Molecular Cloning and Expression inSaccharomyces cerevisiaeof a Laccase Gene from the AscomyceteMelanocarpus albomyces

Abstract

The lac1 gene encoding an extracellular laccase was isolated from the thermophilic fungus Melanocarpus albomyces. This gene has five introns, and it encodes a protein consisting of 623 amino acids. The deduced amino acid sequence of the laccase was shown to have high homology with laccases from other ascomycetes. In addition to removal of a putative 22-amino-acid signal sequence and a 28-residue propeptide, maturation of the translation product of lac1 was shown to involve cleavage of a C-terminal 14-amino-acid extension. M. albomyces lac1 cDNA was expressed in Saccharomyces cerevisiae under the inducible GAL1 promoter. Extremely low production was obtained with the expression construct containing laccase cDNA with its own signal and propeptide sequences. The activity levels were significantly improved by replacing these sequences with the prepro sequence of the S. cerevisiae ␣-factor gene. The role of the C-terminal extension in laccase production in S. cerevisiae was also studied. Laccase production was increased sixfold with the modified cDNA that had a stop codon after the native processing site at the C terminus.Laccases (benzenediol:oxygen oxidoreductases; EC 1.10.3.2) are multicopper enzymes belonging to the group of blue oxidases. They catalyze the oxidation of a variety of phenolic compounds, as well as diamines and aromatic amines, with concomitant reduction of molecular oxygen to water (43). Laccases are widely distributed in higher plants and fungi, and laccase or laccase-like activity has also been demonstrated in some insects and bacteria (18,31,12). In fungi, laccases are involved in several physiological functions, such as plant pathogenesis (3, 11), pigment production (2), and degradation of lignocellulosic materials (6). Because of their surprisingly wide variety of substrates, laccases are considered industrially interesting enzymes for various applications, including textile dye bleaching, pulp bleaching, detergents, and enzymatic conversion of chemical intermediates (47).Despite intensive research, the molecular basis of laccasecatalyzed reactions is still partially unknown. In order to determine the function of laccases and to produce them heterologously in large quantities, several laccase genes have been cloned, especially the genes from basidiomycetous fungi, including Phlebia radiata (39), Cryptococcus neoformans (46), Pleurotus ostreatus (19), Trametes versicolor (25), Trametes villosa (49), Pycnoporus cinnabarinus (13), and Coprinus cinereus (48). Some laccase genes have also been cloned from ascomycetes, including Neurospora crassa (17), Aspergillus nidulans (1), Podospora anserina (14), and Myceliophthora thermophila (5). Generally, the laccase sequences of members of a fungal class exhibit levels of amino acid identity of 50% or more, whereas the levels of identity between sequences of members of different classes are around 30%.Heterologous expression of laccase genes has been studied in Saccharomyces cerevisiae (27,10,29), Trichoderma reesei (38), Aspergillus oryzae (49, 5), Pich...

Expression of Melanocarpus albomyces laccase in Trichoderma reesei and characterization of the purified enzyme

“…N-and C-terminal peptide sequence analyses of the purified recombinant laccase produced by T. reesei showed that this host is able to perform both of the processing steps correctly. When M. albomyces laccase was expressed in S. cerevisiae, the production levels were enhanced both by using yeast alpha-factor prosequence in the expression construct and by introducing a stop codon into the laccase cDNA at the C-terminal processing site (Kiiskinen & Saloheimo, 2004). This indicated indirectly that baker's yeast was not able to perform either of the processing steps of the laccase efficiently.…”

“…It has been shown that M. albomyces laccase is processed at both its N-and C-termini (Kiiskinen & Saloheimo, 2004). The C-terminal processing is of special interest, because the truncated C-terminus protrudes inside the enzyme, potentially forming a plug to an O 2 /H 2 O exchange tunnel leading to the active site (Hakulinen et al, 2002).…”

“…The crystal structure revealed novel properties of M. albomyces laccase concerning molecular oxygen binding to the active site and the C-terminus entering inside the enzyme. Heterologous expression in Saccharomyces cerevisiae was also recently reported for this laccase, and the results showed that the C-terminal end of the protein is of special interest also with respect to production of this enzyme (Kiiskinen & Saloheimo, 2004). Because these results further emphasized the potential of this laccase for research and industrial purposes, we have now expressed the laccase gene in T. reesei, a filamentous fungus that is well known for its ability to produce high amounts of extracellular enzymes (Mäntylä et al, 1998).…”

“…The T. reesei expression vector pAMH110 (Saloheimo et al, 1989) was digested with KspI and NdeI to remove a spacer fragment between the cbh1 promoter and terminator sequences. M. albomyces lac1 cDNA was released from the plasmid pLLK4 (Kiiskinen & Saloheimo, 2004) by SacI and EcoRI digestion and ligated into pAMH110 by blunt-end ligation, to obtain the plasmid pLLK8. The expression cassette for hygromycin resistance consisting of the Aspergillus nidulans gpdA promoter and trpC terminator and the E. coli hygromycin resistance gene was taken from the plasmid pBluekan7-1.NotI (from P. J. Punt, TNO Nutrition and Food Research, the Netherlands) by NotI digestion.…”

“…This was also verified by N-and C-terminal sequencing of the purified laccase. Both termini were similar to those of native M. albomyces laccase (Kiiskinen & Saloheimo, 2004). After papain digestion, CBHI and laccase were separated by hydrophobic interaction chromatography, because laccase was bound on Phenyl-Sepharose.…”