Fusion of farnesyldiphosphate synthase and epi-aristolochene synthase, a sesquiterpene cyclase involved in capsidiol biosynthesis in Nicotiana tabacum.
Forskningsoutput: Tidskriftsbidrag › Artikel i vetenskaplig tidskrift
A clone encoding farnesyl diphosphate synthase (FPPS) was obtained by PCR from a cDNA library made from young leaves of Artemisia annua. A cDNA clone encoding the tobacco epi-aristolochene synthase (eAS) was kindly supplied by J. Chappell (University of Kentucky, Lexington, KY, USA). Two fusions were constructed, i.e. FPPS/eAS and eAS/FPPS. The stop codon of the N-terminal enzyme was removed and replaced by a short peptide (Gly-Ser-Gly) to introduce a linker between the two ORFs. These two fusions and the two single cDNA clones were separately introduced into a bacterial expression vector (pET32). Escherichia coli was transformed with the expression vectors and enzymatically active soluble proteins were obtained after induction with isopropyl thio-beta-d-thiogalactoside. The recombinant enzymes were purified using immobilized metal affinity chromatography on Co2+ columns. The fusion enzymes produced epi-aristolochene from isopentenyl diphosphate through a coupled reaction. The Km values of FPPS and eAS for isopentenyl diphosphate and farnesyl diphosphate, respectively, were essentially the same for the single and fused enzymes. The bifunctional enzymes showed a more efficient conversion of isopentenyl diphosphate to epi-aristolochene than the corresponding amount of single enzymes.
|Enheter & grupper|
Ämnesklassifikation (UKÄ) – OBLIGATORISK
|Tidskrift||European Journal of Biochemistry|
|Status||Published - 2002|
|Peer review utförd||Ja|
Sesquiterpene synthases in Artemisia annua: Cloning, Expression, and Characterisation of the Recombinant EnzymesPer Mercke, 2000, Per Mercke, Spolegatan 3a, 222 20 Lund. 120 s.
Forskningsoutput: Avhandling › Doktorsavhandling (sammanläggning)