生物学杂志

生物学杂志 ›› 2025, Vol. 42 ›› Issue (2): 1-.doi: 10.3969/j.issn.2095-1736.2025.02.001

• 研究报告 •    下一篇

酶法合成（S）-3-羟基四氢呋喃

李润通1,2， 李　旭2,3， 袁　波2,3,4， 郭敬功1， 曲　戈2,3,4， 孙周通2,3,4   

  1. 1. 河南大学 生命科学学院， 开封 475000； 2. 中国科学院天津工业生物技术研究所， 天津 300308；
    3. 国家合成生物技术创新中心， 天津 300308； 4. 中国科学院天津工业生物技术研究所
    低碳合成工程生物学重点实验室， 天津 300308
  • 出版日期:2025-04-18 发布日期:2025-04-14
  • 通讯作者: 郭敬功，博士，副教授，研究方向为植物生理生化与次生代谢，E-mail:jgguo@henu.edu.cn；曲戈，博士，副研究员，研究方向为酶分子进化与生物合成，E-mail:qug@tib.cas.cn；郭敬功和曲戈为共同通信作者
  • 作者简介:李润通，硕士研究生，研究方向为酶定向进化与生物催化，E-mail:lirt@tib.cas.cn；李旭，博士，研究方向为酶理性设计与生物催化，E-mail:lix@tib.cas.cn；李润通和李旭为共同第一作者
  • 基金资助:
    天津市合成生物技术创新能力提升行动项目（TSBICIP-CXRC-053）

Enzymatic synthesis of (S)-3-hydroxytetrahydrofuran

LI Runtong1,2, LI Xu2,3, YUAN Bo2,3,4, GUO Jinggong1, QU Ge2,3,4, SUN Zhoutong2,3,4   

  1. 1. School of Life Sciences, Henan University, Kaifeng 475000, China; 2. Tianjin Institute of Industrial Biotechnology,
    Chinese Academy of Sciences, Tianjin 300308, China; 3. National Center of Technology Innovation for Synthetic
    Biology, Tianjin 300308, China; 4. Key Laboratory of Engineering Biology for Low-Carbon Manufacturing,
    Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China
  • Online:2025-04-18 Published:2025-04-14

PDF (PC)

5

摘要： （S）-3-羟基四氢呋喃是一种重要的医药中间体，其化学法合成面临手性控制不好、成本高等挑战，作为替代方案，醇脱氢酶可一步催化底物二氢-3（2H）-呋喃酮的不对称还原反应，生成相应的醇产物。研究通过筛选醇脱氢酶库，获得来源于Aromatoleum aromaticumEbN1的醇脱氢酶PED，能够不对称催化还原二氢-3（2H）-呋喃酮合成（S）-3-羟基四氢呋喃。通过反应条件优化，PED催化该反应的转化率>99%，催化效率（kcat/Km）为0.40 L/（mmol·s），产物ee值为90%，且维持较好的热稳定性（Tm=67 ℃）。通过计算分析酶关键残基与底物的相互作用，揭示了PED催化底物还原的立体选择性控制机制，即S141、Y151和Y154关键残基与底物形成氢键相互作用网络，促进形成（S）-前手性构象，辅酶NADH中氢负离子转移至羰基碳原子，将底物还原为（S）-产物。此外，放大反应实验表明PED可耐受400 mmol/L底物浓度，并可在5 h内实现完全转化。通过分离纯化，产物收率为74.7%，ee值为90%。研究挖掘到一个新颖醇脱氢酶PED，并对其酶学性能进行表征，拓展了生物法合成（S）-3-羟基四氢呋喃的酶工具箱。

关键词: 醇脱氢酶, 生物催化, 不对称催化还原, （S）-3-羟基四氢呋喃, 辅酶再生

Abstract: (S)-3-Hydroxytetrahydrofuran is an important pharmaceutical intermediate, and its chemical synthesis meets challenges such as low stereoselectivity and high cost. Alternatively, alcohol dehydrogenases catalyze the one-step asymmetric reduction of dihydro-3(2H)-furanone to the corresponding alcohol product. In this study, after screening an alcohol dehydrogenase library, an alcohol dehydrogenase derived fromAromatoleum aromaticumEbN1 (PED) was obtained, which enabled the reduction of dihydro-3(2H)-furanone to (S)-3-hydroxytetrahydrofuran. By optimizing the reaction conditions, PED catalyzed the model reaction with a >99% conversion. The catalytic efficiency (kcat/Km) was measured at 0.40 L/（mmol·s）, with aneevalue of 90%. Additionally, PED demonstrated a good thermostability, maintaining aTmvalue of 67 ℃. Computational analysis revealed the mechanism of stereoselectivity in the reduction of substrates catalyzed by PED. The key residues such as S141, Y151 and Y154 formed a network of hydrogen-bonding interactions with the substrate, which promoted the formation of apro-(S) conformation of the substrate. The (S)-product was then formed by the migration of a hydride from the coenzyme NADH to the carbonyl carbon of the substrate. Furthermore, large-scale reactions showed that PED can completely convert the substrate concentration up to 400 mmol/L in 5 h, with an isolated yield of 74.7% and aneevalue of 90%. In this study, a novel alcohol dehydrogenase PED was identified and characterized with enzymatic performance, expanding the repertoire of enzymes available for the biocatalysis of (S)-3-hydroxytetrahydrofuran.

Key words: alcohol dehydrogenase, biocatalysis, asymmetric reduction, (S)-3-hydroxytetrahydrofuran, cofactor regeneration

中图分类号: