Enhanced genetic stability and expression control in growth-decoupled continuous two-stage E. coli fermentations...

Area of Science:

• Biotechnology
• Microbial Engineering
• Synthetic Biology

Background:

• Continuous two-stage fermentation in E. coli is efficient but challenged by plasmid instability and unintended gene expression.
• Plasmid instability and genetic drift limit the reliability of E. coli in long-term bioprocesses.
• Auxotrophic selection systems offer a potential solution for maintaining genetic stability in microbial cultures.

Purpose of the Study:

• To develop and evaluate a plasmid-dependent auxotrophic selection system using thymidine auxotrophy (thyA deletion) for enhanced E. coli fermentation.
• To engineer E. coli strains and plasmids for improved stability, controlled expression, and reduced basal expression.
• To validate the system's efficacy in continuous two-stage fermentations over extended periods.

Main Methods:

• Engineered E. coli strain enGenes-eXpress V2 ΔthyA and modified plasmids containing thyA.
• Conducted continuous two-stage fermentations under carbon-limited conditions.
• Utilized microbioreactor screenings, fed-batch cultivations, and single-cell analyses for validation.

Main Results:

• Demonstrated significant reduction in plasmid loss and improved population homogeneity.
• Observed suppressed basal expression in non-induced phases with modified plasmids.
• Achieved robust performance in continuous chemostat fermentations exceeding 1000 hours with stable GFP titers and cell mass.

Conclusions:

• The thyA-based auxotrophic selection system, coupled with plasmid modifications, substantially enhances genetic stability and productivity in E. coli continuous bioprocesses.
• This approach offers a sustainable, antibiotic-free platform for industrial biotechnology, suitable for long-term continuous fermentations.
• Optimized constructs maintain homogeneous producing populations and suppress non-producing cells, ensuring process reliability.