Regulation Of Isoprenoid Biosynthesis Research Articles

Molecular cloning and characterization of squalene synthase from Matricaria recutita L.

Matricaria recutita L. has been known as an important medicinal plant for centuries. The main active components are sesquiterpenes. Squalene synthase (SQS) is an enzyme that plays an important role in regulation of isoprenoid biosynthesis. In this study, the full-length cDNA of the M. recutita SQS gene was cloned and analyzed for the first time. The clone, designated MrSQS (Matricaria recutita L. squalene synthase gene), consisted of 1580 bp, including a 1230-bp-coding sequence for a predicted protein of 409 amino acids with 46.8 kDa molecular weight. MrSQS shows higher homology to the SQS from Artemisia annua than to SQSs from other plants based on its amino acid sequence. Recombinant MrSQS, produced in a bacterial expression system, showed squalene synthase activity in vitro. In M. recutita, the expression level of MrSQS is determined by real-time quantitative PCR that was highest in disk florets and lowest in stems. These results provide basis and foundation for the study and development of regulation of valuable products from M. recutita L.

Molecular Cloning and Characterization of Two Key Enzymes involved in the Diterpenoid Biosynthesis Pathway of Isodon rubescens

Isodon rubescens, an important medical plant, contains various terpenoids. This plant’s active compounds are primarily oridonin with antitumor properties. As the precursor for oridonin biosynthesis, are synthesized by MEP pathway. On the basis of our earlier studies, we isolated and cloned two important genes catalyzing diterpenoid biosynthesis in the MEP pathway. 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase and 4-hydroxy-3- methylbut-2-enyl diphosphate reductase are the fifth enzymes and the last step key enzyme for the methylerythritol phosphate (MEP) pathway, respectively, which is important for the regulation of isoprenoid biosynthesis. Sequence analysis revealed that DcIspF (accession no. KT948057) was 966 bp, contains a gene open reading frame (ORF) of 708 bp belonging to the MECDP-synthase superfamily and DcIspH (accession no KT948058) contains a 1389 bp ORF encoding a predicted 462 amino acid polypeptides as a member of the lytB_ispH superfamily. The deduced DcIspF and DcIspH amino acid sequences shared high similarity with DcIspF and DcIspH of other plant respectively, each of them exhibiting an N-terminal transit peptide and conserved amino acid sites. Quantitative real-time PCR analysis showed that the expression of DcIspF was considerably higher in leaves, the lowest in callus. These results indicate that we have identified functional DcIspF and DcIspH enzymes, which may play a pivotal role in the biosynthesis of diterpenoid in I. rubescens.

Comprehensive molecular evolution and gene expression analyses of the ABC1 atypical kinase family in rice and Arabidopsis

The ABC1 atypical kinases (aPKs) have been extensively studied in bacteria, yeast and human, where their mutation causes a deficiency of ubiquinone, an isoprenoid compound in the respiratory electron transfer chain. Considerably less is known, however, about their evolution and function in higher plants. In this study, we identified 16 ABC1-domain containing genes from model plant rice by comprehensive genome analysis and gene cloning. Detection of positive selection showed that purifying selection was the major force underlying the evolution of most rice and Arabidopsis ABC1s. However, the expression profiles of plant ABC1s appear to have diverged based on microarray data analysis and realtime PCR; some of them are primarily expressed in developing leaves, some in stamens and/or mature pollen, whereas others in diverse tissues and organs. The possible functions of plant ABC1s were identified using genome-wide coexpression analysis. This analysis suggested that many of them might be involved in the regulation of isoprenoid biosynthesis. In conclusion, the plant ABC1 aPKs, which harbor distinct expression patterns, might have conserved functions in modulating isoprenoid metabolism.

ERG9 and COQ1 disruption reveals isoprenoids biosynthesis is closely related to mitochondrial function in Saccharomyces cerevisiae

