Co‐expression of N‐terminal truncated 3‐hydroxy‐3‐methylglutaryl CoA reductase and C24‐sterol methyltransferase type 1 in transgenic tobacco enhances carbon flux towards end‐product sterols

Abstract

SummaryThe enzymes 3-hydroxy-3-methylglutaryl CoA reductase (HMGR) and C24-sterol methyltransferase type 1 (SMT1) have been proposed to be key steps regulating carbon¯ux through the sterol biosynthesis pathway. To further examine this hypothesis, we co-expressed the catalytic domain of Hevea brasiliensis HMGR (tHMGR) and Nicotiana tabacum SMT1 in tobacco, under control of both constitutive and seed-speci®c promoters, resulting in increased accumulation of total sterol in seed tissue by 2.5-and 2.1-fold, respectively. This enhancement is greater than when tHMGR and SMT1 were expressed singularly where, for example, seed-speci®c expression enhanced total sterols by 1.6-fold. Signi®cantly, the relative level of 4-desmethyl sterols (end-product sterols) was higher in seed co-expressing tHMGR and SMT1 from seedspeci®c promoters (79% of total sterols) than when co-expressed from constitutive promoters (59% of total sterols) and similar to wild-type seed (80% of total sterols). These results demonstrate that HMGR and SMT1 work in concert to control carbon¯ux into end-product sterols and that the sterol composition can be controlled by the temporal activity of the promoters driving transgene expression. In addition, constitutive expression of the transgenes resulted in elevated accumulation of substrates for C4-demethylation reactions, which indicates that one or several enzymes catalysing such reactions limit carbon¯ow to endproduct sterols, at least in a physiological situation when the carbon¯ow is upregulated.

Molecular characterization and functional analysis of Glycine max sterol methyl transferase 2 genes involved in plant membrane sterol biosynthesis

“…The ABRE sequences are also present in the SMT2 gene promoters and they are possibly involved in the observed weak ABA responsiveness of the SMT2 genes. The first committed step in the isoprenoid biosynthesis reactions catalyzed by HMGR and the C-24 methyl transfer reaction catalyzed by SMT1 have been clearly elucidated to be two crucial control points in regulating sterol accumulation in plant systems (Chappell et al 1995;Harker et al 2003;Holmberg et al 2002Holmberg et al , 2003Schaeffer et al 2000). The potential role of GmSMT2-2 gene in sterol synthesis and allocation of the campesterol: sitosterol ratio in seeds was investigated for the first time in transgenic Arabidopsis.…”

Strategies for strain improvement in Fusarium fujikuroi: overexpression and localization of key enzymes of the isoprenoid pathway and their impact on gibberellin biosynthesis

“…4a, b) indicating that increased FDP pools do not stimulate GA biosynthesis. Instead, elevated formation of precursors of ergosterol biosynthesis seem to activate a negative feedback regulation lowering HmgR enzyme activity or gene expression mediated by end products or intermediates of the isoprenoid pathway as shown in yeast and plants (Holmberg et al 2003;Verwaal et al 2007). …”

Homology Modeling and Site-Directed Mutagenesis Reveal Catalytic Key Amino Acids of 3β-Hydroxysteroid-Dehydrogenase/C4-Decarboxylase from Arabidopsis

“…The C4-demethylation involves, sequentially, the tight cooperation of three individual enzymes, including 3bHSD/D, which proceeds without accumulation of the transient intermediate products. Several enzymes of the C4-demethylation step control carbon flow to sterol end products (Maillot-Vernier et al, 1991;Holmberg et al, 2003).…”