Cloning and heterologous expression of early genes in gibberellin and steviol biosynthesis via the methylerythritol phosphate pathway in Stevia rebaudiana

Abstract

The ent-kaurene skeleton of chloroplast diterpene glycosides, which are produced in large quantities in the leaves of Stevia rebaudiana Bertoni, is formed via the recently discovered 2-C-methyl-D-erythritol 4-phosphate pathway. The enzymes catalyzing the first two steps of this pathway, 1-deoxy-D-xylulose-5-phosphate synthase (DXS) and 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR) were characterized. Using reverse transcriptase-PCR, the dxs and dxr cDNAs were cloned, which comprise ORFs of 2148 and 1422 nucleotides, respectively. The cDNA-derived amino acid sequences for DXS and DXR contain 716 and 474 residues, encoding polypeptides of about 76.6 and 51 kDa, respectively. DXS and DXR from Stevia both contain an N-terminal plastid targeting sequence and show high homology to other known plant DXS and DXR enzymes. Furthermore, we demonstrated through heterologous expression in Escherichia coli that the cloned cDNAs encode functional proteins.Key words: Stevia rebaudiana Bertoni, Asteraceae, isoprenoids, 2-C-methyl-D-erythritol 4-phosphate pathway, 1- deoxy-D-xylulose-5-phosphate synthase, 1-deoxy-D-xylulose-5-phosphate reductoisomerase.