Understanding the metabolic burden of protein overproduction in Escherichia coli using multi-step directed evolution

The recombinant protein production market has been growing since its advent in the 1980s with the introduction of the first recombinant protein - based pharmaceutical, insulin. Today, recombinant proteins are crucial not only in the biopharmaceutical industry but are widely used in everyday products such as cosmetics and detergents. Out of many available hosts for recombinant protein production, including yeast, plant, insect, and mammalian cells, bacterial hosts remain favoured by many manufacturers. Escherichia coli, one of the most common bacterial host choices for recombinant protein production, is a well-studied organism with ample techniques available for further genetic modification. However, producing heterologous proteins in E. coli comes with unique challenges; its rapid evolution and adaptation to stressful environments makes it an unsuitable candidate for some biomanufacturing processes, such as continuous fermentation. The work presented here not only showcases some of the
evolutionary metabolic burden escape mechanisms employed by plasmid carrying E. coli under high metabolic stress of recombinant protein production, but also introduces a novel Fluorescent Assisted Cell Sorting (FACS) screening protocol that can direct the experimental evolution of plasmid-carrying E. coli cells towards a more stable and more productive phenotype. Next generation long-read sequencing (Oxford Nanopore) was used to identify some of the genetic changes associated with these novel
evolved phenotypes. This research highlights genes as a potential targets for modification in industrial E. coli strains, thus providing valuable insights for the biopharmaceutical and biomanufacturing industries.