化工学报 ›› 2022, Vol. 73 ›› Issue (7): 3202-3211.doi: 10.11949/0438-1157.20220490

• 生物化学工程与技术 • 上一篇    下一篇

甘草黄酮合酶Ⅱ催化甘草素特异性合成7，4′-二羟基黄酮

孙甲琛1(),孙文涛2,孙慧1,吕波3(),李春1,2,3()   

  1. 1.石河子大学化学化工学院/新疆兵团绿色化工过程重点实验室，新疆 石河子 832003
    2.清华大学化学工程系 生物化工研究所/工业生物催化教育部重点实验室，北京 100084
    3.北京理工大学化学与化工学院 生物化工研究所/医药分子科学与制剂工信部重点实验室，北京 100081
  • 收稿日期:2022-04-06 修回日期:2022-05-12 出版日期:2022-07-05 发布日期:2022-08-01
  • 通讯作者: 吕波,李春 E-mail:sunjiachen_synbio@163.com;lv-b@ bit.edu.cn;lichun@tsinghua.edu.cn
  • 作者简介:孙甲琛（1996—），男，硕士研究生，sunjiachen_synbio@163.com

Licorice flavone synthase Ⅱ catalyzes liquiritigenin to specifically synthesize 7,4′-dihydroxyflavone

Jiachen SUN1(),Wentao SUN2,Hui SUN1,Bo LYU3(),Chun LI1,2,3()   

  1. 1.Key Laboratory for Green Processing of Chemical Engineering of Xinjiang Bingtuan, School of Chemistry and Chemical Engineering, Shihezi University, Shihezi 832003, Xinjiang, China
    2.Key Laboratory for Industrial Biocatalysis, Ministry of Education, Department of Chemical Engineering, Tsinghua University, Beijing 100084, China
    3.Key Laboratory of Medical Molecule Science and Pharmaceutics Engineering, Ministry of Industry and Information Technology, Institute of Biochemical Engineering, School of Chemistry and Chemical Engineering, Beijing Institute of Technology, Beijing 100081, China
  • Received:2022-04-06 Revised:2022-05-12 Published:2022-07-05 Online:2022-08-01
  • Contact: Bo LYU,Chun LI E-mail:sunjiachen_synbio@163.com;lv-b@ bit.edu.cn;lichun@tsinghua.edu.cn

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摘要：

利用同源序列比对和分子进化树分析从胀果甘草转录组数据库中挖掘并成功克隆到2个黄酮合酶基因：Gur.gene26505和Gur.gene26116。经表征Gur.gene26505为黄酮合酶Ⅱ，具有催化甘草素特异性合成7,4′-二羟基黄酮的特性，而Gur.gene26116为黄烷酮2位羟化酶，可催化甘草素生成包括7,4′-二羟基黄酮在内的三种产物。进一步通过蛋白质结构预测、分子对接和分子动力学模拟探究了黄酮合酶Ⅱ(Gur.gene26505)催化合成7,4′-二羟基黄酮特异性的原因。由于Gur.gene26505活性口袋附近特有的刚性结构β片层使大位阻苯丙氨酸残基翻转至羟化中心下方，消除了羟化产物2-羟基甘草素进入脱水中心的阻力进而发生C2-C3位的脱水反应特异性生成7,4′-二羟基黄酮。最后通过基因过表达、反应条件优化和强化菌体生长建立了7,4′-二羟基黄酮特异性合成的最佳细胞催化工艺，使甘草素转化率达到了76.67%。

关键词: 甘草黄酮合酶Ⅱ, 甘草素, 7,4′-二羟基黄酮, 特异性转化, 生物催化

Abstract:

Using homologous sequence alignment and molecular phylogenetic tree analysis, two flavonoid synthase genes are successfully cloned from the Glycyrrhiza inflata: Gur.gene26505 and Gur.gene26116. Gur.gene26505 is characterized as a flavonoid synthase Ⅱ, catalyzing liquiritigenin to specifically synthesize 7,4'-dihydroxyflavone, while Gur.gene26116 is a flavanone 2-hydroxylase that catalyzes liquiritigenin to synthesize three products including 7,4'-dihydroxyflavone. The reason for the specificity of 7,4'-dihydroxyflavone catalyzed by flavonoid synthase Ⅱ (Gur.gene26505) was further explored through protein structure prediction, molecular docking, and molecular dynamics simulations. Due to the unique rigid structure β-sheet near the active pocket of Gur.gene26505, the large sterically hindered phenylalanine residue is turned over to the lower part of the hydroxylation center, which eliminates the resistance of the hydroxylated product 2-hydroxyliquiritigenin to enter the dehydration center, occurring dehydration at C2-C3 positions and generating 7,4'-dihydroxyflavone. Finally, the optimal cell catalysis process for the specific synthesis of 7,4'-dihydroxyflavone is established through gene overexpression, optimization of reaction conditions and enhancement of bacterial growth, and the conversion rate of liquiritigenin reaches 76.67%.

Key words: licorice flavonoid synthase Ⅱ, liquiritigenin, 7,4'-dihydroxyflavone, specific transformation, biocatalysis

中图分类号: 

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