title: Expression in Pichia pastoris and characterization of two novel dirigent proteins for atropselective formation of gossypol.
abstract: 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® SuperMan5strain 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-1of 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 Man5GlcNAc2with, however, a significant contribution of Man4-10GlcNAc2. 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.
authors.affiliation: Institute of Plant Physiology and Biotechnology, University of Hohenheim, 70593, Stuttgart, Germany.
authors.forename: Isabelle
authors.initials: I
authors.lastname: Effenberger
authors.affiliation: Institute of Plant Physiology and Biotechnology, University of Hohenheim, 70593, Stuttgart, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Harport
authors.affiliation: Core Facility Hohenheim, Mass Spectrometry Unit, University of Hohenheim, 70593, Stuttgart, Germany.
authors.forename: Jens
authors.initials: J
authors.lastname: Pfannstiel
authors.affiliation: Core Facility Hohenheim, Mass Spectrometry Unit, University of Hohenheim, 70593, Stuttgart, Germany.
authors.forename: Iris
authors.initials: I
authors.lastname: Klaiber
authors.affiliation: Institute of Plant Physiology and Biotechnology, University of Hohenheim, 70593, Stuttgart, Germany. andreas.schaller@uni-hohenheim.de.
authors.forename: Andreas
authors.identifier: http://orcid.org/0000-0001-6872-9576
authors.initials: A
authors.lastname: Schaller
mesh_terms: D003001:Cloning, Molecular; D006031:Glycosylation; D003368:Gossypium / Q000235:genetics / Q000378:metabolism*; D006072:Gossypol / Q000378:metabolism*; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D010940:Plant Proteins; D011994:Recombinant Proteins; C575905:dirigent protein, cotton; D006072:Gossypol
keywords: Atropselectivity; Biaryls; Dirigent protein; Fermentation; N-glycosylation; Pichia pastoris