Engineering a Cytidine Aminotransferase for Biocatalytic Production of the Antiviral Molnupiravir


The COVID-19 pandemic highlights the urgent need for cost-effective processes to rapidly manufacture antiviral drugs at scale. Here we report a concise biocatalytic process for Molnupiravir, a nucleoside analogue currently in phase 3 clinical trials as an orally available treatment for SARS-CoV-2. Key to the success of this process was the development of a cytidine aminotransferase for the production of N-hydroxy-cytidine through evolutionary adaption of the hydrolytic enzyme cytidine deaminase. This engineered biocatalyst performs >100,000 turnovers in less than 30 minutes, operates at 180 g/L substrate loading and benefits from in situ crystallization of the N-hydroxy-cytidine product (>90% yield), which can be converted to Molnupiravir by a selective 5’-acylation using Novozym® 435.

Version notes

We have gained more experimental data, in particular a more highly evolved version of the enzyme cytidine deaminase has been developed and we now publish its sequence. The new enzyme is much more effective for the conversion of cytidine to N-hydroxycytidine.