Expression in yeast of a Bacillus alpha-amylase gene by the ADH1 promoter

Abstract

A DNA fragment of Bacillus amyloliquefaciens encoding alpha-amylase was cloned and combined with the yeast ADH1 promoter on Escherichia coli/Saccharomyces cerevisiae shuttle vectors and transformed into S. cerevisiae laboratory strains. The Bacillus gene expression in yeast was found essentially to depend on activity of the yeast ADH1 promoter in plasmid pAAH5, which also contained the ADH1 terminator sequence. The yeast transformants were characterized as to the presence of the recombinant plasmid, identity of the cloned Bacillus DNA fragment, mitotic stability and significance of the alpha-amylase activity. By Northern blotting of total RNA isolated either from E. coli or yeast transformants, 1 or 3 transcripts, respectively, were identified by hybridization with the ClaI/ BamHI Bacillus DNA fragment containing the amylase gene. Variable length of amylase gene transcripts is suggested to be due to not strictly determined transcription (probably termination) within Bacillus DNA. Alpha-amylase was determined by enzymographic techniques after isoelectric focusing of yeast cellular proteins and found to differ in its isoelectric point from alpha-amylase of B. amyloliquefaciens. Incubation of pAAH5-amy transformed yeast cells on starch media showed, after a prolonged period of time, hydrolytic activities as well as cell division, thus indicating substrate utilization.