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

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® SuperMan 5 strain to prevent hyperglycosylation. The two (His) 6 -tagged pro...

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Bibliographic Details
Published in:Applied microbiology and biotechnology 2017-03, Vol.101 (5), p.2021-2032
Main Authors: Effenberger, Isabelle, Harport, Michael, Pfannstiel, Jens, Klaiber, Iris, Schaller, Andreas
Format: Article
Language:English
Subjects:
Alcohol
Biomedical and Life Sciences
Biosynthesis
Biotechnologically Relevant Enzymes and Proteins
Biotechnology
Cloning, Molecular
Cotton
Cytochrome
Enzymes
Fermentation
Glycosylation
Gossypium - genetics
Gossypium - metabolism
Gossypium barbadense
Gossypol
Gossypol - metabolism
Life Sciences
Mass spectrometry
Metabolism
Microbial Genetics and Genomics
Microbiology
Morphine
Natural products
Oxidation
Phenols
Phylogenetics
Physiological aspects
Pichia - genetics
Pichia - metabolism
Pichia pastoris
Plant Proteins - genetics
Plant Proteins - metabolism
Proteins
Recombinant Proteins - genetics
Recombinant Proteins - metabolism
Studies
Substrates
Toxicity
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Summary: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® 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.
ISSN:0175-7598
1432-0614
DOI:10.1007/s00253-016-7997-3