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 E. coli that were purified by immobilized metal affinity chromatography. As part of the cascade, ATP is in‐situ regenerated from polyphosphate to reduce synthesis costs. The enzymatic cascade was characterized at laboratory scale and the products 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 was 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.

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