Cloning and characterization of baker's yeast alpha-glucosidase: over-expression in a yeast strain devoid of vacuolar proteinases.
Two alpha-glucosidase (maltase) genes, designated GLUCPI and GLUCPII, have been cloned from an industrial strain of baker's yeast (Saccharomyces cerevisiae) by complementation of a maltase-negative mutant strain. The different genes were identified according to their alternatively expressed isoenzymes PI and PII in transformants after isoelectric focusing and activity staining in separated cell lysates. The gene encoding alpha-glucosidase PI (GLUCPI), which was not present in laboratory strains of S. carlsbergensis with a defined MAL1, 2, 3, 4 or 6 locus, was sequenced and compared with the recently published MAL6S gene. This comparison revealed single amino acid deviations at three positions in the predicted polypeptide sequence. In addition, the divergent promoter region of GLUCPI differed from MAL6S by a triple repeated 147-bp DNA segment. Maltose induction and glucose repression of alpha-glucosidase PI were not affected by the deletion of the repeated DNA segment. However, the absolute expression of alpha-glucosidase PI increased two- to four-fold. In addition, a two-fold increase in the maltase synthesis occurred when the cloned positive regulator gene MAL2-8ep was on the same plasmid. Furthermore, stability of the alpha-glucosidase in cultures in the stationary growth phase was greatly enhanced using a host strain lacking the proteinases A and B and the carboxypeptidases Y and S. Promoter trimming, MAL2-8cp stimulation and the use of a host strain deficient in four vacuolar proteinases resulted in alpha-glucosidase PI expression of about 13% of the soluble protein.
Kopetzki
E
E
Boehringer Mannheim GmbH, Department of Genetics, Penzberg, F.R.G.
Buckel
P
P
Schumacher
G
G
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004271:DNA, Fungal / Q000235:genetics; D005786:Gene Expression Regulation; D005800:Genes, Fungal; D005947:Glucose / Q000378:metabolism; D007525:Isoelectric Focusing; D008320:Maltose / Q000378:metabolism; D008969:Molecular Sequence Data; D010957:Plasmids; D011401:Promoter Regions, Genetic; D012091:Repetitive Sequences, Nucleic Acid; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D012689:Sequence Homology, Nucleic Acid; D014617:Vacuoles / Q000201:enzymology; D000520:alpha-Glucosidases / Q000096:biosynthesis / Q000235:genetics*
D004271:DNA, Fungal; D008320:Maltose; D000520:alpha-Glucosidases; D005947:Glucose