Transcriptome and Biochemical Analyses of Fungal Degradation of Wood

Lignocellulosic accounts for a large percentage of material that can be utilized for biofuels. The most costly part of lignocellulosic material processing is the initial hydrolysis of the wood which is needed to circumvent the lignin barrier and the crystallinity of cellulose. Enzymes will play an increased role in this conversion in that they potentially provide an alternative to costly and caustic high temperature and acid treatment. The increasing use of enzymes in biotechnology is facilitated by both continued improvements in enzyme technology but also in the discovery of new and novel enzymes. The present proposal is aimed at identifying the enzymes which are known to depolymerize woody biomass. Fundamental understanding of how nature gains access to cellulose and hemicellulose will impact all applications. Because fungi are the only known microbes capable of circumventing the lignin barrier, knowledge of the enzyme they use is of great potential for biofuel processing. Nature has evolved different fungal mechanisms for enzymatic hydrolysis of wood. Most notable are the white-rot fungi (wrf) and the brown-rot fungi (brf). This proposed research aims at determining the complete transcriptome of three wrf and two brf to determine the enzymes involved in lignocellulose degradation. The transcriptome work will be supported by enzyme characterization (and zymograms) and finally analysis of the lignin component to determine the mode of lignin modification. In this proposed research, we hypothesize that: 1) Determination of the complete transcriptome of closely related white and brown rot fungi will lead to knowledge of the relevant enzymes involved in wood degradation. 2) Knowledge of the extracellular transcriptome and the mechanism of wood decay can only be obtained if the products of the decay are known. As such, characterization of the lignin oxidation products will correlate the enzymes involved (obtained from the transcriptome) to the lignin oxidation products. The Department of Energy has sequenced the P. chrysosporium genome and has approved the sequencing of the genome of the closely-related brown rot fungus P. placenta. This comparative genomics approach will yield important information on differences between these two fungi. Analysis of gene unique to each fungus (which have been lost or gained) can potentially lead to determining the enzymes which are responsible for each type of decay. This comparison, however, would not be complete without comparing the transcriptome and the proteome/enzymes. Comparative genomics may tell us which genes may be important, but it will not tell us when these genes are expressed, at what levels and will not necessarily tell us what these genes do