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.