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

Abstract

Laccases are copper-containing enzymes which oxidize phenolic substrates and transfer the electrons to oxygen. Many filamentous fungi contain several laccase-encoding genes, but their biological roles are mostly not well understood. The main interest in laccases in biotechnology is their potential to be used to detoxify phenolic substances. We report here on a novel application of laccases as a reporter system in fungi. We purified a laccase enzyme from the ligno-cellulolytic ascomycete Stachybotrys chartarum. It oxidized the artificial substrate 2,2-azino-di-(3-ethylbenzthiazolinsulfonate) (ABTS). The corresponding gene was isolated and expressed in Aspergillus nidulans, Aspergillus niger, and Trichoderma reesei. Heterologously expressed laccase activity was monitored in colorimetric enzyme assays and on agar plates with ABTS as a substrate. The use of laccase as a reporter was shown in a genetic screen for the isolation of improved T. reesei cellulase production strains. In addition to the laccase from S. charatarum, we tested the application of three laccases from A. nidulans (LccB, LccC, and LccD) as reporters. Whereas LccC oxidized ABTS (K m ‫؍‬ 0.3 mM), LccD did not react with ABTS but with DMA/ADBP (3,5-dimethylaniline/ 4-amino-2,6-dibromophenol). LccB reacted with DMA/ADBP and showed weak activity with ABTS. The different catalytic properties of LccC and LccD allow simultaneous use of these two laccases as reporters in one fungal strain.

Alternaria alternata transcription factor CmrA controls melanization and spore development

“…This raises the question of how the two processes could be linked. One explanation for the (Mander et al, 2006). (b) Strains were tested under repressing and inducing conditions.…”

“…As negative control we used 500 ml 100 mM sodium acetate buffer (pH 3.5), 200 ml 5 mM ABTS, 200 ml H 2 O and 100 ml medium. Absorption was measured after 1 h of incubation at 420 nm in a Pharmacia LKBUltrospec III spectrophotometer (Mander et al, 2006).…”

“…nidulans because the activity can easily be followed through the colorimetric change of ABTS after oxidation. Electrons are transferred to oxygen (Mander et al, 2006). From 80 transformants 20 were chosen for the analysis on medium containing ABTS.…”

Heterologous laccase production and its role in industrial applications

“…Heterologous expression of laccases in bacteria, yeasts, filamentous fungi and plants has been reported, along with examples of homologous expression. [25][26][27][28][29][30] Laccase heterologous expression in Escherichia coli has been often used as a strategy to get around the problem of obtaining laccases not easily producible in natural hosts. The recombinant expression of Bacillus subtilis CotA in E. coli has allowed its deep characterization, structure solving, and functional evolution.…”

Wood production, wood technology, and biotechnological impacts

“…Although laccases usually have a wide substrate spectrum, enzymes can differ in the range of substances they attack , Mander et al 2006. In consequence, enzymes may be differentially suitable for specific tasks in biotechnology.…”