The isolation of strains of Saccharomyces cerevisiae showing altered plasmid stability characteristics by means of selective continuous culture.
A recombinant strain of Saccharomyces cerevisiae containing a plasmid-encoded lacZ gene from Escherichia coli was grown for 420 generations under selective conditions in glucose-limited continuous culture. A ura3-based auxotrophic system was used to apply selection in favour of plasmid-containing organisms. A similar strategy had previously proved successful at evolving clones of Bacillus subtilis, showing improved plasmid stability characteristics. In this study a series of clones were isolated which exhibited large variation in their ability to retain the recombinant plasmid. Clones showed both significantly increased and reduced capacity to maintain the recombinant plasmid. The probabilities of obtaining clones in either category were essentially equal so that selection was not seen to enrich for more stable clones. Periodic selection events appeared to exert a greater influence on the distribution of stability characteristics amongst clones than did the applied selective pressure. Alterations in plasmid retention characteristics could be associated with host or plasmid. The most stable clone isolated exhibited a approximately 30% improvement of its overall stability (sigma(N+)) and an 80% improvement in productivity, when compared to the parental strain CGpLG. This improved stability was associated with alterations in the plasmid genome.
O'Kennedy
R D
RD
Department of Microbiology, National University of Ireland, Galway, Ireland.
Patching
J W
JW
D003470:Culture Media; D004926:Escherichia coli / Q000235:genetics; D005818:Genetic Engineering; D005947:Glucose / Q000378:metabolism; D007763:Lac Operon; D010957:Plasmids / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000254:growth & development / Q000302:isolation & purification*; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
D003470:Culture Media; D001616:beta-Galactosidase; D005947:Glucose