解淀粉芽胞杆菌HZ-12中ysnE参与吲哚-3-乙酸合成的代谢途径研究

Metabolic pathways of ysnE involved in indole-3-acetic acid synthesis in Bacillus amyloliquefaciens HZ-12

【目的】吲哚-3-乙酸是调控植物生长发育和生理活动的重要激素,吲哚-3-乙酸N-乙酰转移酶YsnE在吲哚-3-乙酸合成中发挥重要作用,本研究拟解析解淀粉芽胞杆菌中YsnE参与吲哚-3-乙酸合成的代谢途径。【方法】通过基因ysnE缺失和强化表达,分析ysnE对吲哚-3-乙酸合成影响,结合吲哚-3-乙酸合成中间物(吲哚丙酮酸、吲哚乙酰胺、色胺和吲哚乙腈)添加和体外酶转化实验,解析ysnE参与吲哚-3-乙酸合成的代谢途径。【结果】明确了YsnE在解淀粉芽胞杆菌HZ-12吲哚-3-乙酸合成中发挥重要作用。发现ysnE缺失菌株中的吲哚丙酮酸、吲哚乙酰胺和吲哚乙腈利用显著降低,揭示了YsnE主要发挥吲哚丙酮酸脱羧酶YclB和吲哚乙酰胺水解酶/腈水解酶/腈水合酶YhcX的功能,并通过参与吲哚丙酮酸、吲哚乙酰胺和吲哚乙腈途径来影响吲哚-3-乙酸合成。【结论】初步揭示了YsnE通过影响吲哚丙酮酸、吲哚乙酰胺和吲哚乙腈途径参与吲哚-3-乙酸合成的代谢机理,为吲哚-3-乙酸合成途径解析和代谢工程育种构建吲哚-3-乙酸高产菌株奠定了基础。

[Objective]Indole-3-acetic acid is crucial for plant growth and development and physiological activities.Indole-3-acetic acid N-acetyltransferase YsnE plays an important part in indole-3-acetic acid synthesis.This study aims to analyze the metabolic pathways of YsnE involved in indole-3-acetic acid synthesis of Bacillus amyloliquefaciens.[Methods]Through deletion and overexpression of ysnE,the role of ysnE in indole-3-acetic acid synthesis was elucidated.Combined with the addition of indole-3-acetic acid synthesis intermediates(indole pyruvic acid(IPA),indole-3-acetamide(IAM),tryptamine(TAM),indole-3-acetonitrile(IAN))and in vitro enzymatic conversion experiments,the metabolic pathways of ysnE involved in indole-3-acetic acid synthesis were clarified.[Results]YsnE played an important role in indole-3-acetic acid synthesis in B.amyloliquefaciens HZ-12.The amount of IPA,IAM,and IAN consumed in ysnE deletion strain was significantly reduced,and YsnE functioned as indolepyruvate decarboxylase YclB,and indoleacetamide hydrolase/nitrilase/nitrile hydratase YhcX,and affected indole-3-acetic acid synthesis by participating in IPA,IAM,and IAN pathways.[Conclusion]YsnE influences the IPA,IAM,and IAN pathways to involve in indole-3-acetic acid synthesis,which lays a foundation for analysis of indole-3-acetic acid synthesis pathway and breeding strains with high yield of indole-3-acetic acid by metabolic engineering.