Modified expression of multi-cellulases in a filamentous fungus Aspergillus oryzae

Abstract

Aspergillus oryzae, a filamentous fungus, can secrete large amounts of enzymes extracellularly. We constructed a genetically engineered A. oryzae that simultaneously produced cellobiohydrolase, endoglucanase, and β-glucosidase by integrating multiple copies of the genes encoding these cellulases into fungal chromosomes. The resulting strain possessed 5–16 copies of each cellulase gene within the chromosome and showed approximately 10-fold higher activity versus single integration strains. Copy number polymorphisms were attributed to differences in flanking region sequence for the integrated gene fragments. Furthermore, we found that the P-sodM/T-glaB set demonstrated the strongest transcription levels per gene copy number. We therefore modified promoter/terminator set and cellulase gene combinations based on this polymorphism and transcription level data, with the resulting transformant showing 40-fold higher cellulolytic activity versus the single integration strain. This designed expression method could be useful for the overexpression of multiple enzymes and pathway flux control-mediated metabolic engineering in A. oryzae.