Low Metabolic Burden Fermentation
Constitutive high plasmid copy number throughout the fermentation process is not necessary or desirable. Maintaining high copy numbers during biomass accumulation creates an environment in which plasmid-free cells have a significant growth advantage.
Plasmid-mediated metabolic burden can inhibit biomass growth and may lead to stability or quality problems (e.g., deletions or dimerization) with many plasmids. Thus, maintaining low cell stress/metabolic burden during biomass accumulation, and inducing high plasmid copy numbers for only the final portion of the process leads to higher volumetric plasmid yields while preserving plasmid quality.
This has been achieved by using a temperature-inducible, fed-batch process (Figure 2A) with an optimized semi-defined medium. The initial temperature setpoint is 30°C to keep the plasmid copy number at its minimum. Feed medium containing concentrated glycerol is added according to an exponential feeding strategy to control biomass growth at about 0.12 h-1.
After sufficient biomass growth, the temperature is shifted to 42°C and growth continues for up to about one doubling of cell mass. This process has resulted in volumetric plasmid yields up to 2.2 g/L, and specific plasmid DNA yields up to 51 mg/g DCW (5% of the total dry cell weight). Figure 2B shows a time profile from a fermentation that reached a plasmid yield of 2.1 g/L.