Periodic Reporting for period 1 - SI-MEPSI (When Biotechnology Meets Chemistry: Metabolic and Chemical Stable Isotope Labelling of RNA Building Blocks for the Modular Synthesis of (2H/13C/15N)-N1-Methylpseudouridine (m1Ψ) compounds)
Reporting period: 2022-07-01 to 2024-06-30
The conversion of renewable resources into valuable products is one of the main challenges of our society. In this project, we started from inorganic reagents and utilised CO2 fixation to convert relatively inexpensive materials into fine chemicals by combining the strengths of biotechnology, chemistry, and enzymatic synthesis. In the end, we produced valuable stable-isotope-labelled (SI-labelled) ribonucleic acid (RNA) derivatives as the main products for the applications in nuclear magnetic resonance (NMR) spectroscopy. For example, labelling RNAs with stable isotopes can remove unwanted signals and reduce the complexity of NMR data. As a result, it is much easier to study biological systems and structures relevant for advancing the field of RNA therapeutics. However, the accessibility of SI-labelled RNA biomolecules is limited because the common strategies of obtaining them are often very expensive, especially for RNAs containing modifications. Here, we thus focused on developing an affordable method of isotopically labelling two in-demand RNA modifications: pseudouridine and N1-methylpseudouridine. These two RNA modifications were the key players in developing effective mRNA vaccines during the COVID-19 pandemic. In particular, N1-methylpseudouridine enabled high vaccine efficiency and decreased immunogenicity. To facilitate prospective biological NMR studies of these modified RNAs, the overall target of this project was to profitably label pseudouridine-based derivatives with stable isotopes, produce them on a gram scale, and introduce them to the market.
To achieve our objectives, we first investigated H2/O2/CO2-based autotrophic fermentations with the bacterium Cupriavidus necator as a way to profitably label biomolecules with stable isotopes. Since C. necator can grow on a mixture of gases in an aqueous medium containing only inorganic salts, even isotopically labelled analogues of these materials are relatively inexpensive compared to the value of the obtained products. However, such fermentations are traditionally performed under explosive mixtures of H2/O2 gases and require very expensive equipment and extensive safety measures. Hence, only small bioreactors can be used and only multi-gram quantities of SI-labelled biomass can be produced under these conditions. We thus optimised the gas ratios in the fermentation using more sustainable, non-explosive conditions to allow the upscaling of our production of SI-labelled biomolecules. By fulfilling this aim, we produced enough SI-labelled biomass for obtaining multi-gram quantities of SI-labelled RNAs. We then converted these RNA molecules to individual RNA building blocks (nucleotides) and in turn used these as starting materials in a straightforward, versatile, and convergent chemo-enzymatic synthesis of pseudouridine-based derivatives.
To achieve our objectives, we first investigated H2/O2/CO2-based autotrophic fermentations with the bacterium Cupriavidus necator as a way to profitably label biomolecules with stable isotopes. Since C. necator can grow on a mixture of gases in an aqueous medium containing only inorganic salts, even isotopically labelled analogues of these materials are relatively inexpensive compared to the value of the obtained products. However, such fermentations are traditionally performed under explosive mixtures of H2/O2 gases and require very expensive equipment and extensive safety measures. Hence, only small bioreactors can be used and only multi-gram quantities of SI-labelled biomass can be produced under these conditions. We thus optimised the gas ratios in the fermentation using more sustainable, non-explosive conditions to allow the upscaling of our production of SI-labelled biomolecules. By fulfilling this aim, we produced enough SI-labelled biomass for obtaining multi-gram quantities of SI-labelled RNAs. We then converted these RNA molecules to individual RNA building blocks (nucleotides) and in turn used these as starting materials in a straightforward, versatile, and convergent chemo-enzymatic synthesis of pseudouridine-based derivatives.
We produced uniformly SI-labelled biomass metabolically with C. necator under non-explosive conditions, keeping O2 levels under 3.5% in the bioreactor, while preserving good growth rates. To introduce isotopic labels, we used SI-labelled [2H]-H2, [2H]-H2O, [13C]-CO2, and [15N]-NH4Cl. We then hydrolysed the RNA biomolecules present in the biomass into nucleotides, which we isolated pure after extraction, chromatographic separation, and precipitation. We subsequently used these nucleotides as the main starting materials for the chemo-enzymatic synthesis of our target SI-labelled pseudouridine-based derivatives. The first step of this synthesis involved their hydrolysis, giving ribose 5’-monophosphate (rMP) as the desired product in higher than 75% yield. We developed a chemical as well as an enzymatic method for this purpose, each having its own merit depending on the target biomolecule. We then coupled rMP enzymatically to uracil to form pseudouridine 5’-monophosphate in yields higher than 80%. Here, if we needed uracil to be SI-labelled, we synthesised it chemically with site-specific labelling patterns from propiolic acid and urea. Finally, we either transformed pseudouridine 5’-monophosphate enzymatically into other pseudouridine derivatives in one step with yields higher than 90%, or chemo-enzymatically into N1-methylpseudouridine derivatives in 2–4 steps. We confirmed the high quality of all isolated products by NMR, mass spectrometry, and high-performance liquid chromatography. Overall, we successfully produced more than 30 novel [2H/13C/15N]-labelled pseudouridine and N1-methylpseudouridine derivatives on a multi-gram scale with chemical and isotopic purities higher than 95%. Going beyond what we initially proposed, we also enzymatically incorporated our pseudouridine-based derivatives into modified RNA fragments for use in RNA-based research.
In summary, we upscaled the chemolithoautotrophic fermentations under non-explosive conditions from a 1-L laboratory scale to 16 L, with current efforts aiming at a 100-L fermenter. This accomplishment allowed us to produce kilogram quantities of SI-labelled biomass. The chemo-enzymatic synthesis of the target pseudouridine-based derivatives originating from this biomass was then developed to be efficient and profitable, and more than 30 novel compounds were obtained in 3–5 steps and 33–59% yields based on the most expensive reagent. The compounds have been added to the company’s webshop and are currently being commercialised. All achievements were in line with the goals that were set out at the start of the project and in addition, the synthesis of RNA fragments employing our novel compounds was carried out beyond the initial proposed scope of the project.
The project breaks ground in several research areas. Establishing chemolithoautotrophic fermentations with C. necator to produce kilogram quantities of biomass under non-explosive conditions is unprecedented and has potential to enhance the competitiveness of sustainable industrial autotrophic processes in the European Biotechnology sector. In this context, important research that relies on the use of SI-labelled biomolecules would become more accessible. However, further uptake would require expanding the infrastructure and ensuring the market prices can accommodate it. The project also goes beyond the state-of-the-art in the field of RNA technology by introducing a few dozens of novel SI-labelled pseudouridine-based derivatives and RNA building blocks to the market. Further success would be ensured by commercialising these new products and validating their great promise for future studies in the fields of biological NMR and RNA therapeutics.
The project breaks ground in several research areas. Establishing chemolithoautotrophic fermentations with C. necator to produce kilogram quantities of biomass under non-explosive conditions is unprecedented and has potential to enhance the competitiveness of sustainable industrial autotrophic processes in the European Biotechnology sector. In this context, important research that relies on the use of SI-labelled biomolecules would become more accessible. However, further uptake would require expanding the infrastructure and ensuring the market prices can accommodate it. The project also goes beyond the state-of-the-art in the field of RNA technology by introducing a few dozens of novel SI-labelled pseudouridine-based derivatives and RNA building blocks to the market. Further success would be ensured by commercialising these new products and validating their great promise for future studies in the fields of biological NMR and RNA therapeutics.