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研究论文

基于吲哚酸单元的那西肽生物合成途径中酶底物容忍度研究

范亚飞a, 张鄂a, 郭恒b, 牟柠a, 陈单丹b, 王文贵a, 王守锋a, 刘文b,c   

  1. a 济南大学化学化工学院 山东省氟化学化工材料重点实验室 济南 250022;
    b 中国科学院上海有机化学研究所 生命有机化学国家重点实验室 上海 200032;
    c 中国科学院上海有机化学研究所 湖州 生物制造中心 湖州 313000
  • 收稿日期:2020-06-28 修回日期:2020-08-06 出版日期:2030-01-01 发布日期:2020-09-09
  • 通讯作者: 王守锋, 刘文 E-mail:chm_wangsf@ujn.edu.cn;wliu@mail.sioc.ac.cn
  • 基金资助:
    国家自然科学基金(No.31972850,21750004、21520102004)、山东省重点研发计划(No.2019GSF108223)、中国科学院(No.QYZDJ-SSW-SLH037、XDB20020200)、上海市科委(No.17JC1405100)、中国科学院青年创新促进会(No.2017303)、微生物技术国家重点实验室开放课题基金(No.M2020-05)和王宽诚教育基金会资助项目.

Insights into the Substrate Tolerance of Enzymes Involved in the Nosiheptide Biosynthesis Pathway Based on Indolic Acid Moiety

Fan Yafeia, Zhang Ea, Guo Hengb, Mu Ninga, Chen Dandanb, Wang Wenguia, Wang Shoufenga, Liu Wenb,c   

  1. a Shandong Provincial Key Laboratory of Fluorine Chemistry and Chemical Materials, School of Chemistry and Chemical Engineering, University of Jinan, Jinan 250022;
    b State Key Laboratory of Bioorganic and Natural Products Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai 200032;
    c Huzhou Center of Bio-Synthetic Innovation, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Hu-zhou 313000
  • Received:2020-06-28 Revised:2020-08-06 Online:2030-01-01 Published:2020-09-09
  • Supported by:
    Project supported by the grants from the National Natural Science Foundation of China (31972850, 21750004 and 21520102004), the Shandong Key Research Program (2019GSF108223), Chinese Academy of Sciences (QYZDJ-SSW-SLH037 and XDB20020200), Science and Technology Commission of Shanghai Municipality (17JC1405100), Youth Innovation Promotion Association of the Chinese Academy of Sciences (2017303), State Key Laboratory of Microbial Technology Open Projects Fund(M2020-05) and K. C. Wong Education Foundation.

那西肽(NOS)是硫肽类抗生素的典型代表,具有非常好的抗菌活性,但水溶性差以及生物利用度低等问题限制了其临床上的应用。由于其结构复杂,采用化学全合成方式获得理化性质改善的类似物难以进行。本文在前期那西肽生物合成研究的基础上,以侧环3-甲基-2-吲哚酸(MIA)类似物作为化学探针,通过探针分子与突变株共同发酵并结合发酵产物的高分辨质谱数据,探究那西肽生物合成酶的底物容忍度。研究结果表明那西肽生物合成酶对-F,-Cl,-CH3取代的MIA类似物有一定的耐受能力,对大位阻取代基(-NO2,-CF3,-Ph)的MIA类似物不能耐受,同时MIA上取代基团的结构大小和性质也影响了NOS生物合成途径中相关酶蛋白对其识别、转运和上载等步骤。本文的研究不仅探究了NOS生物合成途径中相关酶蛋白的底物容忍度,有望通过生物合成途径工程获得NOS的类似物;同时为采用酶的定向进化技术以改善NOS生物合成过程中限速步骤酶的底物容忍度,拓展利用NOS产生菌获得更多类似物提供了借鉴。

关键词: 那西肽, 3-甲基-2-吲哚酸, 突变生物合成, 化学小分子探针, 酶底物容忍度

As a typical representative of thiopeptide antibiotics, nosiheptide (NOS) possesses very good antibacterial activity. However, due to poor water solubility and low bioavailability, its clinical application is hampered. Due to its complex structure, it is difficult to obtain analogues with improved physical and chemical properties via total chemical synthesis. In this paper, based on the previous studies on the biosynthesis of nosiheptide, the side-ring 3-methyl-2-indoleic acid (MIA) analogues were used as chemical small molecule probes to explore the substrate tolerance of enzymes involved in NOS biosynthesis pathway in NOS-producing bacteria via the co-fermentation of probe molecules with mutant strain and the combination of high resolution mass spectrometry data of fermentation products. The results showed that enzymes involved in NOS biosynthesis pathway had a considerable tolerance to MIA analogues substituted by -F, -CL and -CH3, however, MIA analogues substituted by large steric hindrance group, such as-NO2, -CF3 and -ph, were not tolerated.The position, the size and the property of the substituted groups of MIA also affected the steps of identification, transport and upload of the related enzymes involved in NOS biosynthesis. The present study not only explored the substrate tolerance of enzymes involved in NOS biosynthesis pathway, but also was expected to obtain NOS analogues via biosynthetic pathway engineering. What's more, it provides valuable information for using directed evolution technology to improve the substrate tolerance of enzymes in the rate-limiting steps of NOS biosynthesis and to expand the use of NOS-producing bacteria to obtain more analogues.

Key words: nosiheptide, 3-methyl-2-indolic acid, mutational biosynthesis, chemical small molecule probe, substrate tolerance of enzymes