Use of Laccase as a Novel, Versatile Reporter System in Filamentous Fungi

Filamentous fungi play important roles in biotechnology as producers of low-molecular-weight compounds and enzymes. Strain improvement and antifungal compound screenings require different versatile assay systems. Another demand for those genetic tools comes from the continuously increasing number of genomic sequences of filamentous fungi (http://www.broad.mit.edu/annotation/) and the big challenge to assign gene functions. Important information can be gained by measuring promoter activity, subcellular localization of proteins, or protein-protein interactions. Whereas the last two issues can be nicely addressed by the use of green fluorescent protein, other variants of fluorescent proteins, or the two-hybrid system in Saccharomyces cerevisiae, the determination of promoter activities is often hindered (25, 31). Beta-galactosidase (lacZ) or glucuronidase (uidA) work in fungi but generally produce significant background because many fungi contain endogenous enzymes (4). Another reporter system used frequently in fungi is glucose oxidase (12, 16). This assay is based on a coupled enzyme assay, with peroxidase as a helper enzyme. If glucose oxdiase is secreted, the reporter system can be used on agar plates but requires that the colonies be incubated with glucose, a chromogenic substrate, and the peroxidase enzyme. Therefore, we have established laccases as alternative reporter systems. Laccases are copper-containing enzymes whose biological roles are largely unknown (17, 23). Most fungi contain several endogenous genes (at least 8 in Coprinopsis cinerea), but their expression is generally tightly controlled (9, 17). In basidiomycetes, a role of laccases in lignin degradation was discussed previously (5). Furthermore, specific laccases appear to play a role in fruiting body development. In ascomycetes, some laccases are induced upon exposure to phenolic substances (9). In Aspergillus nidulans, YA has a well-characterized role during asexual development (2). If yA is mutated, conidiospores are not green pigmented, but pigment biosynthesis is blocked at a yellow precursor. A second laccase was identified during the sexual cycle, and a third one, TilA, was detected in vegetative cells at the hyphal tip (14, 27). However, the expression of tilA was extremely low. Although laccases are widely distributed enzymes, their expression is usually very low in vegetative hyphae and thus should not interfere much with heterologously expressed laccases. Here, we show the use of laccases as reporter systems in A. nidulans, Aspergillus niger, and Trichoderma reesei. (Part of this work was patented [1, 20a].)