leucine-free medium, beta-GUS activity was at a high, constant level, independent of growth phase. In transformants grown on complete medium, beta-GUS activity was reduced about three-fold, but doubled during logarithmic growth. No intrinsic beta-GUS activity was detectable in untransformed Y. lipolytica and no effect of beta-GUS expression on growth was observed. beta-GUS-producing Y. lipolytica cells could be directly detected on media plates containing X-gluc (5-bromo-4-chloro-3-indolyl-beta-D-glucuronide).
Bauer
R
R
Institut für Biochemie und Lebensmittelchemie, Technische Universität Graz, Austria.
Paltauf
F
F
Kohlwein
S D
SD
D001483:Base Sequence; D003001:Cloning, Molecular; D003470:Culture Media; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005966:Glucuronidase / Q000096:biosynthesis / Q000235:genetics*; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D004718:Saccharomycetales / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D014170:Transformation, Genetic
D003470:Culture Media; D005966:Glucuronidase