Genetic construction of starch-utilizing strain of Saccharomyces cerevisiae and stabilization of its recombinant plasmid by immobilization
Kim, Chung Gyu
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Permalink
https://hdl.handle.net/2142/19335
Description
Title
Genetic construction of starch-utilizing strain of Saccharomyces cerevisiae and stabilization of its recombinant plasmid by immobilization
Author(s)
Kim, Chung Gyu
Issue Date
1990
Doctoral Committee Chair(s)
Witter, Lloyd D.
Department of Study
Food Science and Human Nutrition
Discipline
Food Science
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Biology, Molecular
Agriculture, Food Science and Technology
Biology, Microbiology
Language
eng
Abstract
DNA containing the nucleotide sequence for $\alpha$-amylase activity has been isolated from Bacillus stearothermophilus and shot-gun cloned into E. coli host using a plasmid vector pUC19 by electroporation-induced transformation. The hybrid plasmid (designated as pCK101) containing the amylase gene, after purification of the gene by subcloning (i.e., the trimming and manipulation of the amy$\sp{+}$ fragment from pUC101), was transformed from the host organism to S. cerevisiae using E. coli/yeast shuttle vector, pMF$\alpha$8 by electroporation transformation procedure. When the purified B. stearothermophilus $\alpha$-amylase gene was fused to the pMF$\alpha$8 just after S. cerevisiae MF$\alpha$1 promoter and secretion signals coding sequence, the transformants of S. cerevisiae cells were successfully able to synthesize and secrete functional $\alpha$-amylase efficiently hydrolyzing starch present in the culture medium. Further studies on the characterization of cloned gene was done.
When plasmid stability was studied in continuous air bubble bioreactor, there was a generally observed loss of plasmid with time. The plasmid containing whole cells of S. cerevisiae was immobilized in k-carrageenan and allowed to react for varying period time. Cells were then released from immobilization and examined for plasmid stability, cell mass production and $\alpha$-amylase production. The transformed S. cerevisiae was then compared for free and immobilized system with and without selection pressure. Data showed that the immobilization of cells carrying recombinant plasmid could improve the plasmid stability. The significantly higher cell concentration and consequently higher productivity of the plasmid-coded $\alpha$-amylase product was also obtained in the comparison to free cell system.
This research project was initiated in the hope of developing a heterogeneous gene expression and secretion system in S. cerevisiae by choosing the enzyme $\alpha$-amylase as a model system. Also, it was great concern to associate the methods of both genetic and immobilization engineering in this project. Those results showed good prospectives on its practical use of immobilization technique as a new strategy for maintaining a stable population of plasmid-carrying S. cerevisiae.
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