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 (Km = 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.

FIG. 1.

Sodium dodecyl sulfate gel of purified S. chartarum laccase. Lane 1 is the molecular mass marker. Lanes 2, 3, and 4 were loaded with 50 ng, 25 ng, and 10 ng of purified laccase, respectively. Proteolytic fragments of the laccase are indicated by an asterisk.

FIG. 2.

Expression of S. chartarum laccase in A. nidulans (A, B), A. niger (C), and T. reesei (D). (A) The A. nidulans wild type (WT) (FGSC4) and one positive transformant (G6) were grown on glucose-, glycerol-, and ethanol-containing media supplemented with 2 mM ABTS. (B) Quantification of laccase activity of five transformants with pCF1. Strains were grown on 50 mM glucose (Glu), 2.2 mM glucose plus 100 mM glycerol (Gly), or 100 mM threonine (Thr) plus 100 mM glycerol as indicated. Activity was determined in the supernatant of the culture extract and is given as μmol of ABTS per min and mg of dry mycelium. The average of two measurements is shown. (C) The A. niger wild type (dgr246p2) and transformant 14-5 were grown on maltose- and xylose-containing media with 2 mM ABTS. (D) The T. reesei wild type (1A52) and transformant VHtr6 were grown on glucose- and lactose-containing media with 2 mM ABTS.

FIG. 3.

Application of laccase for strain improvement. (A) A. niger strain improvement for laccase production. Strain 14-5 was mutagenized, and strains were selected which produced bigger ABTS halos on agar plates. Strain 14-5W was selected, and laccase production was compared to that of the wild type in liquid culture. Activities are given in units per ml of culture supernatant. (B) LccC-expressing A. nidulans and wild-type A. nidulans were inoculated in liquid culture (threonine medium supplemented with 1 mM ABTS) in microtiter plates.

FIG. 4.

T. reesei strain improvement for cellulase production. (A) Scheme of mutagenesis and strain selection. (B) Agar plate with a lawn of mutated strains on ABTS-containing plates. The arrow points to a colony with derepressed laccase expression. For details, see the text.

FIG. 5.

Phylogenetic tree of different plant and fungal laccases and ascorbate oxidases. The tree was generated with the ClustalW algorithm from the ClustalX 1.8 program with standard parameters. The gene names and accession numbers are indicated as follows: A. fumigatus, Af Abr2 (AF104823); A. nidulans, An TilA (AJ305224), An YA (X52552), An0878.1, An5397.1, An6830.1, An7389.1, An8581.1, An9170.1; Arabidopsis thaliana, At Lac1 (NP_565881), At Lac2 (NP_195739); Cryptococcus neoformans, Cn Lac1 (A36962); Gaeumannomyces graminis var. graminis, Gg Lac1 (AJ437319), Gg Lac2 (AJ437320); Hortaea acidophila, Ha Lac1 (AAY33971); Lentinus tigrinus, Lt Lac1 (AAX07469); Neurospora crassa, Nc LccA (AAA33591), Nc LccB (AAA33592), Nc LccC (AAA33590); Nicotiana tabacum, Nt Lac1 (AAC49538), Nt Lac2 (AAC49536); Trametes versicolor, Tv LacA (A35883); Zea mays, Zm Lac1 (AAX83113); S. chartarum, Sc Lac1 (AAY23005). The dendrogram was visualized by TreeView. The scale is about 10% calculated sequence divergence. The arrows indicate the four laccases studied in this paper.

FIG. 6.

Substrate specificity of LccB, LccC, and LccD of A. nidulans. Strains were grown on glucose medium (upper panel) or glycerol medium (middle panel) supplemented with ABTS. The ABTS staining of the middle panel was documented, and subsequently, the colonies overlaid with ADBP/DMA (14, 28). The conversion of the substrates is displayed in the lower panel. ABTS staining of the middle colony is lost upon incubation with ADBP/DMA. WT, wild type.