### 9. 英文概要

[Development of Microbial Conversion Technologies Related to Utilization of Waste Mushroom Bed as the Local Biomass Resources]

Lignocellulosic biomass has received much attention in recent years as renewable feedstock due to the exacerbation of energy crisis and global warming. Niigata prefecture is one of the major mushrooms producing areas in Japan, and mainly produces maitake mushroom (Grifola frondosa). Maitake mushroom is cultivated on the medium mainly consisting of hard wood sawdust (containing about 46% of cellulose) thus it can be used as a biomass feedstock for biorefinery. Therefore we are focusing on the use of the waste mushroom bed as excellent local biomass, and developing a bioconversion system of the biomass into valuable chemicals. This system comprises mainly two-steps of conversion: (I) hydrolysis for the production of fermentable sugars by well known cellulolytic fungi Trichoderma reesei, and (II) production of 2-deoxy-scyllo-inosose (DOI), which is a precursor of dihydric phenol, by fermentation using recombinant Escherichia coli. However plant biomass has a complicated structure showing resistance to biodegradation, this bioconversion process needs pre-treatment procedure to increase saccharification efficiency. Previous studies showed that storage of waste mushroom bed after harvesting fruit bodies was an effective pretreatment for saccharification. In this process, it was revealed that the composition of lignin in the waste mushroom bed decreased. The purpose of this study are 1) to clarify which enzymes are produced by Maitake mushroom during storage of mushroom bed, 2) to create Maitake strain enhancing its pretreatment ability, 3) to develop biomass efficient pretreatment – saccharification process, 4) to utilize saccharides derived from mushroom bed for DOI production, and 5) to enhance the DOI productivity.

To analyze which enzymes are produced by G frondasa, genome database was created by using next generation sequencer. In the G. frondasa genome, at least 16,000 genes were annotated and DNA microchip arrayed them are designed. DNA microarray analysis of gene expressed in mushroom bed from 0, 30, 60, and 90 days storage were carried out. Consequently, specifically expressed genes during storage periods were identified. Majority of them were encoding lignin degrading enzymes and carbohydrate degrading enzymes.

Mutagenize of G. frondasa was carried out by using UV irradiation. To screen mutant strain having enhanced lignolytic activity, enzyme assay method on plate medium was established. As a result of screening of mutated G. frondasa strain, 3 strains showed higher lignolytic activity than parental strain.

Enzyme addition to cellulase degradation of mushroom bed for long term storage enhanced the degree of saccharification. In addition, alkaline pretreatment made mushroom bed susceptible to degradation by cellulase, resulting in 80% of saccharification ratio. These data also imply that efficient removal of lignin component from mushroom bed brings more effective saccarification. Although enzyme amounts in mushroom bed was thought not to be enough, lignolytic enzymes highly expressed in mushroom bed were expressed in Trichoderma reesei which is cellulolytic fungus having potent protein production ability. Consequently, lignolytic enzymes from G. frondasa were successfully expressed in T. reesei but expression amount was faint. When endogenous lignolytic enzymes were over-expressed in T. reesei, laccase expressing strain was successfully constructed. To utilize enzyme preparation produced this strain, it is possible to carry out efficient saccharification of the waste mushroom bed of G. frondasa.