Two-stage continuous operation of recombinant Escherichia coli using the bacteriophage lambda Q- vector

Area of Science:

• Biotechnology
• Molecular Biology
• Microbial Engineering

Background:

• Plasmid instability is a common challenge in recombinant fermentation using Escherichia coli.
• Bacteriophage lambda systems offer a potential solution but require optimization for stability and expression.

Purpose of the Study:

• To overcome plasmid instability in Escherichia coli recombinant fermentation.
• To enhance the expression of a cloned gene using a bacteriophage lambda system.
• To determine optimal operational parameters for a two-stage continuous culture.

Main Methods:

• Utilized a two-stage continuous culture of Escherichia coli with a phage lambda vector (Q(-) mutation).
• Optimized substrate concentration, dilution rate, and mean residence time.
• Assessed gene expression and plasmid stability in batch and continuous cultures.

Main Results:

• Full induction of the cloned gene occurred 4 hours post-temperature shift across all culture modes.
• Two-stage continuous culture maximized overproduction at a dilution rate of 0.25 h(-1) with 1.5 S(0) medium supply.
• Achieved a maximum beta-galactosidase productivity of 16.3x10^6 U l(-1) h(-1), seven times higher than single-copy lysogenic cultures.
• Recombinant cells remained stable in the lysogenic state for over 260 hours, compared to 40 hours in the lytic state.

Conclusions:

• The optimized two-stage continuous culture system significantly enhances recombinant protein production and plasmid stability.
• Instability in the lytic stage is attributed to lysis protein accumulation from vector leakage.
• This approach provides a robust method for high-yield, stable recombinant fermentation.