Exploring laccase-like multicopper oxidase genesfrom the ascomycete Trichoderma reesei:a functional, phylogenetic and evolutionary study

摘要

Background: The diversity and function of ligninolytic genes in soil-inhabiting ascomycetes has not yet beenelucidated, despite their possible role in plant litter decay processes. Among ascomycetes, Trichoderma reesei is amodel organism of cellulose and hemicellulose degradation, used for its unique secretion ability especially forcellulase production. T. reesei has only been reported as a cellulolytic and hemicellulolytic organism althoughgenome annotation revealed 6 laccase-like multicopper oxidase (LMCO) genes. The purpose of this work was i) tovalidate the function of a candidate LMCO gene from T. reesei, and ii) to reconstruct LMCO phylogeny and performevolutionary analysis testing for positive selection.Results: After homologous overproduction of a candidate LMCO gene, extracellular laccase activity was detectedwhen ABTS or SRG were used as substrates, and the recombinant protein was purified to homogeneity followedby biochemical characterization. The recombinant protein, called TrLAC1, has a molecular mass of 104 kDa. Optimaltemperature and pH were respectively 40-45°C and 4, by using ABTS as substrate. TrLAC1 showed broad pHstability range of 3 to 7. Temperature stability revealed that TrLAC1 is not a thermostable enzyme, which was alsoconfirmed by unfolding studies monitored by circular dichroism. Evolutionary studies were performed to shed lighton the LMCO family, and the phylogenetic tree was reconstructed using maximum-likelihood method. LMCO andclassical laccases were clearly divided into two distinct groups. Finally, Darwinian selection was tested, and theresults showed that positive selection drove the evolution of sequences leading to well-known laccases involved inligninolysis. Positively-selected sites were observed that could be used as targets for mutagenesis and functionalstudies between classical laccases and LMCO from T. reesei.Conclusions: Homologous production and evolutionary studies of the first LMCO from the biomass-degradingfungus T. reesei gives new insights into the physicochemical parameters and biodiversity in this family.