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 adenylationdomain in Zwittermicin A biosynthesis gene cluster

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℃，0.1 mmol/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。

Abstract:

[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 thuringien-sis 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 b-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.