Q-183. Isolation and Heterologous Expression of Manganese Peroxidase Gene from Lentinula edodes

I. S. W. Kwok, W. W. Y. Chum, T. C. H. Au, H. S. Kwan;
The Chinese Univ. of Hong Kong, Hong Kong SAR, HONG KONG.

Background: Lentinula edodes (shiitake) is an edible mushroom that can degrade lignocellulose efficiently. During vegetative growth of mycelia, various lignocellulolytic enzymes are actively secreted but only a few of them have been identified in shiitake. These enzymes have potential applications in biofuel production, thus we are interested in cloning and characterizing lignocellulolytic enzymes from shiitake and producing recombinant enzymes in yeast. Methods: ESTs were obtained from random sequencing of a normalized mycelial cDNA library and large-scale sequencing-by-synthesis of cDNA from lignocellulose-grown mycelia. They were annotated by BLASTX and categorized by Gene Ontology. Genes encoding lignocellulolytic enzymes were identified and the full-length sequence was obtained by RACE (Rapid Amplication of cDNA Ends). The transcript levels of the candidate genes in mycelia grown in media with or without lignocellulose were compared using real time RT-PCR. One of the candidates was cloned and heterologously expressed in Kluyveromyces lactis for further characterization. Results: More than 6000 ESTs were obtained. About 2600 ESTs were annotated, one third of which got significant E-values. Among the ESTs, a putative ligninolytic enzyme, manganese peroxidase (MnP2), was identified. Full-length mnp2 encodes 376 amino acids, which is led by a 19 amino-acid signal peptide. Its nucleotide and amino acid sequences show high similarity to MnP1 of Dichomitus squalens (66%) and MnP3 of Phanerochaete chrysosporium (65%). Transcript level of mnp2 was higher in mycelia grown in lignocellulose. With addition of heme-containing compounds, enzyme activity of the recombinant MnP was increased by more than 2 folds. Conclusion: From ESTs of mycelia growing in lignocellulosic medium, we identified mnp2, a putative ligninolytic enzyme-coding gene. By heterologous expression in yeast, Mnp2 may be useful in bioconversion of lignocellulosic waste to biofuels in the future.