Da Silva, Nancy Anderson (1988) Host-plasmid interactions and regulation of cloned gene expression in recombinant cells. Dissertation (Ph.D.), California Institute of Technology. http://resolver.caltech.edu/CaltechETD:etd-11062007-104408
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Host-plasmid interactions and the regulation of cloned gene expression have been studied in recombinant cells, primarily Saccharomyces cerevisiae. The plasmids employed contain the inducible yeast GAL1, GAL10, or hybrid GAL10-CYC1 promoter cloned upstream of the Escherichia coli lacZ gene (for [beta]-galactosidase). The promoters are controlled by the galactose regulatory circuit, and the yeast strains contain a reg1 mutation which interferes with catabolite repression. Therefore, cloned lacZ gene expression could be induced by galactose addition to glucose-containing medium. Batch and continuous fermentations were performed to study the effects of cloned gene product synthesis on the host cell, and to determine the important factors influencing the kinetics of growth, induction, and cloned gene product formation.
The inducer (galactose) level had a strong influence on cloned lacZ gene expression. In batch culture, the initial specific rate of [beta]-galactosidase synthesis increased with galactose concentration. A galactose concentration of 0.4% (in 0.4% glucose medium) was sufficient for high levels of expression that were approximately proportional to plasmid number (to an average plasmid number of at least 10). The relative galactose level, or galactose/glucose ratio, was also important, and cloned gene expression increased as this ratio increased.
Plasmid stability and promoter strength were also important factors. [Beta]-galactosidase production from an ARS1 plasmid was an order of magnitude lower than for the same plasmid with the [...] origin. The effects of promoter strength were studied in both batch and continuous fermentations. Although at the expense of a growth rate reduction of 16%, the rate of expression from the stronger GAL1 promoter in batch culture was 3-5 times higher than for the GAL10 or GAL10-CYC1 promoters. In continuous culture, steady-state, [beta]-galactosidase specific activity with the GAL1 promoter was 2-4 times greater than that for the GAL10-CYCI promoter. Despite lower plasmid stability (72% versus 86%) and lower biomass concentration (0.98 versus 1.2 g/L), productivity was approximately 3 times higher with the stronger GAL1 promoter system.
Carbon-limited continuous fermentations were utilized to study the effects of dilution rate and induction of cloned gene expression on the recombinant system. Selection for faster growing cells, and the concentrations of glucose and galactose, were important in determining chemostat dynamics. For the GAL10-CYCI promoter system, biomass concentration and [beta]-galactosidase specific activity increased with decreasing dilution rate. The biomass trend is apparently due to the growth efficiency obtained at the various dilution rates, and the utilization of the inducer as an additional carbon source. Plasmid stability dropped after induction of lacZ gene expression and decreased with decreasing dilution rate. Despite lower plasmid stability and flow rate, overall productivity (activity/L/hr) was substantially higher at low dilution rate.
A temperature-sensitive (ts) strain of S. cerevisiae was constructed and characterized. In this strain, cloned lacZ gene expression can be induced by either a temperature-shift (T-shift) from 30°C to 35°C or by galactose addition. In batch culture at 35°C, cloned gene expression induced by T-shift was comparable or better than that observed for galactose induction under the same conditions. Higher levels were observed, however, for galactose induction at the preferred temperature of 30°C. At either temperature, the best levels of [beta]-galactosidase synthesis were obtained for the ts strain in galactose-containing medium.
Theoretical yield factors have been estimated for recombinant cells to investigate the effect of plasmid-directed synthesis on metabolic stoichiometry. The analysis is based upon detailed accounting of ATP utilization for nutrient uptake and biosynthesis. The microorganism considered was E. coli; however, the method can be extended to other organisms for which metabolic ATP requirements can be reasonably estimated.
|Item Type:||Thesis (Dissertation (Ph.D.))|
|Degree Grantor:||California Institute of Technology|
|Major Option:||Chemical Engineering|
|Thesis Availability:||Restricted to Caltech community only|
|Defense Date:||27 April 1988|
|Default Usage Policy:||No commercial reproduction, distribution, display or performance rights in this work are provided.|
|Deposited By:||Imported from ETD-db|
|Deposited On:||27 Nov 2007|
|Last Modified:||26 Dec 2012 03:08|
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