Expression in Pichia pastoris and characterization of two novel dirigent proteins for atropselective formation of gossypol

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• 作者：Isabelle Effenberger ; Michael Harport…
• 关键词：Atropselectivity ; Biaryls ; Dirigent protein ; Fermentation ; N ; glycosylation ; Pichia pastoris
• 刊名：Applied Microbiology and Biotechnology
• 出版年：2017
• 出版时间：March 2017
• 年：2017
• 卷：101
• 期：5
• 页码：2021-2032
• 全文大小：
• 刊物类别：Chemistry and Materials Science
• 刊物主题：Microbiology; Microbial Genetics and Genomics; Biotechnology;
• 出版者：Springer Berlin Heidelberg
• ISSN：1432-0614
• 卷排序：101

文摘

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 GlycoSwitch® SuperMan₅ 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 Man₅GlcNAc₂ with, however, a significant contribution of Man_4–10GlcNAc₂. 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/O₂) 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.