Expression of Neurospora crassa laccase under the control of the copper-inducible metallothionein-promoter

Abstract

Laccase from the ascomycete Neurospora crassa is an inducible secretory enzyme. In vegetatively growing cultures its biosynthesis is repressed but can be induced by different protein synthesis inhibitors. Transformation of the N. crassa wild-type strain Singapore with a fusion gene consisting of the N. crassa copper-metallothionein promoter and the laccase gene are described in this report. Correct integration of the 3.6 kilobase (kb) promoter-fragment fused with the laccase gene containing a 5' consensus region leads to copper-dependent expression of the enzyme during the vegetative growth phase. The enzyme is glycosylated and secreted, and high amounts of extracellular activity can be detected. The regulation of laccase biosynthesis of one examined transformant, followed at both the transcriptional and the translational level, indicates co-induction of both copper-metallothionein and laccase. The data presented show that expression of the recombinant laccase gene is exclusively regulated by the transformed N. crassa metallothionein-promoter.