Establishment of an efficient one-step enzymatic synthesis of cyclic-2,3-diphosphoglycerate

Christina Stracke^1*Benjamin Meyer¹Simone Antonio De Rose²Erica Elisa Ferrandi³Ilya Kublanov⁴Michail N Isupov²Nicholas James Harmer⁵Daniela Monti³Jennifer Ann Littlechild²Felix Mueller⁶Jacky L. Snoep⁷Christopher Braesen^1*Bettina Siebers^1*

• ¹Molecular Enzyme Technology and Biochemistry (MEB), Biofilm Centre, Centre for Water and Environmental Research (CWE), Faculty of Chemistry, University of Duisburg-Essen, Essen, North Rhine-Westphalia, Germany
• ²Henry Wellcome Building for Biocatalysis, University of Exeter, United Kingdom, Exeter, England, United Kingdom
• ³Consiglio Nazionale delle Ricerche, Area della Ricerca Milano 1, Milan, Lombardy, Italy
• ⁴Winogradsky Institute of Microbiology, Russian Academy of Sciences (RAS), Moscow, Moscow Oblast, Russia
• ⁵Faculty of Health and Life Sciences, University of Exeter, Exeter, England, United Kingdom
• ⁶Evonik Industries (Germany), Darmstadt, North Rhine-Westphalia, Germany
• ⁷Department of Biochemistry, Faculty of Science, Stellenbosch University, Matieland, South Africa

Extremolytesunique compatible solutes produced by extremophiles -protect biological structures like membranes, proteins, and DNA under extreme conditions, including extremes of temperature and osmotic stress. These compounds hold significant potential for applications in pharmaceuticals, healthcare, cosmetics, and life sciences. However, despite their considerable potential, only a limited number of extremolytesmost notably ectoine and hydroxyectoinehave achieved commercial relevance, primarily due to the absence of efficient production strategies for the majority of other extremolytes . Cyclic 2,3-diphosphoglycerate (cDPG), a unique metabolite found in certain hyperthermophilic methanogenic Archaea, plays a key role in thermoprotection and is synthesized from 2phosphoglycerate (2PG) through a two-step enzymatic process involving 2-phosphoglycerate kinase (2PGK) and cyclic-2,3-diphosphoglycerate synthetase (cDPGS). In this study, we present the development of an efficient in vitro enzymatic approach for the production of cDPG directly from 2,3-diphosphoglycerate (2,3DPG), leveraging the activity of the cDPGS from Methanothermus fervidus (MfcDPGS). We optimized the heterologous production of MfcDPGS in Escherichia coli by refining codon usage and expression conditions. The purification process was significantly streamlined through an optimized heat precipitation step, coupled with effective stabilization of MfcDPGS for both usage and storage by incorporating KCl, Mg 2+ , reducing agents and omission of an affinity tag. The recombinant MfcDPGS showed a Vmax of 38.2 U mg -1 , with KM values of 1.52 mM for 2,3DPG and 0.55 mM for ATP. The enzyme efficiently catalyzed the complete conversion of 2,3DPG to cDPG. Remarkably, even at a scale of 100 mM, it achieved full conversion of 37.6 mg of 2,3DPG to cDPG within 180 minutes, using just 0.5 U of recombinant MfcDPGS at 55°C. These results highlight that MfcDPGS can be easily produced, rapidly purified, and sufficiently stabilized while delivering excellent conversion efficiency for cDPG synthesis as value-added product. Additionally, a kinetic model for MfcDPGS activity was developed, providing a crucial tool to simulate and scale up cDPG production for industrial applications. This streamlined process offers significant advantages for the scalable synthesis of cDPG, paving the way for further biochemical and industrial applications of this extremolyte.