Cloning and over-expression of a cDNA encoding a polyketide synthase from Cannabis sativa.
A polyketide synthase has been suggested to play an important role in cannabinoid biosynthesis in Cannabis sativa L. This enzyme catalyzes the biosynthesis of olivetolic acid, one of the precursors for cannabinoid biosynthesis. Using a reverse transcriptase-polymerase chain reaction (RT-PCR) based on the DNA homology of chalcone synthase (EC 2.3.1.156) and valerophenone synthase (EC 2.3.1.156) of hop (Humulus lupulus), a cDNA encoding a polyketide synthase in C. sativa was identified. The coding region of the gene is 1170 bp long encoding a 389 amino acid protein of a predicted 42.7 kDa molecular mass and with a pI of 6.04. The gene shares a high homology with a chalcone synthase gene of H. lupulus, 85% and 94% homology on the level of DNA and protein, respectively. Over-expression of the construct in Escherichia coli M15 resulted in a 45 kDa protein. The protein has chalcone synthase activity as well as valerophenone synthase activity, a chalcone synthase-like activity. Using n-hexanoyl-CoA and malonyl-CoA as substrates did not give olivetol or olivetolic acid as a product.
Raharjo
Tri J
TJ
Department of Chemistry, Gadjah Mada University, Yogyakarta, Indonesia.
Chang
Wen-Te
WT
Verberne
Marianne C
MC
Peltenburg-Looman
Anja M G
AM
Linthorst
Huub J M
HJ
Verpoorte
Robert
R
D000214:Acyl Coenzyme A / Q000378:metabolism; D000217:Acyltransferases / Q000235:genetics; D000595:Amino Acid Sequence; D001483:Base Sequence; D002186:Cannabinoids / Q000096:biosynthesis; D002188:Cannabis / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics*; D004926:Escherichia coli / Q000378:metabolism; D015870:Gene Expression; D008316:Malonyl Coenzyme A / Q000378:metabolism; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000096:biosynthesis* / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D017386:Sequence Homology, Amino Acid
D000214:Acyl Coenzyme A; D002186:Cannabinoids; D018076:DNA, Complementary; D009097:Multienzyme Complexes; D011994:Recombinant Proteins; C058644:4-coumaroyl-coenzyme A; D008316:Malonyl Coenzyme A; D000217:Acyltransferases; C020141:flavanone synthetase