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 an 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 (MFalpha1P) and the Bacillus amyloliquefaciens alpha-amylase (AMY(P)) 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 (MFalpha1s), B. amyloliquefaciens alpha-amylase (AMY(S)) and Er. chrysanthemi pectate lyase (pelE(S)). The ADC1P-MFalpha1S expression-secretion system proved to be the most efficient control cassette for the expression of pelE and the secretion of PLe in S. cerevisiae.