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Engineering an E. coli -based in vivo mRNA manufacturing platform
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  • Edward Curry,
  • George Muir,
  • Jixin Qu,
  • Zoltán Kis,
  • Martyn Hulley,
  • Adam Brown
Edward Curry
The University of Sheffield Department of Chemical and Biological Engineering
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George Muir
The University of Sheffield Department of Chemical and Biological Engineering
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Jixin Qu
The University of Sheffield Department of Chemical and Biological Engineering
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Zoltán Kis
The University of Sheffield Department of Chemical and Biological Engineering
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Martyn Hulley
AstraZeneca UK Limited
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Adam Brown
The University of Sheffield Department of Chemical and Biological Engineering

Corresponding Author:[email protected]

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Abstract

Synthetic mRNA is currently produced in standardised in vitro transcription systems. However, this one-size-fits-all approach has associated drawbacks in supply chain shortages, high reagent costs, complex product-related impurity profiles and limited design options for molecule-specific optimisation of product yield and quality. Herein, we describe for the first time development of an in vivo mRNA manufacturing platform, utilising an E. coli cell chassis. Coordinated mRNA, DNA, cell and media engineering, primarily focussed on disrupting interactions between synthetic mRNA molecules and host cell RNA degradation machinery, increased product yields >40-fold compared to standard ‘unengineered’ E. coli expression systems. Mechanistic dissection of cell factory performance showed that product mRNA accumulation levels approached theoretical limits, accounting for ~30% of intracellular total RNA mass, and that this was achieved via host-cell’s reallocating biosynthetic capacity away from endogenous RNA and cell biomass generation activities. We demonstrate that varying sized functional mRNA molecules can be produced in this system and subsequently purified in large- or small-scale processes. Accordingly, this study introduces a new mRNA production technology, expanding the solution space available for mRNA manufacturing.
06 Nov 2023Submitted to Biotechnology and Bioengineering
06 Nov 2023Assigned to Editor
06 Nov 2023Submission Checks Completed
06 Nov 2023Review(s) Completed, Editorial Evaluation Pending
14 Nov 2023Reviewer(s) Assigned