Engineering an E. coli -based in vivo mRNA manufacturing platform
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.