Bacillomycin D合成酶系NRPS硫酯酶结构域位移对脂肽合成的影响  

Effect of translocation of NRPS thioesterase domain of Bacillomycin D synthase system on the synthesis of lipopeptides

作　　者：张平 陈美容 马文杰 陆兆新[1] 吕紫岩 吕凤霞[1] 赵海珍[1] 别小妹[1] ZHANG Ping;CHEN Meirong;MA Wenjie;LU Zhaoxin;Lü Ziyan;Lü Fengxia;ZHAO Haizhen;BIE Xiaomei(College of Food Science and Technology,Nanjing Agricultural University,Nanjing 210095,China;College of Pharmacy,China Pharmaceutical University,Nanjing 211198,China)

机构地区：[1]南京农业大学食品科学与技术学院,江苏南京210095 [2]中国药科大学药学院,江苏南京211198

出　　处：《南京农业大学学报》2023年第1期169-178,共10页Journal of Nanjing Agricultural University

基　　金：国家自然科学基金项目(31972174)。

摘　　要：[目的]本文旨在研究解淀粉芽胞杆菌(Bacillus amyloliquefaciens)fmbJ菌株中Bacillomycin D合成酶系非核糖体肽合成酶(NRPS)硫酯酶TE结构域位移对脂肽合成的影响。[方法]通过温敏型质粒pKS2介导的同源重组将fmbJ菌株中Bacillomycin D合成酶系NRPS硫酯酶TE结构域分别前移至模块5和模块6末端,然后对fmbJ突变菌株的发酵产物进行高效液相色谱(HPLC)、液相质谱(LC-MS)以及黄曲霉菌抑菌试验分析,并且使用AlphaFold蛋白结构数据库预测NRPS合成酶系末端PCP-TE双结构域的三维结构,以及分析双结构与两者之间的相互作用关系。[结果]在突变菌株fmbJ-M5-TELong的发酵液中检测出线性脂五肽(C14-15β-NH2FA-Asn-Tyr-Asn-Pro-Glu)和环状脂五肽[C14-15β-NH2FA(Asn-Tyr-Asn-Pro-Glu)],在突变菌株fmbJ-M6-TELong的发酵液中检测出线性脂六肽(C14-17β-NH2FA-Asn-Tyr-Asn-Pro-Glu-Ser)和环状脂六肽[C12-14,16-17β-NH2FA(Asn-Tyr-Asn-Pro-Glu-Ser)]等新型脂肽。此外,NRPS硫酯酶TE结构域易位前移后,PCP-TE双结构域蛋白的构象和相对作用力发生变化,疏水相互作用界面缩小。[结论]fmbJ菌株中Bacillomycin D合成酶系NRPS硫酯酶TE结构域位移导致发酵产物提前环化和水解,合成了截短的新型线性脂五肽、环状脂五肽、线性脂六肽和环状脂六肽等脂肽,为新型脂肽的开发提供了思路。[Objectives]The aim of this study was to study the effect of translocation of nonribosomal peptide synthetases(NRPS)thioesterase domain in Bacillomycin D synthase system on lipopeptides synthesis in Bacillus amyloliquefaciens fmbJ. [Methods]The TE domain at the end of Bacillomycin D synthesis in fmbJ strain was advanced to the end of module 5 and module 6 by homologous recombination mediated by temperature sensitive plasmid pKS2,respectively. Then, the fermentation products of fmbJ mutant strain were analyzed by high performance liquid chromatography(HPLC),liquid chromatography-mass spectrometry(LC-MS)and bacteriostasis experiments with Aspergillus flavus. AlphaFold protein structure database was used to predict the three-dimensional structures of PCP-TE didomains at the end of NRPS synthase system, and the interactions between domains were analyzed. [Results]Linear lipopentapeptides(C14-15β-NH2FA-Asn-Tyr-Asn-Pro-Glu)and cyclic lipopentapeptides[C14-15β-NH2FA(Asn-Tyr-Asn-Pro-Glu)]were detected in the fermentation broth of mutant strain fmbJ-M5-TELong. Linear lipohexapeptides(C14-17β-NH2FA-Asn-Tyr-Asn-Pro-Glu-Ser)and cyclic lipohexapeptides[C12-14,16-17β-NH2FA(Asn-Tyr-Asn-Pro-Glu-Ser)]were detected in the fermentation broth of mutant strain fmbJ-M6-TELong. In addition, after translocation of thioesterase domain, the relative conformation of PCP-TE didomains and the interactions between two domains were changed, and the hydrophobic interaction interface was narrowed. [Conclusions]The translocation of NRPS thioesterase domain of Bacillomycin D synthase system in fmbJ strain made the products hydrolyzed and cyclized in advance, and novel lipopeptides including truncated linear lipopentapeptides, cyclic lipopentapeptides, linear lipohexapeptides and cyclic lipohexapeptides were synthesized, which provided ideas for the development of novel lipopeptides.

关 键 词：Bacilomycin D 硫酯酶 同源重组 液相质谱(LC-MS) 蛋白三维结构 

分 类 号：TS201.3[轻工技术与工程—食品科学]