bagZH编码酪氨酸酶样铜酶并参与bagremycin生物合成  

BagZH encodes tyrosinase-like copper enzyme and participates in bagremycin biosynthesis

作　　者：祁双双 吴海珍[1] 叶江[1] 张惠展[1] Shuangshuang Qi;Haizhen Wu;Jiang Ye;Huizhanhang(State Key Laboratory of Bioreactor Engineering,East Zhina University of Science and Technology,Shanghai 200237,China)

机构地区：[1]华东理工大学生物反应器工程国家重点实验室,上海200237

出　　处：《微生物学报》2018年第12期2229-2239,共11页Acta Microbiologica Sinica

基　　金：国家自然科学基金(31200026)~~

摘　　要：【目的】研究bagremycin产生菌链霉菌Tü4128中编码酪氨酸酶样铜酶的bagZH基因的功能。【方法】基于同源重组技术敲除bagZH基因,利用HPLC和LC-ESI-MS分析其次级代谢产物谱。在E. coli BL21(DE3)中异源表达BagH并分离纯化,分别以邻氨基酚和3,4-AHBA为底物,利用LC-ESI-MS分析BagH催化产物。【结果】HPLC显示,bagZH基因敲除突变株的bagremycin产量显著降低,回补bagZH基因表达盒后bagremycin产量有所上调。LC-ESI-MS分析bagZH基因敲除突变株的次级代谢产物谱,结果显示,保留时间为3.18 min的新产物分子量为286.32 g/mol,与推测的3,4-AHBA在体内酯化合成的产物分子量吻合。体外生化分析显示,BagH能将邻氨基酚的邻位氨基氧化为亚硝基(保护基团)。【结论】本文首次鉴定了bagZH基因编码的酪氨酸酶样铜酶参与bagremycin生物合成。BagH负责将3,4-AHBA的邻位氨基氧化为亚硝基(保护基团)避免自身酯化,待与反式对香豆酸衍生物缩合后,再由胞内的还原酶将保护性亚硝基还原为氨基,最终合成bagremycin A和bagremycin B。我们的研究结果为bagremycin作用机制的深入研究以及高产菌株的理性设计与改造提供了基础和参考。[Objective]We aimed to reveal a novel function ofbagZHin Streptomyces sp.Tü4128,which encoded a tyrosinase-like copper enzyme.[Methods]The bagZH gene was deleted through homologous recombination,and the secondary metabolites were detected and analyzed by HPLC and LC-ESI-MS.The activity of the BagH enzyme expressed by E.coli BL21(DE3)was measured by biochemical assays.The catalytic products of the enzyme were analyzed by LC-ESI-MS,in which o-aminophenol and 3,4-AHBA were used as substrates.[Results] HPLC analysis showed that the production of bagremycin significantly decreased when bagZH was deleted. Complementation of bagZH gene expression cassettes in the mutant increased the accumulation of bagremycin. LC-ESI-MS results showed that the molecular weight of the new product with a retention time of 3.18min was 286.32g/mol,which was consistent with the predicted molecular weight of the product synthesized by esterification of 3,4-AHBA in vivo.In vitro biochemical analysis demonstrated that BagH can catalyze the oxidation of o-aminophenol (protecting group).[Conclusion]Our findings revealed for the first time that bagZH participated in the biosynthesis of bagremycin by encoding a tyrosinase-like copper enzyme.BagH protected the biosynthetic intermediates by catalyzing the oxidation of 3,4-AHBA to a nitroso derivative (protecting group).After condensation with p-coumanic acid,the nitroso-group is reduced by a reductase in vivo to generate bagremycin A and B.The results obtained in this study provide a basis and reference for in-depth study of the mechanism of bagremycin and rational design and transformation of high-yielding strains.

关 键 词：bagremycin bagZH 酪氨酸酶样铜酶 亚硝基化 

分 类 号：Q933[生物学—微生物学]