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PubMed2000

Changes in isoprenoid lipid synthesis by gemfibrozil and clofibric acid in rat hepatocytes.

We studied whether gemfibrozil and clofibric acid alter isoprenoid lipid synthesis in rat hepatocytes. After incubation of the cells with the agent for 74 hr, [(14)C]acetate or [(3)H]mevalonate was added, and the cells were further incubated for 4 hr. Gemfibrozil and clofibric acid increased ubiquinone synthesis from [(14)C]acetate and [(3)H]mevalonate. The effect of gemfibrozil was greater than that of clofibric acid. Also, gemfibrozil decreased dolichol synthesis from [(14)C]acetate and [(3)H]mevalonate. However, clofibric acid increased dolichol synthesis from [(3)H]mevalonate. Gemfibrozil decreased cholesterol synthesis from [(14)C]acetate and [(3)H]mevalonate. Clofibric acid decreased cholesterol synthesis from [(14)C]acetate, but did not affect synthesis from [(3)H]mevalonate. These results suggest that both agents, at different rates, activate the synthetic pathway of ubiquinone, at least from mevalonate. Gemfibrozil may inhibit the synthetic pathway of dolichol, at least from mevalonate. Contrary to gemfibrozil, clofibric acid may activate the synthetic pathway of dolichol from mevalonate. Gemfibrozil may inhibit the synthetic pathway of cholesterol from mevalonate in addition to the pathway from acetate to mevalonate inhibited by both agents.

Acetates↗
PubMed2003

Distinct light-mediated pathways regulate the biosynthesis and exchange of isoprenoid precursors during Arabidopsis seedling development.

Plants synthesize an astonishing diversity of isoprenoids, some of which play essential roles in photosynthesis, respiration, and the regulation of growth and development. Two independent pathways for the biosynthesis of isoprenoid precursors coexist within the plant cell: the cytosolic mevalonic acid (MVA) pathway and the plastidial methylerythritol phosphate (MEP) pathway. In at least some plants (including Arabidopsis), common precursors are exchanged between the cytosol and the plastid. However, little is known about the signals that coordinate their biosynthesis and exchange. To identify such signals, we arrested seedling development by specifically blocking the MVA pathway with mevinolin (MEV) or the MEP pathway with fosmidomycin (FSM) and searched for MEV-resistant Arabidopsis mutants that also could survive in the presence of FSM. Here, we show that one such mutant, rim1, is a new phyB allele (phyB-m1). Although the MEV-resistant phenotype of mutant seedlings is caused by the upregulation of MVA synthesis, its resistance to FSM most likely is the result of an enhanced intake of MVA-derived isoprenoid precursors by the plastid. The analysis of other light-hyposensitive mutants showed that distinct light perception and signal transduction pathways regulate these two differential mechanisms for resistance, providing evidence for a coordinated regulation of the activity of the MVA pathway and the crosstalk between cell compartments for isoprenoid biosynthesis during the first stages of seedling development.

Alleles↗
PubMed1997

Post-translational regulation of mevalonate kinase by intermediates of the cholesterol and nonsterol isoprene biosynthetic pathways.

To assess the potential for feedback inhibition by isoprene intermediates in the cholesterol and nonsterol isoprene biosynthetic pathway, we expressed human cDNAs encoding mevalonate kinase (MKase), phosphomevalonate kinase (PMKase), and mevalonate diphosphate decarboxylase (MDDase) as fusion proteins in Escherichia coli DH5alpha, and purified these proteins by affinity chromatography. Several phosphorylated and non-phosphorylated isoprenes were analyzed as inhibitors of the enzymes using a standard spectrophotometric assay. Of the three proteins, only MKase was inhibited through competitive interaction at the ATP-binding site. The intermediates studied (and their relative inhibitory capacity) were: geranylgeranyl-diphosphate (GGPP, C20) > farnesyl-diphosphate (FPP, C15) > geranyl-diphosphate (GPP, C10) > isopentenyl-diphosphate (IPP, C5) > or = 3,3-dimethylallyl-diphosphate (DMAPP, C5) > farnesol (C15) > dolichol-phosphate (DP, C(80-100)). Mevalonate-diphosphate, geraniol, and dolichol were not inhibitors. Our data further define the spectrum of physiologic inhibitors of MKase, and provide the first evidence for feedback inhibition of MKase by a nonsterol isoprene produced by the branched pathway, dolichol-phosphate. These results provide additional evidence that MKase may occupy a central regulatory role in the control of cholesterol and nonsterol isoprene biosynthesis.

Carboxy-Lyases↗
PubMed2004

Biosynthesis of the irregular monoterpene artemisia ketone, the sesquiterpene germacrene D and other isoprenoids in Tanacetum vulgare L. (Asteraceae).

The incorporation of [1-13C]-labeled glucose into the irregular monoterpene artemisia ketone, the regular monoterpenes camphor and beta-thujone, the sesquiterpene germacrene D, the diterpene trans-phytol and beta-sitosterol and isofucosterol has been studied in axenic cultures of Tanacetum vulgare L. (Asteraceae). Quantitative 13C NMR spectroscopic analysis of the resulting labeling patterns showed that the isoprene units of the monoterpenes and the diterpene are formed via the methylerythritol phosphate (MEP) pathway, whereas the isoprene building blocks of the sesquiterpene and the sterols originate from the mevalonic acid (MVA) pathway.

Bicyclic Monoterpenes↗
PubMed2003

Metabolic cross talk between cytosolic and plastidial pathways of isoprenoid biosynthesis: unidirectional transport of intermediates across the chloroplast envelope membrane.

