Optimizing initial plasmid copy number distribution for improved protein activity in a recombinant fermentation.

Area of Science:

• Biotechnology
• Molecular Biology
• Biochemical Engineering

Background:

• Recombinant bacterial cells often exhibit heterogeneous plasmid copy numbers, deviating from simplified assumptions.
• Previous studies highlight the limitations of assuming uniform plasmid distribution in fermentation processes.

Purpose of the Study:

• To model the macroscopic distribution of plasmid copy numbers per cell in a Gaussian form.
• To optimize glyceraldehyde-3-phosphate dehydrogenase (GAPDH) production in Escherichia coli by considering plasmid number variance.

Main Methods:

• Representing plasmid copy number distribution using a Gaussian function for biomass fraction.
• Applying an experimentally validated kinetic model to GAPDH synthesis in Escherichia coli with plasmid pBR Eco gap.
• Analyzing batch fermentation parameters to determine optimal conditions for protein yield.

Main Results:

• GAPDH production is maximized by a specific initial (non-zero) plasmid copy number variance.
• An optimal fermentation duration was identified for maximizing protein yield.
• The study demonstrates the impact of plasmid heterogeneity on recombinant protein synthesis.

Conclusions:

• A Gaussian distribution effectively models plasmid copy number heterogeneity in recombinant bacteria.
• Optimizing plasmid variance and fermentation time enhances GAPDH production.
• Discretizing the plasmid distribution and preparing corresponding inocula can improve bioreactor implementation.