Cloning and characterisation of the gene encoding HMG-CoA reductase from Taxus media and its functional identification in yeast.

Abstract

In plants, the first committed step in the pathway for biosynthesis of isoprenoids is catalysed by 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR, EC: 1.1.1.34). Here we report for the first time the cloning of a full-length cDNA encoding HMGR (Tm-HMGR) from a taxol-producing gymnosperm, Taxus media Rehder. The full-length cDNA of Tm-HMGR (GenBank accession number: AY277740) was 2307 base pairs (bp), with a 1791-bp open reading frame (ORF) encoding a 596-amino-acid polypeptide. Bioinformatic analysis revealed that Tm-HMGR contained two trans-membrane domains and a catalytic domain, and showed high homology to other plant HMGRs. Phylogenetic analysis indicated that Tm-HMGR was more ancient than other plant HMGRs. The structural modelling showed that Tm-HMGR had the typical spatial structure of HMGRs whose catalytic domains could be folded and divided into three spatial domains, L-domain, N-domain and S-domain. Southern blot analysis revealed that Tm-HMGR belonged to a small HMGR gene family. Northern blot analysis showed that Tm-HMGR was expressed in roots, stems and needles, with higher expression in stems and needles than in roots. Functional complementation of Tm-HMGR in a HMGR-deficient mutant yeast demonstrated that Tm-HMGR mediated the biosynthesis of mevalonate and provided the general precursor for taxol biosynthesis.