Final Report Summary - FUNBIO (Fungi in white biotechnology: Expression of novel lignocellulose degrading enzymes)
Plant biomass degradation is of major importance for many fungi, as lignocellulose represents the most abundant renewable organic resource. For the efficient decomposition of lignocelluloses, fungi utilise a set of hydrolytic and oxidative enzymes recognised as economically viable and environmentally friendly industrial biocatalysts. This project focuses on the science underlying the use of lignocellulosic materials in biotechnological applications. The important aspect of the project has been to find an efficient and rapid production system for basidiomycete lignin modifying enzymes to make various applications feasible. The main objective of this project was to enhance our understanding of decomposition of lignocellulose. New lignocellulose modifying fungal enzymes were produced heterologously and characterised biochemically. In addition, this project resulted in setting-up the recombinant protein expression techniques for basidiomycete fungal enzymes in the host laboratory.
Different white-rot fungal species have been used as model organisms for lignocellulose degradation. Physisporinus rivulosus is highly selective fungal species for degradation of softwood lignin, which makes this fungus suitable for biopulping application. This white-rot basidiomycete degrades lignin efficiently and is promising for use in biopulping of softwood. We have previously isolated two laccase (Lac) isozymes of P. rivulosus 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 previously undescribed thermotolerant P. rivulosus laccases which were heterologously expressed in the methylotrophic yeast Pichia pastoris. Both recombinant laccases demonstrated low pH optima for phenolic substrates. Recombinant Lac1 showed thermal activation for oxidation of phenolic substrates that has been earlier described only for a few laccases. Comparison of the substrate-binding pockets in the structural models of Lac1 and Lac2 showed the differences in amino acid composition which may explain the distinct catalytic properties of enzymes.
The lignocellulose degrading enzymes of two other white-rot fungal species, Phlebia radiata and Dichomitus squalens, were studied at enzyme and transcript level, respectively, when the fungi were grown on natural substrate. Production of lignin peroxidase, manganese peroxidase and laccase were significantly promoted by P. radiata when wood was used as a carbon source suggesting synergistic catalytic functions of oxidoreductases on lignocellulose. Cellulose degrading machinery and oxalate metabolism of D. squalens was studied at transcript level by quantitative RT-PCR when the fungus was grown on spruce wood.
Agaricus bisporus is the most common commercial edible fungus and also the model fungus for growth on agricultural lignocellulosic wastes and in soil litter. The ability to grow in the humic rich leaf-litter environment is an advantage over other saprobes. The whole genome of A. bisporus was sequenced as the first litter decomposing species and we participated in in silico analyses for enzymes involved in lignin degradation. The expansion of heme-thiolate peroxidases was distinctive from wood-degrading fungi and suggests a broad attack on lignin and related metabolites which can be found in soil litter. To expand our knowledge in the genus Agaricus, six wild type isolates of Agaricus spp. were studied on their nutritional demands for enzyme production and mycelial growth. In accordance to transcriptome data obtained from compost cultures of A. bisporus, the determination of enzyme activities showed the importance of both manganese peroxidase and multicopper oxidases in Agaricus spp. while growing on lignocelluloses.
The socio-economical impact of this project has been significant. This funding has enabled a successful reintegration and the building up an active research group to the host organisation. In addition, this project has educated undergraduate and doctoral female students. The international visibility of the group has increased and enabled collaboration with new international contacts.
Different white-rot fungal species have been used as model organisms for lignocellulose degradation. Physisporinus rivulosus is highly selective fungal species for degradation of softwood lignin, which makes this fungus suitable for biopulping application. This white-rot basidiomycete degrades lignin efficiently and is promising for use in biopulping of softwood. We have previously isolated two laccase (Lac) isozymes of P. rivulosus 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 previously undescribed thermotolerant P. rivulosus laccases which were heterologously expressed in the methylotrophic yeast Pichia pastoris. Both recombinant laccases demonstrated low pH optima for phenolic substrates. Recombinant Lac1 showed thermal activation for oxidation of phenolic substrates that has been earlier described only for a few laccases. Comparison of the substrate-binding pockets in the structural models of Lac1 and Lac2 showed the differences in amino acid composition which may explain the distinct catalytic properties of enzymes.
The lignocellulose degrading enzymes of two other white-rot fungal species, Phlebia radiata and Dichomitus squalens, were studied at enzyme and transcript level, respectively, when the fungi were grown on natural substrate. Production of lignin peroxidase, manganese peroxidase and laccase were significantly promoted by P. radiata when wood was used as a carbon source suggesting synergistic catalytic functions of oxidoreductases on lignocellulose. Cellulose degrading machinery and oxalate metabolism of D. squalens was studied at transcript level by quantitative RT-PCR when the fungus was grown on spruce wood.
Agaricus bisporus is the most common commercial edible fungus and also the model fungus for growth on agricultural lignocellulosic wastes and in soil litter. The ability to grow in the humic rich leaf-litter environment is an advantage over other saprobes. The whole genome of A. bisporus was sequenced as the first litter decomposing species and we participated in in silico analyses for enzymes involved in lignin degradation. The expansion of heme-thiolate peroxidases was distinctive from wood-degrading fungi and suggests a broad attack on lignin and related metabolites which can be found in soil litter. To expand our knowledge in the genus Agaricus, six wild type isolates of Agaricus spp. were studied on their nutritional demands for enzyme production and mycelial growth. In accordance to transcriptome data obtained from compost cultures of A. bisporus, the determination of enzyme activities showed the importance of both manganese peroxidase and multicopper oxidases in Agaricus spp. while growing on lignocelluloses.
The socio-economical impact of this project has been significant. This funding has enabled a successful reintegration and the building up an active research group to the host organisation. In addition, this project has educated undergraduate and doctoral female students. The international visibility of the group has increased and enabled collaboration with new international contacts.