Understanding in vivo regulation of isoprenoid biosynthesis is important for identifying strategies to improve carbon fluxes to desirable endproducts. To study the relevance of gene functions, substrate availability, metabolic flux, and the energetics of resource utilization for isoprenoids biosynthesis, Saccharomyces cerevisiae was co-engineered with endogenous deregulated erg9 and/or coq1 and exogenous plant genes that will lead to sesqui- and diterpene production. The flow of substrates through the MVA pathway statistically showed isoprenoids precursor levels were tightly regulated by erg9 and coq1 genes in S. cerevisiae. Physiological analysis by fluorescent probes and labeling techniques revealed elimination of the competing and/or degrading pathways by gene knockout can increase cellular stresses and change the energy metabolism. To further evaluate the role of erg9 and coq1 in the regulation of isoprenoids synthesis, heterologous terpene synthases from plants were introduced into S. cerevisiae, including the wild-type and mutant strains along with feeding of various metal cofactors under anoxic/oxic conditions. The product distribution in the strains indicated that the energy barrier could be the reason that different metal ion-assisted electrophilic attacks initiated carbocation formation, carbocation stabilization, and rearrangement, incorporating exquisite regio- and stereospecificity, to generate structurally diverse products. The yeast altered in the profile of terpenoids and their precursor pools aided the mechanisms of terpenoid biosynthesis and its regulation, highlighting the need for mitochondrial function when engineering metabolic flux using assumptions based on a comprehensive network of the mitochondrial system.

배추 유래 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase 관련 Brmecp 유전자의 발현 및 분자적 특성

Abstract In plants, the fifth step of the plastidial 2-C- methyl-D-erythritol 4-phosphate (MEP) pathway is catalyzed by 2-C-Methyl-D-erythritol 2,4-cyclodiphosphate synthase (MECP; EC: 4. 6. 1. 12), an enzyme proposed to play a key role in the regulation of isoprenoid biosynthesis. Here we report the isolation and functional characterization of a 823 bp Brassica rapa MECP ( Brmecp ) cDNA encoding a deduced polypeptide of 230 amino acid residues. Tran-scription levels of Brmecp were two-fold higher in petal compared to leaves. In addition, Brmecp expression in cabbage seedlings treated with ABA, H 2 O 2 and drought was higher than control seedlings. These results were consistent with changes in chlorophyll contents in transgenic Arabidopsis . Thus, the Brmecp may contribute to the production of primary (chlorophylls and carotenoids) isoprenoid end-products in chloroplasts. Keywords Brassica rapa , 2-C-methyl-D-erythritol 2,4-cyclo-diphosphate synthase, isoprenoid biosynthesis, 2-C- methyl-D- erythritol 4-phosphate pathway, transgenic

TheYTA7gene is involved in the regulation of the isoprenoid pathway in the yeastSaccharomyces cerevisiae

The isoprenoid pathway in yeasts is important not only for sterol biosynthesis but also for the production of nonsterol molecules, deriving from farnesyl diphosphate (FPP), implicated in N-glycosylation and biosynthesis of heme and ubiquinones. FPP formed from mevalonate in a reaction catalyzed by FPP synthase (Erg20p). In order to investigate the regulation of Erg20p in Saccharomyces cerevisiae, we searched for its protein partners using a two-hybrid screen, and identified five interacting proteins, among them Yta7p. Subsequently, we showed that Yta7p was a membrane-associated protein localized both to the nucleus and to the endoplasmic reticulum. Deletion of YTA7 affected the enzymatic activity of cis-prenyltransferase (the enzyme that utilizes FPP for dolichol biosynthesis) and the cellular levels of isoprenoid compounds. Additionally, it rendered cells hypersensitive to lovastatin, an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) that acts upstream of FPP synthase in the isoprenoid pathway. While HMGR is encoded by two genes, HMG1 and HMG2, only HMG2 overexpression was able to restore growth of the yta7Delta cells in the presence of lovastatin. Moreover, the expression level of the S. cerevisiae YTA7 gene was altered upon impairment of the isoprenoid pathway not only by lovastatin but also by zaragozic acid, an inhibitor of squalene synthase. Altogether, these results provide substantial evidence of Yta7p involvement in the regulation of isoprenoid biosynthesis.

Molecular and functional characterization of a cDNA encoding 4-hydroxy-3-methylbut-2-enyl diphosphate reductase from Dunaliella salina

In green algae, the final step of the plastidial methylerythritol phosphate (MEP) pathway is catalyzed by 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (HDR; EC: 1.17.1.2), an enzyme proposed to play a key role in the regulation of isoprenoid biosynthesis. Here we report the isolation and functional characterization of a 1959-bp Dunaliella salina HDR (DsHDR) cDNA encoding a deduced polypeptide of 474 amino acid residues. Phylogenetic analysis implied a cyanobacterial origin for plant and algal HDR genes. Steady-state DsHDR transcript levels were higher in D. salina cells submitted to nutritional depletion, high salt and/or high light, suggesting that DsHDR may respond to the same environmental cues as genes involved in carotenoid biosynthesis.

