Expression in Pichia pastoris and characterization of two novel dirigent proteins for atropselective formation of gossypol
Expression in Pichia pastoris and characterization of two novel dirigent proteins for atropselective formation of gossypol
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DOI：
10.1007/s00253-016-7997-3
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发表时间：
2017-03-01
影响因子：
5
通讯作者：
Schaller, Andreas
中科院分区：
文献类型：
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作者：
Effenberger, Isabelle;Harport, Michael;Schaller, Andreas
利用毕赤酵母GlycoSwitchA(R)Superman(5)工程菌株，建立了一种高效的补料分批发酵工艺，以防止高糖基化，从而从棉花中获得两种新的转导蛋白：海岛棉GbDIR2和陆地棉GhDIR3。两种(His)(6)标记的蛋白经金属螯合亲和层析纯化后，分别获得了12和15 mg的培养体积L-1。通过质谱学鉴定了天然蛋白和酶去糖化蛋白的糖基化位点，证实了NxS/T序列中预测的七个糖基化位点中的五到六个。主要的糖链结构是Man(5)GlcNAc(2)，而Man(4-10)GlcNAc(2)贡献很大。这两种引导性蛋白(DIRS)都通过半棉酚自由基的反式选择性偶联来介导(+)-棉酚的形成。GbDIR2和GhDIR3与以前的DIRS相似，缺乏氧化活性，依赖于底物自由基氧化体系(漆酶/O-2)的产生。与参与木脂素生物合成的DIRS不同，糖基化不是功能所必需的。定量酶促脱糖反应可获得高纯度的活性GbDIR2和GhDIR3。所描述的发酵过程与酶促脱糖作用相结合，将为机理和结构研究铺平道路，并最终将棉花DIRS应用于仿生合成阿托品选择性联芳基。
We established an efficient fed-batch fermentation process for two novel dirigent proteins from cotton plants, GbDIR2 from Gossypium barbadense and GhDIR3 from G. hirsutum, using the engineered Pichia pastoris GlycoSwitchA (R) SuperMan(5) strain to prevent hyperglycosylation. The two (His)(6)-tagged proteins were purified by metal-chelate affinity chromatography and obtained in quantities of 12 and 15 mg L-1 of culture volume, respectively. Glycosylation sites were identified for the native and for the enzymatically deglycosylated proteins by mass spectrometry, confirming five to six of the seven predicted glycosylation sites in the NxS/T sequence context. The predominant glycan structure was Man(5)GlcNAc(2) with, however, a significant contribution of Man(4-10)GlcNAc(2). Both dirigent proteins (DIRs) mediated the formation of (+)-gossypol by atropselective coupling of hemigossypol radicals. Similar to previously characterized DIRs, GbDIR2 and GhDIR3 lacked oxidizing activity and depended on an oxidizing system (laccase/O-2) for the generation of substrate radicals. In contrast to DIRs involved in the biosynthesis of lignans, glycosylation was not essential for function. Quantitative enzymatic deglycosylation yielded active GbDIR2 and GhDIR3 in excellent purity. The described fermentation process in combination with enzymatic deglycosylation will pave the way for mechanistic and structural studies and, eventually, the application of cotton DIRs in a biomimetic approach towards atropselective biaryl synthesis.