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

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

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.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,探究了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.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.

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. 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.