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<p>Continuous two-stage <i>E. coli</i> fermentations offer potential for high-efficiency bioprocesses but are often limited by plasmid instability, genetic mutations, and unintended expression during seed phases. In this study, we aimed to overcome these limitations by employing a plasmid-dependent auxotrophic selection system, specifically using a thymidine auxotrophy (<i>thyA</i> deletion), in conjunction with a series of plasmid modifications to enhance stability and expression control. We engineered the <i>E. coli</i> strain enGenes-e<sup>X</sup>press V2 Δ<i>thyA</i> and constructed modified plasmids containing <i>thyA</i> 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. Our results indicate significant reduction in plasmid loss, improved population homogeneity, and suppressed basal expression in non-induced phases. The addition of elements such as the <i>cer</i> (ColE1 resolution) site and a modified T7 promoter further enhanced plasmid stability and reduced basal expression levels. High-throughput screenings in microbioreactor setups confirmed that optimized constructs maintained a homogeneous producing population and suppressed non-producing cells over extended periods, which was validated by fed-batch cultivations and single-cell analyses. Finally, the two most promising constructs demonstrated high robustness in continuous two-stage chemostat fermentations lasting over 1000&#xa0;h, maintaining stable GFP titers, plasmid concentrations, and cell dry mass throughout the process. Our findings demonstrate that the auxotrophic marker <i>thyA</i>-based selection system, combined with strategic plasmid modifications, can substantially improve the genetic stability and productivity of <i>E. coli</i> in continuous bioprocesses. This approach provides a robust platform for sustainable, antibiotic- free production in industrial biotechnology, highlighting its potential for scale-up in long-term continuous fermentations.</p>

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Enhanced genetic stability and expression control in growth-decoupled continuous two-stage E. coli fermentations using plasmid-dependent thyA auxotrophic selection

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年12月26日

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  • Florian Simon,
  • Patrick Stargardt,
  • Natalia Danielewicz,
  • Florian Weiß,
  • Johanna Berein,
  • Gerald Striedner,
  • Juergen Mairhofer

摘要

Continuous two-stage E. coli fermentations offer potential for high-efficiency bioprocesses but are often limited by plasmid instability, genetic mutations, and unintended expression during seed phases. In this study, we aimed to overcome these limitations by employing a plasmid-dependent auxotrophic selection system, specifically using a thymidine auxotrophy (thyA deletion), in conjunction with a series of plasmid modifications to enhance stability and expression control. We engineered the E. coli strain enGenes-eXpress V2 ΔthyA and constructed modified 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. Our results indicate 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. High-throughput screenings in microbioreactor setups confirmed that optimized constructs maintained a homogeneous producing population and suppressed non-producing cells over extended periods, which was validated by fed-batch cultivations and single-cell analyses. Finally, the two most promising constructs demonstrated high robustness in continuous two-stage chemostat fermentations lasting over 1000 h, maintaining stable GFP titers, plasmid concentrations, and cell dry mass throughout the process. Our findings demonstrate that the auxotrophic marker thyA-based selection system, combined with strategic plasmid modifications, can substantially improve the genetic stability and productivity of E. coli in continuous bioprocesses. This approach provides a robust platform for sustainable, antibiotic- free production in industrial biotechnology, highlighting its potential for scale-up in long-term continuous fermentations.