Authors
Andrej Ribar, MSc (Technische Universität Graz), Martin Pfeiffer, PhD (Institute for Biotechnology and Bioengineering) and Univ.-Prof. DI DDr. hc Bernd Nidetzky (Technische Universität Graz)
This work presents an efficient one-pot, multi-enzyme cascade reaction for the synthesis of C-nucleoside 5′-monophosphates, exemplified by the production of pseudouridine 5′-phosphate (Psi5P), a key building block for therapeutic mRNA, from D-ribose (Rib) and uracil (Ura). The cascade integrates pentose phosphorylation with C-C bond formation using ribokinase (RbsK), acetate kinase (AcK), and Psi5P C-glycosidase (YeiN).
A critical step involves the efficient phosphorylation of Rib (~1.0 M) at the O5 position. While phosphatase-catalyzed phosphorylation was explored using acetyl phosphate (AcP) as a donor, it proved unsuitable due to poor site selectivity, yielding both Rib5P and Rib1P. The kinase-based route, using RbsK coupled with AcK for ATP regeneration from AcP (1.15 M) and catalytic ATP (5 mM), demonstrated high selectivity for Rib5P. Optimization of this step focused on cofactor concentration (Mg2+ optimal at ~60 mM) and enzyme stability. Despite AcK instability (half-life ~20 min), minimizing reaction time through optimized enzyme ratios (RbsK:AcK) allowed for high conversion (>90%) of Rib to Rib5P (~670 mM final concentration) with a productivity of ~150 g/L/h.
The subsequent condensation step utilizes YeiN to react the Rib5P intermediate with Ura. To overcome the low solubility of Ura (~22 mM), process intensification was achieved by supplying Ura as a solid (~1 M total), enabling reaction concentrations far exceeding solubility limits. Under optimized agitation and using 0.75 mg/mL YeiN, near-quantitative conversion to Psi5P (~650 mM) was achieved with a productivity of 38 g/L/h for the overall cascade (10 mL scale) and a mass-based total turnover number (TON) of 150 g/g enzymes. The reaction can be performed sequentially or simultaneously, with the sequential mode offering slightly higher productivity.
The synthetic flexibility of the cascade was demonstrated by successfully using alternative pentoses (D-arabinose, 2-deoxy-Rib, D-Xylose) and uracil analogs (6-amino, 2-thio, 4-thio), albeit with varying efficiencies, yielding corresponding C-nucleoside 5′-phosphate analogs. This highlights the potential for diversity-oriented synthesis.
This biocatalytic cascade represents a significant advancement over previous methods relying on uridine as a starting material or less efficient phosphorylation strategies. The high efficiency, productivity, use of inexpensive starting materials (Rib, Ura, AcP), and demonstrated flexibility position this enzymatic approach as a highly promising, greener alternative for the industrial production of Psi5P and related C-nucleosides, crucial for RNA therapeutics and other applications.
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