CLONING AND CHARACTERIZATION OF 3 ASPERGILLUS-NIGER PROMOTERS

An Aspergillus niger (An) genomic library was constructed using the promoter-trap vector, pLX2A, which contains a hygromycin B (Hy) phosphotransferase-encoding gene (hph) for selection of DNA fragments with promoter activity. This library was transformed in Escherichia coli and 80000 colonies were obtained, 94% of which contained inserts. Transformations of plasmid DNA from the library into An resulted in 53 Hy-resistant (Hy(R)) colonies. Southern blot analysis of 21 transformants confirmed the integration of hph into the Art genome. Using the sib selection procedure, three functional promoters, PX6, PX18 and PX21, were identified from this library. Both DNA strands of all three fragments were sequenced and their sequences showed no significant homology to those in the database. Comparison of the sequences of all known promoters from An suggested that C+T-rich stretches are probably important for promoter structures. The promoter activity was analysed further using beta-galactosidase (beta Gal) as a quantitative marker. The results suggest that while PX21 is a much stronger promoter than the known or-amylase promoter of A. oryzae, PX6 promotes only weak expression of beta Gal.