Functional expression of bacterial β‐glucuronidase and its use as a reporter system in the yeast Yarrowia lipolytica
Abstract
The use of β‐glucuronidase (β‐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 β‐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, β‐GUS activity was at a high, constant level, independent of growth phase. In transformants grown on complete medium, β‐GUS activity was reduced about three‐fold, but doubled during logarithmic growth. No intrinsic β‐GUS activity was detectable in untransformed Y. lipolytica and no effect of β‐GUS expression on growth was obseved. β‐GUS‐producing Y. lipolytica cells could be directly detected on media plates containing X‐gluc (5‐bromo‐4‐chloro‐3‐indolyl‐β‐D‐glucuronide).
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