By enGenes Biotech…
Subscribe to Supplier
Subscribe to Supplier
Keeping E.coli production strain productive over 1,000 hours
Vienna, Austria: – Florian Simon, a doctorate researcher at recombinant proteins and plasmid DNA specialist CDMO enGenes Biotech, has helped pioneer a new method that overcomes one of biomanufacturing’s biggest challenges – maintaining genetic stability and recombinant protein production over extended cultivation periods.
Florian is lead author of a study recently published in Microbial Cell Factories that involved a joint research team from enGenes and Vienna University’s Institute of Bioprocess Science and Engineering.
Bringing stability to continuous fermentations
The study ‘Enhanced genetic stability and expression control in growth-decoupled continuous two-stage E. coli fermentations using plasmid-dependent thyA auxotrophic selection’ aimed to overcome problems of plasmid instability, genetic mutations and unintended expression in continuous Escherichia coli fermentations by employing a plasmid-dependent auxotrophic selection system, using a thymidine auxotrophy (thyA deletion), in conjunction with a series of plasmid modifications to enhance stability and expression control.
A crucial part of the answer was to base the fermentation on the enGenes-eXpress™ E. coli strain, modified with plasmids containing thyA along with regulatory elements to maintain plasmid stability and minimize basal expression. The modified strains were evaluated in continuous two-stage fermentations under carbon-limited conditions.
Simplified batch-fed cultivation
EnPresso’s EnPump® 200 and Reagent A were used to control enzymatic glucose release during the high-throughput screening phase. The technology enabled controlled carbon-limited, fed-batch-like cultivation conditions in microbioreactors without pumps or complex feeding equipment. This allowed the researchers to evaluate strain stability, basal expression, and population dynamics under process-relevant conditions before transferring the most promising candidates to long-term bioreactor studies.
The team’s results indicated significant reduction in plasmid loss, improved population homogeneity, and suppressed basal expression in non-induced phases. The addition of elements such as the cer (ColE1 resolution) site and a modified T7 promoter further enhanced plasmid stability and reduced basal expression levels.
Extending continuous cultivation boundaries
The optimized strains maintained stable biomass, plasmid retention, and GFP production for more than 1,000 hours (over six weeks) of continuous cultivation without antibiotic selection.
“To our knowledge, this represents one of the longest-running E. coli-based processes without the addition of antibiotics reported to date,” the study notes.
The study demonstrates that combining growth-decoupled protein production using an E. coli strain with antibiotic-free plasmid stabilization via plasmid-dependent thymidine auxotrophy in conjunction with improved plasmid architecture can dramatically increase the stability of continuous two-stage fermentations.
The study is particularly interesting in the way it directly demonstrates rapid plasmid loss and the emergence of non-producing subpopulations as root causes of typical failure in continuous E. coli processes: While the original system gradually lost productivity, the optimized constructs largely prevented this population takeover and remained productive throughout the experiment.
About enGenes Biotech
enGenes Biotech GmbH (enGenes) is a contract research, development and manufacturing company that provides leading-edge technologies and production services focused on recombinant proteins in bacteria. The company’s mission is to provide cost-effective and scalable production of recombinant proteins at a fraction of the current cost, allied to a vision of developing a world-class portfolio of cutting-edge protein production technologies, relevant to a broad spectrum of application fields.
enGenes has developed advanced technologies to drive more cost-effective recombinant protein production processes, including its proprietary enGenes-eXpress™ E. coli platform that achieves outstanding yields of soluble and active recombinant protein. enGenes- eXpress™ has been successfully applied for the manufacturing of enzymes and biopharmaceutical products that failed to give economically feasible yields in a conventional expression host.
enGenes Biotech offers development and manufacturing services tailored to the needs of pharmaceutical and industrial biotech companies. The services include expression strain and vector development, fermentation process development and optimization, downstream process development, production of purified protein, technology transfer and scale-up support with technology out-licensing and co-development opportunities.
Find out more at: https://www.engenes.cc
Resources
See Microbial Cell Factories to read the full study.
Florian Simon, study lead author.
Study workflow illustrated.
Related Articles
