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

Abstract

Background: The diversity and function of ligninolytic genes in soil-inhabiting ascomycetes has not yet been elucidated, despite their possible role in plant litter decay processes. Among ascomycetes, Trichoderma reesei is a model organism of cellulose and hemicellulose degradation, used for its unique secretion ability especially for cellulase production. T. reesei has only been reported as a cellulolytic and hemicellulolytic organism although genome annotation revealed 6 laccase-like multicopper oxidase (LMCO) genes. The purpose of this work was i) to validate the function of a candidate LMCO gene from T. reesei, and ii) to reconstruct LMCO phylogeny and perform evolutionary analysis testing for positive selection.

Results: After homologous overproduction of a candidate LMCO gene, extracellular laccase activity was detected when ABTS or SRG were used as substrates, and the recombinant protein was purified to homogeneity followed by biochemical characterization. The recombinant protein, called TrLAC1, has a molecular mass of 104 kDa. Optimal temperature and pH were respectively 40-45°C and 4, by using ABTS as substrate. TrLAC1 showed broad pH stability range of 3 to 7. Temperature stability revealed that TrLAC1 is not a thermostable enzyme, which was also confirmed by unfolding studies monitored by circular dichroism. Evolutionary studies were performed to shed light on the LMCO family, and the phylogenetic tree was reconstructed using maximum-likelihood method. LMCO and classical laccases were clearly divided into two distinct groups. Finally, Darwinian selection was tested, and the results showed that positive selection drove the evolution of sequences leading to well-known laccases involved in ligninolysis. Positively-selected sites were observed that could be used as targets for mutagenesis and functional studies between classical laccases and LMCO from T. reesei.

Conclusions: Homologous production and evolutionary studies of the first LMCO from the biomass-degrading fungus T. reesei gives new insights into the physicochemical parameters and biodiversity in this family.

Figure 1

SDS-PAGE of extracellular proteins and purified TrLAC1. Total proteins from wild-type strain (lane a) and TrLAC1 transformant (lane b) were loaded onto an SDS-PAGE (11% polyacrylamide) gel. Gel was stained with Coomassie blue. L, molecular mass standards.

Figure 2

Effect of temperature (a) and pH (b) on the activity of the purified TrLAC1. Various temperatures and pH values were tested under standard conditions by using ABTS "blue diamond" and SRG "pink square" as substrates.

Figure 3

Effect of temperature (a) and pH (b) on the stability or the purified TrLAC1. Selected temperatures were 30°C "blue diamond", 35°C "pink square", 40°C "yellow triangle", 45°C "blue cross", 50°C "purple star" and the selected pH values were 3 "blue diamond", 4 "pink square", 5 "yellow triangle", 6 "blue cross", 7 "purple star".

Figure 4

CD spectra of TrLAC1. measured under native (solid line) and thermal denaturing conditions (dashed line).

Figure 5

Phylogenetic reconstruction of homologous sequences for TrLAC1. Basidiomycotina laccases branch is labelled as a and is considered as the foreground branch for branch-site models.