游动放线菌Actinoplanes sp.SE50/110中阿卡波糖脱氧氨基糖单元的生物合成  

Biosynthetic pathway of deoxyaminosugar moiety in acarbose from Actinoplanes sp. SE50/110

作　　者：张丹 赵芹芹 蒋明 康前进[1] 白林泉[1] Dan Zhang;Qinqin Zhao;Ming Jiang;Qianjin Kang;Linquan Bai(State Key Laboratory of Microbial Metabolism,School of Life Sciences and Biotechnology,Shanghai Jiao Tong University,Shanghai 200240,China)

机构地区：[1]上海交通大学生命科学技术学院微生物代谢国家重点实验室

出　　处：《微生物学报》2020年第1期118-134,共17页Acta Microbiologica Sinica

基　　金：国家自然科学基金(31830104)~~

摘　　要：【目的】解析Actinoplanes sp.SE50/110(简称SE50/110)中阿卡波糖脱氧氨基糖单元的生物合成机制。【方法】经过BLASTp分析,推测了Acb A、Acb B和Acb V负责阿卡波糖脱氧氨基糖单元的生物合成。首先,本研究在SE50/110中分别构建了acb A、acb B和acb V的同框缺失和回补突变株。然后,利用大肠杆菌BL21(DE3)/p Gro7分别对Acb A、Acb B和Acb V成功实现了可溶性表达。最后,以D-葡萄糖-1-磷酸为起始底物,通过体外催化反应,研究脱氧氨基糖单元的生物合成过程和相关蛋白的酶学性质。【结果】在SE50/110中分别缺失acb A、acb B和acb V基因后,相应突变株均丧失了阿卡波糖的合成能力,将acb A、acb B和acb V基因分别回补后,各菌株又恢复了阿卡波糖的合成能力,证明了它们均为阿卡波糖生物合成的必需基因。在体外酶促反应中,D-葡萄糖-1-磷酸-胸腺嘧啶转移酶Acb A催化D-葡萄糖-1-磷酸和d TTP合成d TDP-D-葡萄糖,对D-葡萄糖-1-磷酸的Km值为(0.185±0.053)mmol/L,Vmax为(2.366±0.217)μmol/(min·mg);对d TTP的Km值为(4.964±1.089)mmol/L,Vmax为(60.310±5.419)μmol/(min·mg)。d TDP-D-葡萄糖-4,6-脱水酶Acb B催化d TDP-D-葡萄糖转化为d TDP-4-酮基-6-脱氧-D-葡萄糖,Km值和Vmax分别为(0.353±0.089)mmol/L和(306.401±28.740)μmol/(min·mg)。氨基转移酶Acb V催化d TDP-4-酮基-6-脱氧-D-葡萄糖生成d TDP-4-氨基-4,6-双脱氧-D-葡萄糖,Km值和Vmax分别为(1.411±0.293)mmol/L和(3.447±0.279)μmol/(min·mg)。【结论】本研究阐明了阿卡波糖脱氧氨基糖单元的生物合成过程,为全面解析阿卡波糖生物合成途径奠定了基础。同时,测定了相关酶的动力学参数,为代谢工程改造SE50/110,提高阿卡波糖产量提供了重要的理论依据。[Objective]Elucidation of the biosynthetic mechanism of the deoxyaminosugar moiety in acarbose from Actinoplanes sp.SE50/110.[Methods]On the basis of Blast P analysis,Acb A,Acb B and Acb V were proposed to be associated with the biosynthesis of deoxyaminosugar moiety.Firstly,the in-frame deletion and trans-complementation of acb A,acb B and acb V were performed in SE50/110 to investigate their involvement in acarbose biosynthesis.Then,Acb A,Acb B and Acb V were heterologously expressed in E.coli BL21(DE3)/p Gro7 and purified by Ni affinity chromatography.Finally,using D-glucose-1-phosphate as the starting substrate,the biosynthetic process of deoxyaminosugar moiety was elucidated by enzymatic assay.In addition,the properties of these involved enzymes were characterized.[Results]Deletion mutants of acb A,acb B and acb V in SE50/110 were named as ZD03,ZD04 and ZD05,respectively,all of which lost the productivity of acarbose.And then,the production of acarbose was recovered through trans-complementation of acb A,acb B and acb V in ZD03,ZD04 and ZD05,respectively.In vitro enzymatic analysis suggested that Acb A,a D-glucose-1-phosphate thymidylyltransferase,is responsible for the biosynthesis of d TDP-D-glucose from D-glucose-1-phosphate and d TTP.It showed a Km of(0.185±0.053)mmol/L and a Vmax of(2.366±0.217)μmol/(min·mg)with D-glucose-1-phosphate,as well as a Km of(4.964±1.089)mmol/L and a Vmax of(60.310±5.419)μmol/(min·mg)with d TTP.Acb B,a TDP-D-glucose-4,6-dehydratase,catalyzed the dehydration of d TDP-D-glucose to d TDP-4-keto-6-deoxy-D-glucose.The Km and Vmax of Acb B are(0.353±0.089)mmol/L and(306.401±28.740)μmol/(min·mg),respectively.Acb V is a d TDP-4-keto-6-deoxy-D-glucose-aminotransferase and catalyzes the transamination of d TDP-4-keto-6-deoxy-D-glucose to d TDP-4-amino-4,6-dideoxy-D-glucose using glutamic acid as amino donor.The Km and Vmax of Acb V are(1.411±0.293)mmol/L and(3.447±0.279)μmol/(min·mg),respectively.[Conclusion]This study elucidated the biosynthetic pathway of deoxyaminosug

关 键 词：阿卡波糖 脱氧氨基糖 生物合成 酶促反应 

分 类 号：Q936[生物学—微生物学]