Introduction: Extremolytes - unique compatible solutes synthesized exclusively by extremophiles - hold substantial promise for applications in pharmaceuticals, healthcare, and cosmetics. Cyclic-2,3-diphosphoglycerate (cDPG) has been identified as an extremolyte within hyperthermophilic methanogenic Archaea. However, the potential of cDPG for application as well as its ability to protect cellular components, such as membranes, proteins, and DNA, from damage under extreme environmental stress remains largely unexplored since feasible production procedures are missing.
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: Codon optimization enhanced the heterologous production of cDPGS in Escherichia coli. The purification process has been optimized by implementing a streamlined heat precipitation step, followed by optional size-exclusion chromatography for further refinement. Enzyme assays and 31P-NMR analysis were used to assess kinetic parameters and conversion efficiencies. Based on these data a kinetic model for the production of cDPG was implemented.
Results: From 1.7 g (wet weight) of Escherichia coli cells, we purified 3.5 mg of cDPGS. The recombinant protein showed a Vmax of 38.2 U mg-1, with Km values of 1.55 mM and 0.55 mM for 2,3 DPG and ATP, respectively. For enhanced activity and improved stability of the cDPGS, we added 400 mM KCl, 10 mM DTT, 5 mM Mg²⁺, and 25% (v/v) glycerol (for storage at -70°C). This resulted in a retention of 95% enzyme activity after 1.5 months. The optimized in vitro reaction was scaled up (1-100 mM), achieving full conversion of 37.6 mg 2,3-DPG to cDPG at 55°C within 180 minutes.
Conclusion: These results represent an important step towards a simple one-step in vitro approach to produce cDPG. The complete substrate conversion simplifies downstream purification, and the established kinetic model allows further up-scaling of the process. This approach paves the way for cost-effective cDPG production as a value-added product.