A Cell-Free Multi-enzyme Cascade Reaction for the Synthesis of CDP-Glycerol

CDP-glycerol is a nucleotide-diphosphate-activated version of glycerol. In nature, it is required for the biosynthesis of teichoic acid in Gram-positive bacteria, which is an appealing target epitope for the development of new vaccines. Here, a cell-free multi-enzyme cascade was developed to synthetize nucleotide-activated glycerol from the inexpensive and readily available substrates cytidine and glycerol. The cascade comprises five recombinant enzymes expressed in Escherichia coli that were purified by immobilized metal affinity chromatography. As part of the cascade, ATP is regenerated in situ from polyphosphate to reduce synthesis costs. The enzymatic cascade was characterized at the laboratory scale, and the products were analyzed by high-performance anion-exchange chromatography (HPAEC)-UV and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). After the successful synthesis had been confirmed, a design-of-experiments approach was used to screen for optimal operation conditions (temperature, pH value and MgCl₂ concentration). Overall, a substrate conversion of 89 % was achieved with respect to the substrate cytidine.