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

Publikations-Art

Zeitschriftenbeitrag (peer-reviewed)

Autoren

Effenberger, I., Harport, M., Pfannstiel, J., Klaiber, I., Schaller, A.

Erscheinungsjahr

2017

Veröffentlicht in

Appl. Microbiol. Biotechnol.

Band/Volume

101/

Seite (von - bis)

2021–2032

We established an efficient fed-batch fermentationprocess for two novel dirigent proteins from cotton plants,GbDIR2 fromGossypium barbadenseand GhDIR3 fromG. hirsutum, using the engineeredPichia pastorisGlycoSwitch® SuperMan5strain to preventhyperglycosylation. The two (His)6-tagged proteins were pu-rified by metal-chelate affinity chromatography and obtainedin quantities of 12 and 15 mg L−1of culture volume, respec-tively. Glycosylation sites were identified for the native andfor the enzymatically deglycosylated proteins by mass spec-trometry, confirming five to six of the seven predicted glyco-sylation sites in the NxS/Tsequence context. The predominantglycan structure was Man5GlcNAc2with, however, a signifi-cant contribution of Man4–10GlcNAc2.Bothdirigentproteins(DIRs) mediated the formation of (+)-gossypol byatropselective coupling of hemigossypol radicals. Similar topreviously characterized DIRs, GbDIR2 and GhDIR3 lackedoxidizingactivityanddependedonanoxidizingsystem(laccase/O2) for the generation of substrate radicals. In con-trast to DIRs involved in the biosynthesis of lignans, glyco-sylation was not essential for function. Quantitative enzymaticdeglycosylation yielded active GbDIR2 and GhDIR3 in ex-cellent purity. The described fermentation process in combi-nation with enzymatic deglycosylation will pave the way formechanistic and structural studies and, eventually, the appli-cation of cotton DIRs in a biomimetic approach towardsatropselective biaryl synthesis.