14-11: Construction of new starch-inducible protein expression system of Acremonium cellulolyticus to produce cellulolytic enzymes

Lignocellulosic biomass is an attractive material to produce alternative fuels and chemicals in biorefineries. Cellulolytic enzymes from filamentous fungi, such as Trichoderma reesei, are widely employed for the hydrolysis process of biomass. Acremonium cellulolyticus CF-2612 is one of several promising fungi that are competitive with T. reesei for the production of cellulolytic enzymes. Suitable protein expression system must be required to better understand the individual cellulolytic enzymes from A. cellulolyticus. Heterologous expression of fungal cellulolytic enzymes often results in the production of proteins having altered properties such as insoluble inclusion body and hyper-glycosylated. To circumvent this problem, in this study, we have constructed a starch-inducible protein expression system to produce recombinant cellulolytic enzymes from self-cloning A. cellulolyticus strains. Amylases (α-amylase, glucoamylase, and α-glucosidase) were produced in the starch culture of A. cellulolyticus, whereas cellulolytic enzymes were hardly detected. Glucoamylase was purified, and its gene including the promoter and terminator regions was identified by amino acid sequence analysis of peptide fragments and genome sequence analysis. An expression plasmid, containing the 1.4-kb promoter and 0.4-kb terminator regions of glucoamylase gene and pyrF (orotate phosphoribosyl transferase gene) as a selection marker, was constructed. The cellulolytic enzymes Cel7A, Cel5A, and Xyl10A were successfully expressed under control of the glucoamylase promoter in starch cultures of self-cloning A. cellulolyticus strains. The recombinant enzymes exhibited the same thermal stability as the corresponding native enzymes from cellulose cultures. This study will provide a new expression system for the production of fungal cellulosic enzymes.