Modifications to the ADH1 promoter of Saccharomyces cerevisiae for efficient production of heterologous proteins.
The promoter of alcohol dehydrogenase I of the yeast Saccharomyces cerevisiae was studied using Bacillus amyloliquefaciens alpha-amylase as a marker protein. On glucose, activity of the original ADH1 promoter decreases during late exponential, ethanol production growth phase. When 1100 bp (from -414 bp to -1500 bp) of the upstream sequence are deleted, activity increases into the late ethanol consumption phase but the promoter becomes active only after ethanol production growth phase (Ruohonen et al. (1991) Yeast 7, 337-346). We have now restored 300 bp (from -414 bp to -700 bp) upstream of the deletion site and obtained expression from the ADH1 promoter throughout the yeast growth cycle. The restored sequence allowed alpha-amylase expression to start during early exponential growth phase indicating that it is required for activation of the ADH1 promoter during ethanol production growth phase, possibly through glucose induction. On ethanol, all the promoters were active, but the short promoter was temporally activated first, suggesting that the restored sequence is not required for promoter activity during early oxidative growth.
Ruohonen
L
L
VTT Biotechnology and Food Research, Espoo, Finland.
Aalto
M K
MK
Keränen
S
S
D000426:Alcohol Dehydrogenase / Q000235:genetics*; D001407:Bacillus / Q000201:enzymology / Q000235:genetics; D015152:Blotting, Northern; D000431:Ethanol / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D005947:Glucose / Q000378:metabolism; D007527:Isoenzymes / Q000235:genetics; D010957:Plasmids; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D017384:Sequence Deletion; D014158:Transcription, Genetic; D000516:alpha-Amylases / Q000096:biosynthesis* / Q000235:genetics
D007527:Isoenzymes; D012333:RNA, Messenger; D000431:Ethanol; D000426:Alcohol Dehydrogenase; D000516:alpha-Amylases; D005947:Glucose