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.