Molecular cloning and expression of tryptophan decarboxylase from Mitragyna speciosa
2013
Charoonratana, Tossaton | Wungsintaweekul, Juraithip | Keawpradub, Niwat | Verpoorte, R.
Tryptophan decarboxylase (TDC) catalyzes the decarboxylation of tryptophan to tryptamine in mitragynine biosynthesis via the shikimate pathway. Using the rapid amplification of cDNA ends (RACE) technique, the gene encoding TDC from Mitragyna speciosa was cloned (designated as MsTDC). The MsTDC cDNA contained an open reading frame (ORF) of 1,521 base pairs (bp) encoding 506 amino acid residues. It had a pyridoxal-phosphate (PLP)-binding site at the amino acid position 313–334 residues. The MsTDC showed homology of 68–76 % to the TDC of other plants. Heterologous expression in Escherichia coli afforded the soluble proteins as an apparent band of 57 kDa as judged by SDS-PAGE. Expression of the MsTDC in M. speciosa hairy roots under the 35S promoter was performed by insertion of MsTDC into pCAMBIA1300-gfp. The transgenic hairy root lines were detected by fluorescence microscopy and showed an increased accumulation of tryptamine.
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