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

Publication Type

Journal contribution (peer reviewed)

Authors

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

Year of publication

2017

Published in

Appl. Microbiol. Biotechnol.

Band/Volume

101/

Page (from - to)

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.