The yeast Saccharomyces cerevisiae has certain advantages as a host for the production of heterologous proteins. It is a eukaryote, in which the production of proteins of higher eukaryotes may be more successful than in bacterial cells. Specific posttranslational modifications of eukaryotic proteins are likely to take place in yeast. Yeast has the capacity to secrete proteins outside the cell, which offers the possibility for the production of secretory proteins. However, the rather modest intrinsic secretory capacity of S. cerevisiae is a drawback of the yeast expression system. The glycosylation pattern of yeast secretory proteins differs from that of higher eukaryotes. Thus it is of interest to study the effects of yeast specific glycans on heterologous proteins. The promoter of the yeast alcohol dehydrogenase 1 gene (ADH1) was among the first regulatory regions used for heterologous gene expression. Characterisation of the original promoter fragment has revealed an additional upstream promoter element, which was suggested to be responsible for the down-regulation of the promoter activity during the later stage of the fermentative growth cycle or during growth on non-fermentable carbon sources. In the present study, the original ADH1 promoter was modified in order to increase the production level of Bacillus amyloliquefaciens a-amylase used as a model secretory protein. The promoter (long ADH1 promoter) was active only during fermentative growth on glucose or prolonged cultivation on ethanol. Two altered promoter constructions were made. Deletion of 1100 bp upstream from the original promoter resulted in activation of the modified promoter (short ADH1 promoter) only after fermentative growth, i.e. during the ethanol consumption phase, or during growth on ethanol. When 300 upstream bp, containing a UAS element, were restored to the deleted promoter construction, activity of the promoter (middle ADH1 promoter) reappeared during fermentative growth and continued until well into the growth cycle. The activation during growth on ethanol was less delayed than with the original promoter. The primary carbon source affected the promoter activity during the growth cycle. The short promoter was active during respiratory growth on ethanol, whereas the original and middle promoter were activated during early growth on glucose. The concomitant appearance of a nonfunctional messenger RNA and disappearance of a-amylase activity during late fermentative growth showed that the upstream promoter element is responsible for the down-regulation of the original promoter. Once this upstream region was deleted the promoter was constitutively expressed. Gluconeogenesis may activate the original and middle promoter during growth on ethanol. The bacterial signal peptide of a-amylase was functional and correctly processed in yeast cells. a-Amylase was transported through the entire yeast secretory route and N-linked core-glycans were added to the a-amylase during export. Interestingly, the presence of glycans did not abolish the enzymatic activity of a-amylase. A significant increase in a-amylase production was obtained by promoter optimisation. The secretion capacity of yeast was enhanced when a component of the yeast secretory machinery, the Sso2 protein, was simultaneously expressed with a-amylase in yeast cells. Varying the Sso2p level during the growth cycle using the original and middle ADH1 promoter revealed a correlation between the amount of Sso2p and the level of secretion. This offers a means to improve secretion efficiency in yeast.
|Award date||14 Dec 1995|
|Place of Publication||Espoo|
|Publication status||Published - 1995|
|MoE publication type||G5 Doctoral dissertation (article)|
- genetic engineering
- Sec proteins
- ADHI proteins