In silicio expression analysis of PKS genes isolated from Cannabis sativa L.

Abstract

Cannabinoids, flavonoids, and stilbenoids have been identified in the annual dioecious plant Cannabis sativa L. Of these, the cannabinoids are the best known group of this plant's natural products. Polyketide synthases (PKSs) are responsible for the biosynthesis of diverse secondary metabolites, including flavonoids and stilbenoids. Biosynthetically, the cannabinoids are polyketide substituted with terpenoid moiety. Using an RT-PCR homology search, PKS cDNAs were isolated from cannabis plants. The deduced amino acid sequences showed 51%-73% identity to other CHS/STS type sequences of the PKS family. Further, phylogenetic analysis revealed that these PKS cDNAs grouped with other non-chalcone-producing PKSs. Homology modeling analysis of these cannabis PKSs predicts a 3D overall fold, similar to alfalfa CHS2, with small steric differences on the residues that shape the active site of the cannabis PKSs.

Cannabis: A Treasure Trove or Pandora's Box?

“…The phenolic moiety (OA) of phytocannabinoids is thought to be formed by the condensation of three molecules of malonyl-CoA and one molecule of n-hexanoyl-CoA into a C 12 polyketide, followed by the cyclization of the latter. The first reaction is driven by a putative type III polyketide synthase (termed as tetraketide synthase, TKS - [119], or olivetol synthase - [120]), the presence and activity of which needs further elucidation [11,107,[119][120][121][122][123][124]. The C2-C7 intramolecular aldol condensation with carboxylate retention (the cyclization) is catalyzed by a recently identified polyketide cyclase, OA cyclase (OAC) [119], which enzyme functions in concert with the TKS to generate carbon scaffolds and OA.…”

“…For instance, the glandcontaining plant parts may be shaked with powdered dry ice and sieved [172]. Alternatively, the glandular hairs can be removed from the frozen female inflorescences or flowers by shaking this material through a tea leaf sieve and collecting it in a mortar containing liquid nitrogen [121] or by the Bead Beater method adapted from [173] to cannabis [119,125]. During this process, the trichome-rich surfaces were immersed in ice-cold water, then abraded with teflon rotor in the presence of glass beads and isolation buffer, then filtered and washed twice with different buffers [119].…”

In silico gene expression profiling in Cannabis sativa

“…Genes involved in the biosynthesis of cannabinoids and their precursors have been shown to be overexpressed in flowers 15 . To validate gene expression profiling, cannabinoid, hexanoate, 2-C-methyl-D-erythritol 4-phosphate ( MEP ) and geranyl diphosphate ( GPP) pathway genes 11 , 16 , together with the olivetol synthase ( OL S) gene 17 , 18 , the (-)-limonene terpene synthase ( TPS ) gene 19 and the polyketide synthase ( PKS ) gene 20 , have been analyzed. As expected, most of these genes were overexpressed in flowers, although many of the genes also displayed high expression in other tissues ( Figure 2 ; Supplementary table 1 ).…”

“…Moreover, expression profiles of the genes belonging to the cannabinoid pathway 11, 16– 20 are provided.…”

“…To this end, a total of 40,224 gene expression profiles taken from cannabis plant tissue at different developmental stages were obtained by exploiting common bioinformatics pipelines 13 . Moreover, expression profiles of the genes belonging to the cannabinoid pathway 11 , 16 – 20 are provided.…”