Optimization of Bacillus alpha-amylase production by Saccharomyces cerevisiae.
Production of Bacillus amyloliquefaciens alpha-amylase by Saccharomyces cerevisiae using the multicopy plasmid pAAH5 and ways of improving the yields of secreted enzyme were studied. In standard non-buffered medium, alpha-amylase was rapidly inactivated but stabilization of the pH at 6 led to stable accumulation of alpha-amylase in the culture medium. Removal of 1100 bp of the upstream sequence of the ADH1 promoter present on pAAH5 resulted in delayed but increased alpha-amylase production: 29-fold in selective medium, two-fold in non-selective medium. With the original ADH1 promoter, accumulation of alpha-amylase in the medium started to level off before the cultures reached stationary phase and was very low when exponentially growing cells were transferred from glucose to ethanol. This coincided with the appearance of a mRNA larger than the alpha-amylase messenger. With the shortened promoter, the normal-size alpha-amylase mRNA was detected under all growth conditions and alpha-amylase was efficiently secreted into the medium also late in stationary phase and after transfer to ethanol. Highest total yields of alpha-amylase were obtained with the short promoter in non-selective glucose-containing medium; this may be explained by the greater final cell density obtained. However, the production of alpha-amylase per cell mass was higher in ethanol-containing selective medium. Seventy to eighty per cent of the alpha-amylase activity was secreted into the medium independent of the total amount produced.
Ruohonen
L
L
Recombinant DNA Laboratory, University of Helsinki, Finland.
Penttilä
M
M
Keränen
S
S
D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D015152:Blotting, Northern; D003470:Culture Media; D000431:Ethanol / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D006863:Hydrogen-Ion Concentration; D016296:Mutagenesis; D010957:Plasmids; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D014158:Transcription, Genetic; D000516:alpha-Amylases / Q000096:biosynthesis* / Q000235:genetics
D003470:Culture Media; D012333:RNA, Messenger; D000431:Ethanol; D000516:alpha-Amylases