Genetic engineering of Saccharomyces cerevisiae for efficient expression and secretion of Erwinia pectate lyase and polygalacturonase

Dissertation (Ph. D.) -- University of Stellenbosch, 1992.One copy microfiche.Full text to be digitised and attached to bibliographic record

A note on the primary structure and expression of an Erwinia carotovora polygalacturonase-encoding gene (peh1) in Escherichia coli and Saccharomyces cerevisiae

A l209-base pair (bp) DNA fragment containing the endopolygalacturonase-encoding gene (peh1) from Erwinia carotovora subsp. carotovora was amplified by the polymerase chain reaction (PCR) technique and expressed in Escherichia coli. The nucleotide sequence of the PCR product was determined and found to be highly homologous to the primary structures of other polygalacturonase-encoding genes. The peh1 DNA fragment encoding the mature polygalacturonase was inserted between two different yeast expression-secretion cassettes and a yeast gene terminator, generating recombinant yeast-integrating shuttle plasmids pAMS10 and pAMS11. These Ylp5-derived plasmids were transformed and stably integrated into the genome of a laboratory strain of Saccharomyces cerevisiae. Transcription initiation signals present in these expression-secretion cassettes were derived from the yeast alcohol dehydrogenase (ADC1(p)) or mating pheromone α-factor (MPα1(p)) gene promoters. The transcription termination signals were derived from the yeast tryptophan synthase gene terminator (TRP5(T)). Secretion of polygalacturonase was directed by the signal sequence of the yeast mating pheromone α-factor (MTα1(S)). Northern blot analysis revealed the presence of peh1 mRNA in the yeast transformants and a polypectate agarose test was used to monitor polygalacturonase production.10 page(s

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 pAMSl 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 α-factor (MFα1P) and the Bacillus amyloliquefaciens α-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 α-factor (MFα1S), B. amyloliquefaciens α-amylase (AMYS) and Er. chrysanthemi pectate lyase (pelES). The ADClP-MFα1S expression-secretion system proved to be the most efficient control cassette for the expression of pelE and the secretion of PLe in S. cerevisiae.11 page(s

Expression of a Bacillus α-amylase gene in yeast

A recombinant plasmid, pSR11.3, containing the α-amylase gene (AMY) of Bacillus amyloliquefaciens was characterized and expressed in Bacillus subtilis. A 2.3 kilobase BamHI-BglII fragment carrying AMY was cloned into pBR322 (pEL322) and in both orientations into a multi-copy Escherichia coli-yeast shuttle vector YEp13 (pAM13) and expressed in E. coli HB101 and various Saccharomyces strains. We report on the successful secretion of an active bacterial enzyme in yeast without using yeast promoter and secretory signals. Enzyme production in B. subtilis 1A297(pSR11.3), E. coli HB101(pEL322) and Saccharomyces JM277315B(pAM13) transformants was measured as 125, 22 and 123 U/ml, respectively. The molecular weight of the purified α-amylase secreted by B. subtilis 1A297(pSR11.3) and Saccharomyces JM2773-15B(pAM13) was estimated to be 55 kDa. The pH and temperature optima for the α-amylase activities of the transformants were 6.5 to 8.0 and 50 to 65 °C, respectively. Amylose hydrolysis profiles of the α-amylases secreted by B. subtilis 1A297(pSR11.3) and Saccharomyces JM2773-15B(pAM13) indicate effective meso-thermostable hydrolytic enzymes with maltotriose and maltose, respectively, as major end products.8 page(s