Synthesis and secretion of an Erwinia chrysanthemi pectate lyase in Saccharomyces cerevisiae regulated by different combinations of bacterial and yeast promoter and signal sequences.
Nine different expression-secretion cassettes, comprising novel combinations of yeast and bacterial gene promoters and secretion signal sequences, were constructed and evaluated. A pectate lyase-encoding gene (pelE) from Erwinia chrysanthemi was inserted between each one of these expression-secretion cassettes and a yeast gene terminator, generating recombinant yeast-integrating shuttle plasmids pAMS1 through pAMS9. These YIp5-derived plasmids were transformed and stably integrated into the genome of a laboratory strain of Saccharomyces cerevisiae, and the pectate lyase production was monitored. Transcription initiation signals for pelE expression were derived from the yeast alcohol dehydrogenase (ADC1P), the yeast mating pheromone alpha-factor (MF alpha 1P) and the Bacillus amyloliquefaciens alpha-amylase (AMYP) gene promoters. The transcription termination signals were derived from the yeast tryptophan synthase gene terminator (TRP5T). Secretion of pectate lyase (PLe) was directed by the signal sequences of the yeast mating pheromone alpha-factor (MF alpha 1S), B. amyloliquefaciens alpha-amylase (AMYS) and Er. chrysanthemi pectate lyase (pelES). The ADC1P-MF alpha 1S expression-secretion system proved to be the most efficient control cassette for the expression of pelE and the secretion of PLe in S. cerevisiae.
Laing
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Department of Microbiology, University of Stellenbosch, South Africa.
Pretorius
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D000426:Alcohol Dehydrogenase / Q000235:genetics; D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology; D001483:Base Sequence; D003001:Cloning, Molecular; D004274:DNA, Recombinant; D016974:Dickeya chrysanthemi / Q000201:enzymology*; D005786:Gene Expression Regulation; D000072235:Mating Factor; D008969:Molecular Sequence Data; D010455:Peptides / Q000235:genetics; D010957:Plasmids; D011133:Polysaccharide-Lyases / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic; D021382:Protein Sorting Signals / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D014170:Transformation, Genetic; D014367:Tryptophan Synthase / Q000235:genetics; D000516:alpha-Amylases / Q000235:genetics
D004274:DNA, Recombinant; D010455:Peptides; D021382:Protein Sorting Signals; D000072235:Mating Factor; D000426:Alcohol Dehydrogenase; D000516:alpha-Amylases; D014367:Tryptophan Synthase; D011133:Polysaccharide-Lyases; C023911:pectate lyase