Development of high expression system with the improved promoter using the cis-acting element in Aspergillus species

Aspergillus species are widely used for the production of useful enzymes. We found that there exist three highly conserved sequences, designated Region I, II and III, in the promoter regions of the genes encoding α-amylase (amyB), glucoamylase (glaA), and α-glucosidase (agdA) of A. oryzae. Deletion analyses of the promoter regions indicated that Region III is mainly involved in high level expression and maltose induction of the amylase genes. Introduction of the multiple copies of the fragment comprising Region III into the agdA promoter resulted in a significant increase in promoter activity at the transcriptional level. In addition, introduction of the multiple copies of Region III into No.8 promoter that was isolated from A. niger also resulted in a remarkable increase. Using the improved promoter, a remarkably high yield of nuclease S1 and 1,2-α-D-mannosidase, of which production levels are low in wild-type strain, could be gained. In such transformants, the production of α-amylase and glucoamylase was significantly reduced, simultaneously. These phenomena could be caused by titration of the common regulatory protein(s) which interact with Region III. In this study, we have shown the presence in A. oryzae of a common regulatory mechanism interacted with Region III in the expression of the amylase genes. Moreover, we have demonstrated the advantage of over-expression of genes controlled by the improved promoter using Region III in various Aspergillus species.