All mRNA products are currently manufactured in in vitro transcription (IVT) reactions that utilize single-subunit RNA polymerase (RNAP) biocatalysts. Although it is known that discrete polymerases exhibit highly variable bioproduction phenotypes, including different relative processivity rates and impurity generation profiles, only a handful of enzymes are generally available for mRNA biosynthesis. This limited RNAP toolbox restricts strategies to design and troubleshoot new mRNA manufacturing processes, which is particularly undesirable given the continuing diversification of mRNA product lines towards larger and more complex molecules. Herein, we describe development of a high-throughput RNAP screening platform, comprising complementary in silico and in vitro testing modules, that enables functional characterisation of large enzyme libraries. Utilizing this system, we identified eight novel sequence-diverse RNAPs, with associated active cognate promoters, and subsequently validated their performance as recombinant enzymes in IVT-based mRNA production processes. By increasing the number of available characterized functional RNAPs by > 130% and providing a platform to rapidly identify further potentially useful enzymes, this work significantly expands the RNAP biocatalyst solution space for mRNA manufacture, thereby enhancing capability to achieve application and molecule-specific optimisation of product yield and quality.

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.