Engineering 2’O-mRNA methyltransferases for industrial biocatalysis
September 24-28, 2017
Eukaryotic messenger RNA (mRNA) are universally modified at their 5’ end into a cap 0 structure consisting of an N7-methylguanosine and an inverted 5’-5’ triphosphate bridge linking the penultimate nucleoside. Multicellular eukaryotes possess the capacity to further modify this cap by 2’O-methylating the ribose of the penultimate nucleotide producing a so-called cap 1 structure1. This methylation seems to be a molecular signature for the discrimination between self and non-self mRNA2. In order to escape the innate immune system of the infected cell, some viruses have also evolved the ability to methylate their cap structures1.
By analogy, therapeutic mRNAs must be non-immunogenic in order to restore or supplement the function of altered genes by mRNA-based therapy3. In this context, we propose to exploit the capacity of Vaccinia virus to produce non-immunogenic mRNAs. More specifically, VP39 is a 39 kDa-enzyme directly involved in the mRNAs’ post-transcriptional modifications. It catalyses the 2’O-methylation in the 5’ cap structure producing the cap 1 mRNA and acts by heterodimerisation as a processivity factor with the poly(A) RNA polymerase4. However, the low expression level of VP39 in Escherichia coli (E. coli) as well as its low in vitro catalytic efficiency have so far limited its use for industrial biocatalysis.
Here, the two above-mentioned limitations are tackled by complementary approaches: i) we use a Split-GFP5 strategy coupled with ultrahigh throughput screening to select for higher soluble expression in E. coli and ii) we design smart libraries seeking to directly improve the catalytic turnover of the enzyme.
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Pierre-Yves Colin, Paul A. Dalby, Mary Stahley, and Jared Davis, "Engineering 2’O-mRNA methyltransferases for industrial biocatalysis" in "Enzyme Engineering XXIV", Pierre Monsan, Toulouse White Biotechnology, France Magali Remaud-Simeon, LISBP-INSA, University of Toulouse, France Eds, ECI Symposium Series, (2017). https://dc.engconfintl.org/enzyme_xxiv/24