Genetic heterogeneity of engineered Escherichia coli Nissle 1917 strains during scale-up simulation

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 m...

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Bibliographic Details
Published in:	Metabolic engineering Vol. 85; pp. 159 - 166
Main Authors:	Munkler, Lara P., Mohamed, Elsayed T., Vazquez-Uribe, Ruben, Visby Nissen, Victoria, Rugbjerg, Peter, Worberg, Andreas, Woodley, John M., Feist, Adam M., Sommer, Morten O.A.
Format:	Journal Article
Language:	English
Published:	Belgium Elsevier Inc 01-09-2024
Subjects:	Advanced microbiome therapeutic (AMT) Escherichia coli Nissle 1917 Genetic stability Scale-up Genetic stability Advanced microbiome therapeutic (AMT) Escherichia coli Nissle 1917 Scale-up
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Summary:	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.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23
ISSN:	1096-7176 1096-7184 1096-7184
DOI:	10.1016/j.ymben.2024.08.001