title: Expression, purification and activity assay of a patchoulol synthase cDNA variant fused to thioredoxin in Escherichia coli.
abstract: Probing a cDNA library extracted from Pogostemon cablin (Indian Patchouli) with gene specific primers, a variant of patchoulol synthase PTS (GenBank acc. No.: AY508730) was amplified, cloned, and sequenced. The amino acid sequence deduced from the cloned cDNA exhibited a sequence variation of 3.4% compared to the annotated sequence. The enzyme variant tended to form inclusion bodies when expressed in Escherichia coli. The coding sequence was fused to the T7-tag, His-tag and to thioredoxin. Constructs were expressed in three different E. coli expression strains, with several strain/construct combinations yielding soluble enzyme. By fusion to thioredoxin and careful codon optimization of the eukaryotic sequence, soluble expression could be improved on average by 42% in comparison to an unoptimized, His-tagged construct. The thioredoxin-fused protein was successfully purified using a one-step Co(2+)-IMAC purification procedure. Bioactivity assays using prepared farnesyl diphosphate (FDP) in milliliter-scale batch reactions, showed activity of the fused enzyme even with thioredoxin attached. The product spectrum of the enzyme was compared to patchouli oil standards by GC-MS and the main products were identified. Interestingly, the variant showed a shift in product spectrum with germacrene A being the most abundant product instead of patchouli alcohol. In silico structural modeling shows a possible chemical and structural change in the active site of the enzyme, which might be responsible for the shift in product composition.
authors.affiliation: Institute of Technical Chemistry, Leibniz University of Hannover, Callinstr. 5, 30167 Hannover, Germany.
authors.forename: S
authors.initials: S
authors.lastname: Hartwig
authors.affiliation: Institute of Technical Chemistry, Leibniz University of Hannover, Callinstr. 5, 30167 Hannover, Germany.
authors.forename: T
authors.initials: T
authors.lastname: Frister
authors.affiliation: Institute of Technical Chemistry, Leibniz University of Hannover, Callinstr. 5, 30167 Hannover, Germany.
authors.forename: S
authors.initials: S
authors.lastname: Alemdar
authors.affiliation: Institute of Technical Chemistry, Leibniz University of Hannover, Callinstr. 5, 30167 Hannover, Germany.
authors.forename: Z
authors.initials: Z
authors.lastname: Li
authors.affiliation: Institute of Food Chemistry, Leibniz University of Hannover, Callinstr. 5, 30167 Hannover, Germany.
authors.forename: U
authors.initials: U
authors.lastname: Krings
authors.affiliation: Institute of Food Chemistry, Leibniz University of Hannover, Callinstr. 5, 30167 Hannover, Germany.
authors.forename: R G
authors.initials: RG
authors.lastname: Berger
authors.affiliation: Institute of Technical Chemistry, Leibniz University of Hannover, Callinstr. 5, 30167 Hannover, Germany.
authors.forename: T
authors.initials: T
authors.lastname: Scheper
authors.affiliation: Institute of Technical Chemistry, Leibniz University of Hannover, Callinstr. 5, 30167 Hannover, Germany. Electronic address: beutel@iftc.uni-hannover.de.
authors.forename: S
authors.initials: S
authors.lastname: Beutel
mesh_terms: D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics*; D007535:Isomerases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D019686:Lamiaceae / Q000201:enzymology* / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011106:Polyisoprenyl Phosphates / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D012717:Sesquiterpenes / Q000378:metabolism; D045788:Sesquiterpenes, Germacrane / Q000378:metabolism; D013879:Thioredoxins / Q000235:genetics*
chemical_list: D018076:DNA, Complementary; D011106:Polyisoprenyl Phosphates; D011993:Recombinant Fusion Proteins; D012717:Sesquiterpenes; D045788:Sesquiterpenes, Germacrane; C471077:germacrene A; D013879:Thioredoxins; C004808:farnesyl pyrophosphate; D007535:Isomerases; C065995:patchoulol synthase
keywords: Farnesyl diphosphate; Germacrene A; Patchoulol; Sesquiterpenes; Terpene synthase; Thioredoxin