Zwittermicin A合成基因簇中腺苷酰化功能域的预测、表达与活性验证  

Prediction,overexpression and activity confirmation of adenylation domain in Zwittermicin A biosynthesis gene cluster

作　　者：宋春旭[1] 赵昌明[1] 喻子牛[1] 孙明[1] 

机构地区：[1]华中农业大学生命科学技术学院,农业微生物学国家重点实验室,武汉430070

出　　处：《微生物学报》2008年第9期1260-1265,共6页Acta Microbiologica Sinica

基　　金：国家自然科学基金(30770020);国家“863计划”(2006AA02Z174;2006AA03A243);国家“973项目”(2003CB114201)~~

摘　　要：【目的】腺苷酰化功能域为非核糖体肽合成反应第一步底物活化的必需酶,本研究目的为验证2,3-二氨基丙酸是Zwittermicin A生物合成的直接前体物之一。【方法】本研究通过PCR的方法从苏云金芽胞杆菌菌株YBT-1520的Zwittermicin A合成基因簇中扩增得到一腺苷酰化功能域,经过双酶切和亚克隆,将此功能域克隆至超量表达载体pGEX-6p-1,将此功能域构建重组质粒pBMB1312;采用不同的IPTG浓度和温度梯度对其进行诱导,摸索了该功能域在大肠杆菌中超量表达纯化的最佳条件。【结果】得出在20℃,0.1mmol/L IPTG,宿主菌为BL21 codon plus RP(DE3)的条件下,该功能域可以被有效纯化。同时,焦磷酸释放实验证实该功能域可以对Zwittermicin A的假想前体物2,3-二氨基丙酸进行腺苷酰化。【结论】进一步支持了2,3-二氨基丙酸是Zwittermicin A生物合成的直接前体物之一的假设。[Objective] The adenylation domain is required for the substrate activation of non-ribosomal peptide synthesis. The objective of this research was to prove that 2, 3-diaminopropionate is one of the presume precursors of Zwittermicin A biosynthesis. [Methods]We cloned the adenylation domain in the Zwittermicin A synthesis cluster of Bacillus thuringiensis strain YBT-1520 with PCR amplification. After a series of enzyme digestions and subclonings, new expression vectors pBMB1312 was obtained. In order to detect the proper condition for overexpression, we tried different Isopropyl β-D-1-thiogalactopyranoside （IPTG） concentration and temperature during overepression. [Results]The overexpression protein of this domain could be purified under 20℃, 0.1 mmol/L Isopropyl β-D-1-thiogalactopyranoside （IPTG）, BL21 codon plus RP （ DE3 ） as the host strain. Then, PPi release assay indicated that 2, 3-diaminopropionate, the presume precursor of Zwittermicin A, could be adenylated by the adenylation domain. [Conclusion] This research confirmed that 2 3-diaminopropionate is one of the presume precursors of Zwittermicin A biosynthesis.

关 键 词：苏云金芽胞杆菌 Zwittermicin A 2 3-二氨基丙酸 腺苷酰化功能域 

分 类 号：Q93[生物学—微生物学]