論文の内容の要旨

氏

名JULIANO DE OLIVEIRA PORCIUNCULA

With the increasing concerns about depletion of petroleum supplies, there has
been an increasing need in explore alternative energy sources. Plant biomass has been
received considerable attention as the most promising alternative energy resource toovercome these problems. Plant biomass must first be converted to fermentable sugars
such as glucose by hydrolytic enzymes before being converted to bioethanol. The
ascomycete Trichoderma reesei is known to produce large amount of hydrolases thatconvert more efficiently plant biomass. Because of the industrial importance andvariety of uses of T. reesei hydrolases, several mutants have been developed byclassical mutagenesis to improve cellulase productivity. In this work, we investigatedby comparative genome analysis single nucleotide polymorphisms (SNPs) that could beinvolved in high cellulase expression in T. reesei mutant. We also analyzed for the
first time sugar transporters that are potentially involved in lactose uptake in T.

reesei.
In Japan, the T. reesei mutant PC-3-7 has been developed by several rounds ofrandom mutagenesis. During the screening, we found that this strain exhibited carbon
catabolite derepressed phenotype and hyper-cellulase productivity in inducing
conditions. To better understand the genetic basis of PC-3-7 phenotype, we sequenced
the genome of this strain using Next Generation Sequencing and identified a total of154 SNPs. Among them, one was found in the cre1 gene, the carbon catabolite repressorof PC-3-7. Gel shift analysis revealed that the mutant CRE1 was unable to bind to its
consensus binding site in the cbh1 promoter in vitro. PC-3-7 strain transformed withthe wild-type cre1 clearly showed a pattern of carbon catabolite repression in inducing
and repressing conditions, while PC-3-7 with the SNP in cre1 exhibited a derepressedphenotype in the same conditions. Cellulase expression was ten to thirty times low whenthe mutated cre1 was replaced by the wild-type cre1 in PC-3-7. These data clearlyindicates that the SNP in cre1 is one of the factors influencing the hyper-cellulolytic
phenotype of PC-3-7.
The expression of cellulases and hemicellulases of T. reesei are induced bycellulose and oligosaccharides, such as sophorose. However these inducers are still
too expensive for economical production of cellulases at the industrial level. Lactose
is a cheap alternative inducer found in large amounts as a by-product of cheese andwhey production. Cellulase yields produced on lactose are still lower compared tocellulose. Previous studies suggested that lactose must first be internalized to induce
cellulase gene expression. To improve cellulase inducibility by lactose, two sugartransporters highly expressed in inducing conditions were investigated. Disruptant

strains of these sugar transporters exhibited a delayed uptake of lactose compared with
PC-3-7. Disruption of these two transporters also affected growth and cellulase
expression. When cultivated in cellobiose or Avicel culture, no significant differencesin cellulase production were observed among these strains. These data clearly indicatesthat these two transporters are specific for lactose uptake and are important forcellulase expression in the same condition.