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).