The nucleotide sequence of a 1.5-kb fragment of the promoter region of the Aspergillus oryzae agdA gene encoding alpha-glucosidase was determined. A comparison with the promoter regions of other Aspergillus amylase genes indicated that there are three highly conserved sequences, designated Regions I, II and III, located at -670 nt, -596 nt and -544 nt relative to the start codon, respectively. The function of these consensus sequences in the agdA promoter was investigated by deletion analysis of a promoter fusion with the Escherichia coli uidA gene, using the niaD homologous-transformation system. Deletion of the upstream half of Region III (IIIa; -544 to -529) resulted in a more than 90% reduction in GUS activity and abolished maltose induction, suggesting that Region IIIa is a functionally essential element for high-level expression and maltose induction. Deletion of Region I and the downstream half of Region III (IIIb; -521 to -511) resulted in a significant reduction in GUS activity, but did not affect maltose induction. This suggested that these two elements most likely contain sequences involved in efficient expression in cooperation with Region IIIa. In addition, deletion of a 340-bp region between Region IIIb and the putative TATA box resulted in a 2-fold increase in activity.

No abstract

A new rapid transformation system for Aspergillus niger that uses electroporation to render intact germinating conidia permeable to DNA is described. The transformant colonies appeared earlier than transformants obtained by the protoplast-forming method. Without pretreatment of the conidia the transformation frequencies were 1.2 colonies per micrograms of integrative vector and 100 colonies per micrograms of plasmid DNA. When the conidia were treated with a dilute solution of fungal cell wall lytic enzyme, the frequency of transformation was increased by approx. 2-fold when using two vectors. Southern blot analysis of genomic DNA and restriction endonuclease-digested DNA from a random sample of transformants showed homologous and nonhomologous integration of the integrative vector into the genome, as is also observed with the protoplast-forming method. In transformation with the plasmid vector, the transformant DNA was shown to be mostly maintained in free form with minimal integration into the chromosome when transformed by either intact electroporation or the conventional method.

No abstract

No abstract