Stabilizing plasmid copy number to improve recombinant protein production

Area of Science:

• Biotechnology
• Molecular Biology
• Microbial Engineering

Background:

• Recombinant protein production in bacteria aims to maximize cellular potential using strong expression vectors.
• High metabolic load from recombinant expression and excessive plasmid replication can lead to host cell exhaustion and reduced protein yield.
• Overstrained transcriptional and translational machineries are critical limitations in current bacterial expression systems.

Purpose of the Study:

• To investigate a method for controlling plasmid replication in bacterial hosts.
• To prevent metabolic overload and maintain host cell productivity during recombinant protein production.
• To optimize bacterial bioprocesses by stabilizing plasmid copy number.

Main Methods:

• A modification was introduced to abolish sequence homology between ColE1 RNA I/RNA II and tRNAs.
• This modification restored the plasmid's native replication control mechanisms.
• Plasmid copy number was maintained at a constant level throughout the culture process.

Main Results:

• The modified plasmids exhibited controlled replication, preventing excessive plasmid amplification.
• Constant plasmid copy number prolonged the metabolic activity and productivity of the bacterial expression system.
• The bacterial host cell's capacity was not exceeded, avoiding system exhaustion.

Conclusions:

• A novel strategy for optimizing bacterial bioprocesses was developed by stabilizing plasmid copy number.
• This method enhances recombinant protein yield by preventing host cell metabolic overload.
• Controlled plasmid replication offers a sustainable approach to bacterial recombinant protein production.