1. Introduction
Synthetic mRNA has potential use in a wide range of applications, including cancer immunotherapy, protein replacement therapy, genome editing, pluripotent stem cell generation, and vaccines against infectious diseases (Baden et al., 2021; Breda et al., 2023; Gan et al., 2019; Qin et al., 2022; Vavilis et al., 2023). In all cases, mRNA molecules are currently produced in standardised in vitrotranscription (IVT) systems, comprising an RNA Polymerase biocatalyst, DNA template, modified nucleosides, magnesium-containing buffer and a capping enzyme/analog (Ouranidis et al., 2022). These simple, modular, cell-free production platforms embed flexibility and predictability in mRNA manufacture, while substantially reducing process-related impurities (Whitley et al., 2022). However, the requirement for purified input components is associated with relatively high costs, and critical reagent shortages (Kis et al., 2021). Moreover, downstream purification processes are complicated by complex product-related impurity profiles, that include immunostimulatory double-stranded RNA and abortive transcripts (Gholamalipour et al., 2018; Rosa et al., 2021). However, despite these drawbacks, expanding product diversification (particularly with respect to size), highly variable intended applications (with associated variability in required production scale, purity, cost, etc), and the increasing pressure placed on reagent/equipment supplies by growing demand for mRNA synthesis, there are currently no alternative technology platforms available for mRNA manufacture.
Cell-based production systems are the dominant choice for manufacture of other bioproducts, such as AAV vectors, recombinant proteins and recombinant DNA plasmids (Agostinetto et al., 2022; Jiang and Dalby, 2023; McElwain et al., 2022). Although they are associated with relatively complex and costly downstream processing steps to remove host-cell impurities, this is somewhat mitigated by the availability of well-characterised chromatographic and membrane-based unit operations (Fan et al., 2023; Sripada et al., 2022). As a relatively simple macromolecule, synthetic mRNA could theoretically be produced in virtually any microbial cell factory. E. coli is a particularly attractive expression host given that decades of use in recombinant plasmid DNA production has led to development of very low-cost, standardised, easy to scale (up to 100,000L) flexible manufacturing platforms (Pontrelli et al., 2018; Yang et al., 2021). Indeed, these benefits have seen E. coli utilised as a biocatalyst for production of RNA aptamers and double stranded RNA (dsRNA) molecules (Delgado-Martín and Velasco, 2021; Ma et al., 2020; Ponchon and Dardel, 2011; Ponchon et al., 2009, 2013)
The primary limitation of mRNA production in microbial expression hosts is endogenous pathways that encode rapid RNA turnover, where the average mRNA half-life in E.coli is ~5 mins (Esquerré et al., 2015; Mohanty and Kushner, 2022). For dsRNA manufacture, multigram per liter yields have been achieved in E. coli bioprocesses by deleting RNase III, a non-essential dsRNA-targeting endonuclease (Pertzev and Nicholson, 2006). However, single stranded mRNA decay is mediated by RNAseE, an essential enzyme required for global RNA metabolism. Although RNAseE has broad substrate specificity, various sequence features have been shown to increase its relative specific activity on individual mRNA species, including unstructured AU rich regions, and, most critically, the presence of a 5’-monophosphate (Bae et al., 2023; Callaghan et al., 2005; Richards and Belasco, 2023). However, other molecule-specific features, such as RNA-binding protein binding sites, codon usage, and secondary structure profiles can reduce RNAse E mediated mRNA turnover (Börner et al., 2023; Roux et al., 2022). More generically, global mRNA half-life is affected by both the relative abundance and activity level of RNAse E (Mohanty and Kushner, 2022). Accordingly, the half-life of a specific mRNA molecule within anE. coli cell chassis is determined by a complex interplay between the mRNA sequence/structure and the host cell’s complement of RNA degradation machinery components.
Herein we report coordinated mRNA, DNA, media and host cell engineering to dramatically increase synthetic mRNA accumulation and maintenance inE. coli cell factories. Achieving mRNA yields >40-fold greater than standard ‘unengineered’ E. coli expression systems, we demonstrate rapid production and purification of a range of functional mRNA products. In doing so, we introduce a new technology platform for mRNA manufacturing solution spaces. This may be particularly useful in contexts where IVT systems are unavailable (e.g., due to reagent shortages), product formats necessitate process optimisation (e.g., production of very large RNA molecules), or manufacturing costs need to be significantly reduced.