Stracke Christina, Meyer Benjamin H, De Rose Simone A, Ferrandi Erica Elisa, Kublanov Ilya V, Isupov Michail N, Harmer Nicholas J, Monti Daniela, Littlechild Jennifer, Müller Felix, Snoep Jacky L, Bräsen Christopher, Siebers Bettina
Molecular Enzyme Technology and Biochemistry, Environmental Microbiology and Biotechnology, Centre for Water and Environmental Research (CWE), University of Duisburg-Essen, Essen, Germany.
Henry Wellcome Building for Biocatalysis, Biosciences, Faculty of Health and Life Sciences, University of Exeter, Exeter, United Kingdom.
Front Microbiol. 2025 May 21;16:1601972. doi: 10.3389/fmicb.2025.1601972. eCollection 2025.
Extremolytes - unique 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 extremolytes - most notably ectoine and hydroxyectoine - have 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 2-phosphoglycerate (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 enzymatic approach for the production of cDPG directly from 2,3-diphosphoglycerate (2,3DPG), leveraging the activity of the cDPGS from (cDPGS). We optimized the heterologous production of cDPGS in by refining codon usage and expression conditions. The purification process was significantly streamlined through an optimized heat precipitation step, coupled with effective stabilization of cDPGS for both usage and storage by incorporating KCl, Mg, reducing agents and omission of an affinity tag. The recombinant cDPGS showed a of 38.2 U mg, with 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 min, using just 0.5 U of recombinant cDPGS at 55°C. These results highlight that cDPGS 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 cDPGS 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.
极端嗜盐菌产生的独特相容性溶质——极端嗜盐菌素，可在极端条件下（包括极端温度和渗透胁迫）保护生物结构，如膜、蛋白质和DNA。这些化合物在制药、医疗保健、化妆品和生命科学领域具有巨大的应用潜力。然而，尽管它们具有相当大的潜力，但只有少数几种极端嗜盐菌素——最显著的是四氢嘧啶和羟基四氢嘧啶——取得了商业应用，主要原因是大多数其他极端嗜盐菌素缺乏有效的生产策略。环状2,3-二磷酸甘油酸（cDPG）是在某些嗜热产甲烷古菌中发现的一种独特代谢产物，在热保护中起关键作用，它由2-磷酸甘油酸（2PG）通过两步酶促过程合成，该过程涉及2-磷酸甘油酸激酶（2PGK）和环状-2,3-二磷酸甘油酸合成酶（cDPGS）。在本研究中，我们利用来自（cDPGS）的cDPGS的活性，开发了一种直接从2,3-二磷酸甘油酸（2,3DPG）生产cDPG的高效酶促方法。我们通过优化密码子使用和表达条件，优化了cDPGS在中的异源生产。通过优化热沉淀步骤，显著简化了纯化过程，并通过加入KCl、Mg、还原剂和省略亲和标签，有效地稳定了cDPGS的使用和储存。重组cDPGS的比活性为38.2 U mg，2,3DPG的Km值为1.52 mM，ATP的Km值为0.55 mM。该酶有效地催化了2,3DPG完全转化为cDPG。值得注意的是，即使在100 mM的规模下，在55°C下仅使用0.5 U的重组cDPGS，它也能在180分钟内将37.6 mg的2,3DPG完全转化为cDPG。这些结果表明，cDPGS可以很容易地生产、快速纯化并充分稳定，同时为作为增值产品的cDPG合成提供优异的转化效率。此外，还建立了cDPGS活性的动力学模型，为模拟和扩大cDPG的工业生产提供了关键工具。这种简化的过程为cDPG的可扩展合成提供了显著优势，为这种极端嗜盐菌素的进一步生化和工业应用铺平了道路。
