3.3 Mechanistic dissection of synthetic mRNA production in E. coli host cell chassis.
In order to understand how E. coli host cells utilise available biosynthetic capacity for mRNA production, we profiled cell biomass, total RNA and product mRNA accumulation/maintenance during a 6hr manufacturing time course. We utilised the previously optimised cell-DNA-media composition (see Fig. 2) to manufacture SARS-COV-2 Spike Protein mRNA. To evaluate mechanistic differences between biosynthesis of circular and linear molecules, we separately manufactured SelfCirc- and TermtRNA-mRNA products. With respect to the latter, we confirmed that the optimal BL21 STAR-Triple terminator-LB(AS2) system combination permitted a 36-fold increase in TermtRNA-GFP yields as compared to the standard control system (see Supplementary data, Fig. S2), similar to the 44-fold increase achieved for SelfCirc-GFP.
As shown in Fig 4A, manufacture of circular and linear synthetic mRNA products induced a significant metabolic burden on the host cell. Producer cells reached maximum cell density 2-3 h post induction of mRNA expression, whereas uninduced cells continued to accumulate biomass up to the 6 h harvest timepoint. Indeed, these cells exhibited a 25% reduction in cell specific growth rate during the first 2 h post expression induction (Fig 4B). Moreover, the final maximum cell density achieved was ~50% lower for producer cells, as compared to non-producers, associated with a ~35% reduction in the integral of cell concentration (cumulative cell hours; Fig. 4B). This indicates that producing substantial amounts of synthetic mRNA forces the cell to reallocate biosynthetic capacity away from cell biomass generation activities. Accordingly, approaches to overcome product biosynthesis-associated burden represent a potentially effective way to enhance total biocatalyst activity and further increase product yields. The simplest method to achieve this may be optimisation of expression induction kinetics, although genetic engineering and/or directed evolution strategies will likely deliver the most significant impact on maximum achievable cell densities (Al’abri et al., 2022; Badran and Liu, 2015; Esvelt et al., 2011). Either way, optimising the biocatalytic capacity available for mRNA product synthesis is critically required to take full-advantage of the ability to scale E. coliproduction processes up to 100,000 L.
As shown in Figure 4C, total RNA synthesised per cell was stable throughout the production process, at ~60 fg/cell, despite Spike Protein-mRNA accumulating over time (Fig. 4D). This is likely due to feedback mechanisms that act to maintain intracellular concentrations of key macromolecules within relatively narrow concentration ranges (Radoš et al., 2022). Accordingly, accumulation of highly-stable product-mRNA forces the cell to reduce biosynthesis and/or induce degradation of endogenous RNA species, with potential associated off-target effects on desirable bioproduction phenotypes such as cell growth rate. These RNA homeostasis mechanisms place a theoretical limit on the total quantity of product-mRNA that can be maintained per cell, above which concentrations of key endogenous RNA molecules will become critically limiting leading to cell death and/or downregulation of product expression. Indeed, as shown in Fig 4D, intracellular concentrations of Spike Protein-mRNA peaked at 4 h, before decreasing slightly at 6 h. At 4 h, SelfCirc-Spike accounted for ~28% of total RNA mass in the host-cell, which is likely approaching the maximum achievable concentration. Although not a direct comparison, during recombinant protein expression in CHO cells, product molecules typically account for ~30% of intracellular protein mass (in-house data). We concluded that engineering efforts to further enhance intracellular product maintenance are unlikely to be beneficial, and instead should focus on maximising product accumulation rates. For SelfCirc-Spike, cell specific productivity (product-mRNA produced per cell per hour) was relatively constant throughout the first 4 hours of the production process, at ~5 fg cell-1 h-1, equating to ~10% of total cellular RNA biosynthetic activity during this time period. DNA vector engineering, for example T7 promoter re-design, may increase synthetic mRNA generation rates, facilitating cells to reach the maximum intracellular product-mRNA concentration level more quickly, permitting shorter production processes with associated benefits in cost and manufacturing flexibility (i.e., ability to rapidly switch between manufacture of different products).
Capillary Gel Electrophoresis analysis clearly shows that product mRNA accumulates intracellularly over time (Fig. 4 F-G). These data exemplify that engineered product molecules are successfully protected from nuclease-mediated decay, facilitating intracellular maintenance over multi-hour time periods, as compared to the typical mRNA half-life inE. coli of ~5 min (Bernstein et al., 2002; Mohanty and Kushner, 2022). Moreover, the presence of a single sharp peak at each sampling point indicates that the cell factory is producing full-length Spike Protein-mRNA that is subject to minimal degradation events. Accordingly, i) further system engineering to disrupt theE. coli degradasome-synthetic mRNA interactome is not required, and ii) E. coli is capable of synthesising homogenous populations of large mRNA molecules, thereby simplifying downstream processing steps. As expected, higher titres were obtained for SelfCirc-Spike than TermtRNA-Spike, where maximum achieved yields were 15 mg/L and 10 mg/L respectively (Fig. 4E). As discussed, we anticipate that significant increases in product yields will be obtained via further DNA/cell/media engineering to increase maximum cell density, integral of cell concentration, and cell specific productivity. Synthetic mRNA yields > 100 mg/L should be relatively straightforward to obtain, however achieving g/L titres, as is standard for recombinant protein production in E. coli , will likely require significant process engineering.