Regulated overproduction of alpha-amylase by transformation of the amylolytic yeast Schwanniomyces occidentalis.
High frequency transformation of a Schwanniomyces occidentalis mutant defective in the last step of tryptophan synthesis was achieved with plasmids containing the tryptophan synthetase gene (TRP5) of Saccharomyces cerevisiae and an autonomous replication sequence from S. occidentalis, which we called "SwARS1". The SwARS1 fragment is also functional in S. cerevisiae. The average copy number of the plasmids in both yeast species was 5-10 per cell under selective conditions. S. occidentalis cells that were transformed with an autonomously replicating plasmid carrying the cloned alpha-amylase gene from S. occidentalis secreted about five times more alpha-amylase than cells without additional copies of the alpha-amylase gene. Both the chromosomal copy and the plasmid-carried copies of the alpha-amylase gene were repressed in the presence of glucose. This transformation system provides a possibility to improve starch degradation by S. occidentalis.
Dohmen
R J
RJ
Institut für Mikrobiologie, Heinrich-Heine-Universität Düsseldorf, Federal Republic of Germany.
Strasser
A W
AW
Zitomer
R S
RS
Hollenberg
C P
CP
D015139:Blotting, Southern; D003001:Cloning, Molecular; D004261:DNA Replication; D004271:DNA, Fungal / Q000235:genetics; D005786:Gene Expression Regulation; D005800:Genes, Fungal; D005816:Genetic Complementation Test; D007700:Kinetics; D009154:Mutation; D010957:Plasmids; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*; D004718:Saccharomycetales / Q000201:enzymology / Q000235:genetics*; D012689:Sequence Homology, Nucleic Acid; D014170:Transformation, Genetic; D014367:Tryptophan Synthase / Q000235:genetics; D000516:alpha-Amylases / Q000096:biosynthesis*
D004271:DNA, Fungal; D000516:alpha-Amylases; D014367:Tryptophan Synthase