Final Report Summary - FUNGEN (Heterologous expression of wood-rotting and litter-decomposing fungal genes involved in lignin degradation)
Lignin is a highly recalcitrant integral polymer in higher plants and the second most abundant organic polymer in nature. Lignin degradation by white-rot fungi has been studied intensively in relation to highly important biotechnological applications such as biopulping, biobleaching and treating of pulp mill effluents. Indeed, lignin-selective fungi that degrade lignin in preference to cellulose are considered the most promising fungi for alternative energy saving applications in pulp and paper industry. Other important biotechnological applications of these fungi or their selected enzymes include biofuel generation and bioremediation, i.e. removal of toxic pollutants from soil and effluents. In this project the main goal has been to find an efficient and rapid production system for basidiomycete lignin modifying enzymes to make various applications feasible.
The white-rot fungus Physisporinus rivulosus T241i produced manganese peroxidase (MnP), laccase, and oxalic acid when it was grown on spruce (Picea abies) wood chips. This white-rot basidiomycete degrades lignin efficiently and is promising for use in biopulping of softwood. We have previously isolated two laccase isozymes of P. rivulosus Lac3.5 and Lac4.8 which both possess interesting features such as thermostability, extremely low pH optima and thermal activation for oxidation of phenolic substrates. In this project we cloned and characterised two novel P. rivulosus laccases which were were heterologously expressed in the methylotrophic yeast Pichia pastoris. The recombinant Lac1 and Lac2 enzymes showed similar low pH optima with phenolic substrates to the previously characterised native P. rivulosus Lac3.5 and Lac4.8 isozymes. Moderate thermotolerance and thermal activation was detected as well.
Litter-decomposing fungi possess a similar lignin modifying enzyme system as wood decaying white-rot fungi but relatively little is known about their potential and significance in the degradation of lignin, humus and organopollutants in soil. Agrocybe praecox is a forest litter-decomposing saprobic basidiomycetous fungal species which is also able to colonize bark mulch and wood chip. The fungus produces extracellular laccase and MnP activities in low-nitrogen liquid medium supplemented with Mn2+ ions. In this project two novel class II haem-containing peroxidases of A. praecox (Ap-Pox1 and Ap-Pox2) have been cloned and heterologously expressed. The recombinant Ap-Pox1 and Ap-Pox2 enzymes were able to oxidize phenolic substrates in the presence of hydrogen peroxide (H2O2). As addition of Mn2+ slightly inhibited the activity of both recombinant peroxidases we suggest that Ap-Pox1 and Ap-Pox2 represented Mn-independent phenol-oxidizing class II peroxidases.
For complete decomposition or partial mineralization of plant lignin additional fungal enzymes are required. Carbohydrate active enzymes (cellulases and hemicellulases) and feruloyl esterases may promote wood lignin reactivity and xylem cell wall accessibility for the fungal lignin modifying enzymes. In addition, enzymes controlling organic acid metabolism play important roles in the wood-decaying fungi. Despite the accumulating genomic sequence data on wood and litter decaying fungi only a few esterase genes have been characterized in basidiomycete fungal species. In this project two novel esterase genes have been cloned and characterized from two litter-decomposing fungal species. Heterologous expression of these esterases has been started for kinetic studies. Also enzymes that regulate fungal organic acid production, oxalate decarboxylase (ODC) and oxalate oxidase, have been studied. ODC1 of white-rot fungus Dichomitus squalens was heterologously expressed in P. pastoris and the properties of this enzyme studied. The cloning of novel ODC-encoding genes has been initiated for expression studies.
The white-rot fungus Physisporinus rivulosus T241i produced manganese peroxidase (MnP), laccase, and oxalic acid when it was grown on spruce (Picea abies) wood chips. This white-rot basidiomycete degrades lignin efficiently and is promising for use in biopulping of softwood. We have previously isolated two laccase isozymes of P. rivulosus Lac3.5 and Lac4.8 which both possess interesting features such as thermostability, extremely low pH optima and thermal activation for oxidation of phenolic substrates. In this project we cloned and characterised two novel P. rivulosus laccases which were were heterologously expressed in the methylotrophic yeast Pichia pastoris. The recombinant Lac1 and Lac2 enzymes showed similar low pH optima with phenolic substrates to the previously characterised native P. rivulosus Lac3.5 and Lac4.8 isozymes. Moderate thermotolerance and thermal activation was detected as well.
Litter-decomposing fungi possess a similar lignin modifying enzyme system as wood decaying white-rot fungi but relatively little is known about their potential and significance in the degradation of lignin, humus and organopollutants in soil. Agrocybe praecox is a forest litter-decomposing saprobic basidiomycetous fungal species which is also able to colonize bark mulch and wood chip. The fungus produces extracellular laccase and MnP activities in low-nitrogen liquid medium supplemented with Mn2+ ions. In this project two novel class II haem-containing peroxidases of A. praecox (Ap-Pox1 and Ap-Pox2) have been cloned and heterologously expressed. The recombinant Ap-Pox1 and Ap-Pox2 enzymes were able to oxidize phenolic substrates in the presence of hydrogen peroxide (H2O2). As addition of Mn2+ slightly inhibited the activity of both recombinant peroxidases we suggest that Ap-Pox1 and Ap-Pox2 represented Mn-independent phenol-oxidizing class II peroxidases.
For complete decomposition or partial mineralization of plant lignin additional fungal enzymes are required. Carbohydrate active enzymes (cellulases and hemicellulases) and feruloyl esterases may promote wood lignin reactivity and xylem cell wall accessibility for the fungal lignin modifying enzymes. In addition, enzymes controlling organic acid metabolism play important roles in the wood-decaying fungi. Despite the accumulating genomic sequence data on wood and litter decaying fungi only a few esterase genes have been characterized in basidiomycete fungal species. In this project two novel esterase genes have been cloned and characterized from two litter-decomposing fungal species. Heterologous expression of these esterases has been started for kinetic studies. Also enzymes that regulate fungal organic acid production, oxalate decarboxylase (ODC) and oxalate oxidase, have been studied. ODC1 of white-rot fungus Dichomitus squalens was heterologously expressed in P. pastoris and the properties of this enzyme studied. The cloning of novel ODC-encoding genes has been initiated for expression studies.