title: Functional expression of a valencene dioxygenase from Pleurotus sapidus in E. coli.
abstract: Valencene dioxygenase (ValOx) from the edible basidiomycete Pleurotus sapidus converted the sesquiterpene (+)-valencene to the valuable grapefruit flavour (+)-nootkatone and to nootkatols through intermediate hydroperoxides. Expression of the enzyme was carried out in the cytosol and periplasm of Escherichia coli. The heterologous production led to high yields of inclusion bodies. The poor yield of soluble recombinant protein was improved by various strategies including cold shock expression, chaperone co-expression, and employment of mutant E. coli strains. Up to 60 mg of the biologically active, soluble ValOx was produced by cold shock under control of the cspA promoter at 8 °C in the BL21(DE3)Star strain and co-expression of the E. coli trigger factor. The recombinant enzyme, purified using the N-terminal His tag, showed the catalytic properties of the wild-type enzyme, as was confirmed by the LC-MS analysis of hydroperoxide intermediates and GC-MS analysis of the volatile products.
authors.affiliation: Gottfried Wilhelm Leibniz University Hannover, Institute of Food Chemistry, Callinstr. 5, D-30167 Hannover, Germany. kateryna.zelena@lci.uni-hannover.de
authors.forename: Kateryna
authors.initials: K
authors.lastname: Zelena
authors.forename: Ulrich
authors.initials: U
authors.lastname: Krings
authors.forename: Ralf G
authors.initials: RG
authors.lastname: Berger
mesh_terms: D002853:Chromatography, Liquid; D049308:Dioxygenases / Q000378:metabolism*; D004926:Escherichia coli; D008401:Gas Chromatography-Mass Spectrometry; D002479:Inclusion Bodies / Q000378:metabolism; D007218:Industrial Microbiology / Q000379:methods*; D013058:Mass Spectrometry; D020076:Pleurotus / Q000201:enzymology*; D000081223:Polycyclic Sesquiterpenes; D012717:Sesquiterpenes / Q000378:metabolism*
chemical_list: D000081223:Polycyclic Sesquiterpenes; D012717:Sesquiterpenes; C506706:valencene; D049308:Dioxygenases; C050302:nootkatone