1. Genetic heterogeneity of engineered Escherichia coli Nissle 1917 strains during scale-up simulation.
- Author
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Munkler, Lara P., Mohamed, Elsayed T., Vazquez-Uribe, Ruben, Visby Nissen, Victoria, Rugbjerg, Peter, Worberg, Andreas, Woodley, John M., Feist, Adam M., and Sommer, Morten O.A.
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RECOMBINANT microorganisms , *DNA polymerases , *ESCHERICHIA coli , *WHOLE genome sequencing , *DELETION mutation - Abstract
Advanced microbiome therapeutics have emerged as a powerful approach for the treatment of numerous diseases. While the genetic instability of genetically engineered microorganisms is a well-known challenge in the scale-up of biomanufacturing processes, it has not yet been investigated for advanced microbiome therapeutics. Here, the evolution of engineered Escherichia coli Nissle 1917 strains producing Interleukin 2 and Aldafermin were investigated in two strain backgrounds with and without the three error-prone DNA polymerases polB, dinB, and umuDC, which contribute to the mutation rate of the host strain. Whole genome short-read sequencing revealed the genetic instability of the pMUT-based production plasmid after serial passaging for approximately 150 generations using an automated platform for high-throughput microbial evolution in five independent lineages for six distinct strains. While a reduction of the number of mutations of 12%–43% could be observed after the deletion of the error-prone DNA polymerases, the interruption of production-relevant genes could not be prevented, highlighting the need for additional strategies to improve the stability of advanced microbiome therapeutics. • Engineered E. coli Nissle 1917 as advanced microbiome therapeutic represents a promising treatment for various diseases. • New modality of living medicine requires a better understanding of genetic stability. • Evolution of engineered E. coli Nissle strains (producing IL-2 and Aldafermin) under industrially relevant conditions. • Whole genome sequencing of populations after ∼150 generations revealed production cassette on pMUT plasmid as mutation target. • Deletion of error-prone DNA polymerases improved genetic stability, but no prevention of the interruption of production genes. [ABSTRACT FROM AUTHOR]
- Published
- 2024
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