Cloning and expression of polyketide synthase genes from cassia alata, plumbago indica and rheum tataricum

Abstract

Studies on cloning and expression of polyketide synthases (PKSs) have been performed using Thai medicinal plants producing anthraquinones (Cassia alata and Rheum tataricum) and naphthoquinones (Plumbago indica). Three cDNAs encoding very similar but unique isoforms of chalcone synthase [EC 2.3.1.74] were isolated from a cDNA library prepared from RNA from root tissue of C. alata. Gene transcript for these three type III polyketide synthases was found to accumulate predominantly in root. The heterologously expressed enzymes accepted a broad starter-coenzyme A (CoA) and together with the co-substrate malonyl-CoA in vitro assays, formed multiple products. C. alata accumulates the flavonoids quercetin, naringenin and kaempferol in roots, suggesting that the in planta function of these enzymes is the biosynthesis of root flavonoids. For P. indica, one cDNA cloned encoding a pyrone synthase, PinPS, was isolated from the root part that correlated with the expression pattern of the PinPS transcripts were found predominantly in roots. Recombinant PinPS was expressed in E. coli and assayed with a various starter-CoA as primers of polyketide synthesis and was found to accepted broad substrate specificity in in vitro assay and produce pyrone product. For R. tataricum, a cDNA encoding a stilbene synthase, RtSTS, was isolated from the rhizomes. Recombinant RtSTS expressed in E. coli and assayed with a various substrate-CoA as primers, could synthesize resveratrol and a trace amount of naringenin chalcone from p-coumaroyl-CoA. These suggested that the enzyme is a resveratrol-type stilbene synthase (EC 2.3.1.95). Moreover, bis-noryangonin and p-coumaroyl triacetic acid lactone (CTAL)-type pyrones were observed in minor amounts in the reaction with p-coumaroyl-CoA. . Using Northern blot analysis, RtSTS transcript was found to be highly expressed in R. tataricum rhizomes.This expression pattern correlated with the occurrence of resveratrol, which was detected in higher amounts in R. tataricum rhizomes compared with leaves and petioles using HPLC