La voie des isoprénoïdes et quelques applications biotechnologiques chez les plantes

Plants produce a large family of natural compounds, isoprénoids, which are essential for a wide range of physiological functions including plant growth, development and reproduction. Most of enzymes involved in isoprenoid biosynthetic pathways have been characterized and their genes cloned. In the past few years, transgenic plants have been used in order to unravel the molecular mechanisms for regulation of isoprenoid biosynthesis. Various examples of such plants, carrying in addition agronomical advantages, have been reported. In spite of a possible horizontal dispersion of transgenes, such transformation experiments should be able to provide significant results on plant physiology.

Molecular cloning, in vitro expression and characterization of a plant squalene synthetase cDNA.

Squalene synthetase (farnesyl-diphosphate:farnesyl-diphosphate farnesyltransferase, EC 2.5.1.21) catalyzes the first committed step for sterol biosynthesis and is thought to play an important role in the regulation of isoprenoid biosynthesis in eukaryotes. Using degenerate oligonucleotides based on a conserved region found in yeast and human squalene synthetase genes, a cDNA was cloned from the plant Nicotiana benthamiana. The cloned cDNA contained an open reading frame of 1234 bp encoding a polypeptide of 411 amino acids (M(r) 47002). Northern blot analysis of poly(A)+ mRNA from N. benthamiana and N. tabacum cv. MD609 revealed a single band of ca. 1.6 kb in both Nicotiana species. The identity and functionality of the cloned plant squalene synthetase cDNA was further confirmed by expression of the cDNA in Escherichia coli and in a squalene synthetase-deficient erg9 mutant of Saccharomyces cerevisiae. Antibodies raised against a truncated form of the protein recognized an endogenous plant protein of appropriate size as well as the full-length bacterially expressed protein as detected by western analysis. Comparison of the deduced primary amino acid sequences of plant, yeast, rat and human squalene synthetase revealed regions of conservation that may indicate similar functions within each polypeptide.

Distribution of prenyltransferases in rat tissues. Evidence for a cytosolic all-trans-geranylgeranyl diphosphate synthase.

The present study describes the presence of two different geranylgeranyl diphosphate (GGPP) synthase activities, one cytosolic and one membrane-associated, in a number of rat tissues. Both enzymes utilize farnesyl diphosphate (FPP) and isopentenyl diphosphate (IPP) as substrates, but they give rise to different products. The membrane-associated activity produces trans,trans,cis-(E,E,Z)-GGPP, involved in the biosynthesis of long-chain polyprenols. The cytosolic activity produces only the all-trans-(E,E,E) isomer of GGPP, which is utilized as substrate in cytosolic protein prenylation reactions. All-trans-GGPP synthase activity was recovered in the cytosolic fraction from all tissues investigated, but the specific activities varied. The highest specific activities were found in brain, spleen, and testis, followed by kidney and liver. The enzyme activity in rat brain cytosol was further characterized and found to exhibit a narrow pH optimum around 5.0-6.0 and to be highly stimulated by Zn2+. Maximal stimulation was attained with 1 mM Zn2+, whereas Mg2+ had no effect on the enzyme activity. The all-trans-GGPP synthase activity exhibited high affinities for its substrates, i.e. the apparent Km values for FPP and IPP were found to be 0.6 and 3.5 microM, respectively. When rats were fed mevinolin (lovastatin), FPP and all-trans-GGPP synthase activities were affected differently in certain tissues. Mevinolin treatment resulted in an increase in FPP but a decrease in all-trans-GGPP synthase activity in rat liver and kidney. In spleen mevinolin treatment caused a greater than 70% decrease in all-trans-GGPP synthase activity, while FPP synthase was almost unaffected. The presence of two different GGPP synthase activities in the cell, together with the fact that FPP and all-trans-GGPP synthesis in the cytosol are regulated independently, may be of significance in the regulation of isoprenoid biosynthesis, as well as of protein isoprenylation.

Studies on isoprenoid biosynthesis with bacterial intact cells

For the study on the regulation of isoprenoid biosynthesis with intact cells, some strains of bacteria capable of growing on mevalonate as a sole carbon source were isolated from soil. Many of them incorporated [ 14C]-mevalonate, [ 14C]isopentenyl- and [ 14C]farnesyl pyrophosphates into the cells. However, radioactivity was found in their degradation products but not in isoprenoids. Addition of [ 14C]isopentenyl pyrophosphate, farnesyl pyrophosphate and Mg 2+ ions in combination to the culture of a strain of Arthrobacter gave rise to 14C-incorporation into isoprenoids. Radioactivity was found in polyprenol, its pyrophosphate, monophosphate and fatty acid esters. The reactions of isopentenyl- and farnesyl pyrophosphates syntheses seemed to be rate-limiting steps.