3.2 Cell, DNA and media engineering to maximise mRNA product yield
We previously described a whole pathway engineering approach that maximised production of recombinant proteins in Chinese Hamster Ovary cells (Brown et al., 2019). We hypothesized that a similar strategy could be applied to mRNA manufacturing in E. Coli by sequentially improving the host cell chassis, DNA expression vector and cell culture media. Commercially available E. coli cell lines have been engineered to reduce RNase E activity to levels that enhance recombinant mRNA stability without impacting global mRNA homeostasis (Heyde and Nørholm, 2021; Miroux and Walker, 1996). Although these strains were originally designed to increase production of ‘easy to express’ recombinant proteins, they theoretically provide a highly permissive cell background for synthetic mRNA manufacture. To directly test this, we compared SelfCirc-mRNA production in previously utilised standard BL21(DE3) cells and engineered BL21 Star™ (DE3) ( (DE3)) cells. As shown in Fig 2A, cells expressing a mutated RNAse E produced ~1.8-fold more GFP mRNA than the unengineered strain. Although circular mRNA is efficiently protected from RNase E mediated degradation, covalent circularisation requires synthesis of the full-length transcript. A reduction in RNase E activity may therefore enhance synthetic mRNA yields by preventing turnover of nascent product mRNA, increasing the pool of mRNA molecules available for circularisation. Product yields may be further enhanced by cell engineering strategies that increase the host cell’s mRNA biosynthesis (E.g., T7 expression level) and/or cell biomass accumulation capacities.
We rationalised that promoter engineering was unlikely to increase product yields, as the expression plasmid already contained a T7 promoter optimised to maximise recombinant mRNA transcription rates. However, enhancing the number of plasmid copies per cell has previously been shown to enhance manufacture of short dsRNA molecules (Ponchon et al., 2013). Accordingly, we tested the effect of using a pUC origin of replication (Ori), which permits very high plasmid copy numbers per cell (~500-700; (Lee et al., 2006; Lin‐Chao et al., 1992)). As shown in Fig 2B, the use of this element did not increase GFP mRNA yields in Star BL21 cells, as compared to the use of the original Rop-ColE1 Ori, despite that construct only encoding maintenance of ~15-20 copies per cell (Bolivar et al., 1977; Lee et al., 2006). This may be caused by the intrinsic metabolic burden associated with replicating and transcribing very high DNA plasmid loads. It is likely that testing a range of synthetic Oris (Joshi et al., 2022; Rouches et al., 2022) will identify a plasmid copy number ‘sweet spot’ that optimises the quantity of DNA templates available for product biosynthesis without negatively impacting other desirable cellular bioproduction phenotypes.
Beyond the promoter and the Ori, the final DNA plasmid element that can be engineered is the transcriptional terminator. The original expression plasmid utilised a standard class I intrinsic late T7 terminator, TΦ, however this is known to encode a termination efficiency of only ~74% (Carter et al., 1981). Replacing TΦ with a previously described novel triple terminator, comprising a combination of T7 TΦ, T3 and E. coli rrnBT1 endogenous terminators, enhanced GFP mRNA yields by ~40% (Fig 2B). This triple terminator has been shown to effectively eliminate read-through transcription by T7 RNA Polymerase (Mairhofer et al., 2015). Accordingly, this terminator facilitates enhanced RNA Polymerase recycling efficiency and increases the total biocatalyst time available for productive synthetic mRNA biosynthesis.
Producing high levels of synthetic mRNA may create product titer-limiting burden/bottlenecks in host cell metabolic pathways. We tested the effect of replacing the commonly utilised protein and plasmid production cell culture media Luria-Bertani broth with other commercially available formulations. Terrific Broth and Bacto CD Supreme Fermentation media were investigated as their use of glycerol, as opposed to oligopeptides, as a carbon source has been reported to increase maximum cell culture densities (Kram and Finkel, 2015). However, both media formulations significantly reduced mRNA product titers (Fig 2C), likely due to the lower cell growth rates achieved (data not shown). We also tested supplementation with L-Glutamine, based on the hypothesis that an additional nitrogen source would enhance mRNA biosynthetic capacity by increasing nucleoside biogenesis, however this did not significantly impact product yields (Fig 2C). Finally, we evaluated the chemical effector design space to identify small molecules that could specifically enhance mRNA production in E. coli . The most promising chemicals identified were a range of RNAse E inhibitors that reduce enzyme activity via interactions with the N-terminal domain. However, only one of these inhibitors was commercially available, and accordingly we tested the effect of supplementing LB media with 3-(4-Hydroxy-5-isopropyl-6-oxo-1,6-dihydro-pyrimidin-2-ylsulfanyl)-propionic acid (AS2). It was determined that 2 mM AS2 was the optimal concentration for maximising mRNA maintenance in the cell chassis (Supplementary data, Fig S1), which has previously been shown to reduce RNase E activity in E. coli by > 80% (Kime et al., 2015; Mardle et al., 2020) . Utilising AS2 at this concentration increased mRNA yield by ~50% (Fig 2C), where higher concentrations reduced cellular productivity. While a similar increase in titer may be possible via BL21 STAR cell engineering to further attenuate RNAse E activity, AS2 supplementation offers a robust mechanism to precisely optimise the synthetic mRNA-RNAse E interactome in a product-specific manner. Similarly, the use of AS2 in combination with a mutated RNAse E permits use of inhibitor concentrations with reduced off-target effects on the host cell.
The optimal combination of engineered mRNA construct (SelfCirc-mRNA), DNA expression plasmid (Triple terminator), cell host (BL21 STAR) and media formulation (LB + AS2), facilitated a 44x increase in mRNA product yield, compared to the standard control system (Fig 2D). Capillary gel electrophoresis analysis confirmed that product mRNA was full-length and constituted a substantial proportion of total cellular RNA (>20%, compared to <1% for the standard control system; Fig3A). Moreover, high yields of full-length synthetic mRNA were maintained when the relatively small GFP coding sequence (720 nt) was substituted for Cypridina Luciferase (cLuc) (1662 nt) or SARS-COV-2 Spike Protein (3783 nt) (Fig 3B), demonstrating that the engineeredin vivo biomanufacturing system can produce larger, more complex molecules. Finally, using oligo-dT magnetic beads, we validated that achieved increases in product yield were maintained following small-scale purification processes (Fig 3C). This also demonstrates that mRNA manufactured in an E. coli cell-host can be purified using simple low-tech methodologies, facilitated by the absence of abundant endogenous mRNAs with PolyA tails > 5 nucleotides in length (Laalami et al., 2014; Mohanty and Kushner, 2019).