Introduction: Extremolytes – compatible solutes exclusively produced by extremophiles – hold great potential for applications in pharmaceuticals, healthcare, and cosmetics. Although cyclic-2,3-diphosphoglycerate (cDPG) has been detected as extremolyte in hyperthermophilic methanogenic Archaea, its potential protective properties in safeguarding membranes, proteins and DNA from damage under stressful conditions remains unused. So far, methods for protein production, stabilization and efficient synthesis of cDPG are missing as no applicable production procedure is available.

Objective: Here, we present a one-step enzymatic in vitro approach for the synthesis of cDPG from 2,3-diphosphoglycerate (2,3-DPG) utilizing the cyclic 2,3-diphosphoglycerate synthetase (cDPGS) from Methanothermus fervidus.

Methods: The heterologous production of cDPGS in Escherichia coli was improved via codon optimization. We implemented a streamlined two-step purification method involving heat precipitation and size exclusion chromatography. Functional enzyme characterization and conversion efficiencies were assessed and analyzed using enzyme assays and 31P-NMR.

Results: Starting from 1.7 g (wet weight) of Escherichia coli cells, we obtained 3.5 mg of pure cDPGS. The recombinant protein showed a Vc of 21 U mg-1, with Km values of 1.4 mM and 1.1 mM for 2,3 DPG and ATP, respectively. To enhance stability for storage at -80°C, we incorporated 400 mM KCl, 5 mM DTT, 2.5 mM Mg2+ and 25% (v/v) glycerol, resulting in the retention of 95% activity even after 1.5 months. The optimized in vitro reaction was successfully scaled up, achieving complete conversion of 37.6 mg 2,3DPG to cDPG at 55°C within 180 minutes.

Conclusion: These results represent an important step towards a streamlined one-step in vitro approach to produce cDPG. The complete substrate conversion to product will simplify downstream cDPG purification, allow further up-scaling and to extend the process to a more complex enzyme cascade including e.g. an ATP recycling system or synthesis from cheaper substrates like glycerate. This paves the way for the cost-effective production of cDPG as a value-added product for application.