Design and optimizing a new CDP‐choline in vitro multienzyme producing process starts from d‐ribose

Abstract

This work designs an in vitro multienzyme system to produce CDP-choline from d-ribose and develop an optimization procedure for one-pot multienzyme catalytic system. The entire process integrated 10 enzymes, and an efficient acetate kinase/acetyl phosphate-based ATP regeneration module was applied. Then, some optimizations to this system were made including selecting optimum enzyme building blocks and improving expression parameters. The process improved the final yield of CDP-choline from 0.2 to 6 g/L (CDP-choline titer 12.2 mM). This new one-pot CDP-choline producing system has a potential for industrial use, and the optimization procedure shed light on improving other one-pot multienzyme system for industrial production of energy rich compounds.

Biocatalytic Production of Bioactive Dipeptides

“…This method was later successfully reproduced by Alissandratos et al (2016), who used E. coli lysate containing endogenous acetate kinase from E. coli (AckA) to regenerate ATP for the generation of nucleoside triphosphates and for cell-free protein synthesis. Other recent examples employing acetate kinase include the production of CDP-choline (Zheng et al, 2021a) and glucose-6-phosphate (Yan et al, 2014). Finally, in the previously mentioned cascade for the production of Islatravir (Chapter 1.1.2), thermostable acetate kinase from Thermotoga maritima was responsible for ATP regeneration (Huffman et al, 2019) and was co-immobilized to the ATP-utilizing enzyme pantothenate kinase.…”