Optimal gene expression and amplification strategies for batch and continuous recombinant cultures
Maximizing the amount of protein produced from a cloned gene in a recombinant organism requires careful consideration of the trade-offs involved between cloned gene expression and host cell growth and biosythetic activity. Numerous experimental studies of recombinant Escherichia coli and Saccharomyces cerevisiae have shown that the presence of plasmids reduces host cell growth rate and, overall protein synthesis activity. Reduction host cell growth rates and biosynthetic activity in the presence of plasmid-directed macromolecular synthesis presumably occurs because of competition between plasmid-directed and host-cell-directed activity for common pools of precursors, chemical energy and electrons, activator species, repressor molecules, transport apparatus, and enzymes and other catalytic assemblies. The use of regulated promoters and plasmid replication controls amenable to environmental manipulation offers the opportunity of reconciling the opposing effects of cloned-gene content and expression level on process productivity. Several promoters are available for E. coli, S. cerevisiae, and other hosts that allow the expression level of the cloned gene to be switched from a relatively low to a relatively high level by a change in the organism environment. Similarly, in a plasmid replicon repressed by a temperature-sensitive molecule, such as the ColE1 origin of replication for E. coli plasmids with a mutant RNA I gene providing temperature-sensitive replication repressor activity, cellular plasmid content can be altered from around 25 to 700 or more copies per cell by increasing the medium temperature. Similar temperature-sensitive replication regulators are known for R1 plasmids.
- Research Organization:
- California Institute of Technology (CalTech), Pasadena, CA (United States)
- OSTI ID:
- 6155733
- Journal Information:
- Biotechnol. Bioeng.; (United States), Vol. 29:3
- Country of Publication:
- United States
- Language:
- English
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09 BIOMASS FUELS
ESCHERICHIA COLI
GENETIC ENGINEERING
SACCHAROMYCES CEREVISIAE
ACTIVATION ANALYSIS
BATCH CULTURE
BIOCHEMICAL REACTION KINETICS
BIOCHEMISTRY
BIOREACTORS
BIOSYNTHESIS
CATALYSIS
CHEMICAL REACTIONS
CLONING
CONTINUOUS CULTURE
ELECTRON TRANSFER
ENERGY
ENVIRONMENT
ENZYMES
GENE AMPLIFICATION
GENE RECOMBINATION
GROWTH
INHIBITION
MATHEMATICAL MODELS
OPTIMIZATION
PERFORMANCE
PLASMIDS
PROCESS CONTROL
SIMULATION
TEMPERATURE DEPENDENCE
BACTERIA
CELL CONSTITUENTS
CHEMICAL ANALYSIS
CHEMISTRY
CONTROL
FUNGI
KINETICS
MICROORGANISMS
PLANTS
REACTION KINETICS
SACCHAROMYCES
SYNTHESIS
YEASTS
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