In higher plants, two independent pathways are responsible for the biosynthesis of isopentenyl diphosphate and dimethylallyl diphosphate, the central five-carbon precursors of all isoprenoids. The cytosolic pathway, which involves mevalonate (MVA) as a key intermediate, provides the precursor molecules for sterols, ubiquinone, and certain sesquiterpenes, whereas the plastidial MVA-independent pathway is involved in the formation of precursors for the biosynthesis of isoprene, monoterpenes, diterpenes, carotenoids, abscisic acid, and the side chains of chlorophylls, tocopherols, and plastoquinone. Recent experiments provided indirect evidence for the presence of an export system for isoprenoid intermediates from the plastids to the cytosol in Arabidopsis thaliana. Here we report that isolated chloroplasts (from spinach, kale, and Indian mustard), envelope membrane vesicles, and proteoliposomes prepared from the solubilized proteins of envelope membranes (from spinach) are capable of the efficient transport of isopentenyl diphosphate and geranyl diphosphate. Lower rates of transport were observed with the substrates farnesyl diphosphate and dimethylallyl diphosphate, whereas geranylgeranyl diphosphate and mevalonate were not transported with appreciable efficiency. Our data suggest that plastid membranes possess a unidirectional proton symport system for the export of specific isoprenoid intermediates involved in the metabolic cross talk between cytosolic and plastidial pathways of isoprenoid biosynthesis.

Biological Transport↗
PubMed1986

Role of the kidneys in the metabolism of circulating mevalonate in humans.

Previous studies in several animal species have demonstrated that the kidneys are the primary site of mevalonate metabolism by the oxidative or shunt pathway. To determine the role of the human kidney in mevalonate oxidation, we studied mevalonate shunt activity in patients undergoing hemodialysis for varying degrees of renal failure. Surprisingly, at least half of the uremic patients and even anephric patients had normal ability to oxidize mevalonate by the shunt pathway. In addition, we found a strong negative correlation (R = -0.94) between mevalonate shunt activity and serum phosphorus levels in uremic patients. The resulting inhibition of mevalonate oxidation by high serum phosphorus levels was reversed by lowering the serum phosphorus in one patient. Finally, a positive correlation was found between mevalonate oxidation and serum PTH levels. The results of this study suggest that, in humans, extrarenal tissues can be major contributors to mevalonate oxidation. It is therefore probable that in humans, in contrast to other animals, the kidney is not the primary site of mevalonate metabolism by this oxidative pathway. Finally, the strong negative correlation between serum phosphorus levels and the ability of uremic patients to oxidize mevalonate suggests a regulatory role for the phosphate ion in the mevalonate shunt pathway.

Adult↗
PubMed1983

The effect of cholesterol on ubiquinone and tetrahymanol biosynthesis in Tetrahymena pyriformis.

The biosynthesis of ubiquinone-8 from radioactive mevalonate by cultures of Tetrahymena pyriformis is demonstrated. Under normal conditions the incorporation of this radioactive precursor into ubiquinone and the triterpenoid alcohol tetrahymanol reflects the amounts of these two compounds in the cell. Growth of T. pyriformis in the presence of cholesterol results in a complete inhibition of incorporation of radioactive mevalonate into tetrahymanol while there is a corresponding increase of radioactive incorporation into ubiquinone. This increased incorporation of mevalonic acid into ubiquinone must reflect a reduced level of mevalonic acid in the cell under these conditions and is not due to increased ubiquinone biosynthesis, indicating tight regulation of the pathway prior to mevalonate formation.

Animals↗
PubMed2007

Squalene synthase inhibitors : clinical pharmacology and cholesterol-lowering potential.

HMG-CoA reductase inhibitors (statins) reduce cardiovascular disease morbidity and mortality with a high level of safety. Nonetheless, there are substantial numbers of people who either do not tolerate statins or whose low-density lipoprotein (LDL) levels are not lowered adequately. For these reasons, there is a need to develop other cholesterol-lowering drugs. A target for these new agents is provided by the enzymes distal to HMG-CoA reductase in the cholesterol biosynthesis pathway. Two classes of drugs have been developed: (i) squalene synthase inhibitors, which act at the first committed step in cholesterol biosynthesis, distal to the mevalonate-farnesyl diphosphate pathway; and (ii) oxidosqualene cyclase inhibitors, which act distal to the squalene intermediate. Of these, squalene synthase inhibitors have received more attention and are the subject of this review. Squalene synthase inhibitors decrease circulating LDL-cholesterol by the induction of hepatic LDL receptors in a similar manner to statins. They have fewer secondary effects mediated by a decrease in non-cholesterol products of mevalonate metabolism distal to HMG-CoA reductase, but have the potential to increase intermediates proximal to squalene. Squalene synthase inhibitors are just now entering clinical trials and data on how effectively they lower LDL-cholesterol and how they compliment the actions of statins and other agents is awaited with considerable interest.

Animals↗
PubMed2004

The biosynthetic pathway to abscisic acid via ionylideneethane in the fungus Botrytis cinerea.

The biosynthetic pathway to abscisic acid (ABA) from isopentenyl diphosphate in the fungus, Botrytis cinerea, was investigated. Labeling experiments with (18)O2 and H2(18)O indicated that all oxygen atoms at C-1, -1, -1' and -4' of ABA were derived from molecular oxygen, and not from water. This finding was inconsistent not only with the known carotenoid pathway via oxidative cleavage of carotenoids, but also with the classical direct pathway via cyclization of farnesyl diphosphate. The fungus produced new C15-compounds, 2E,4E-alpha-ionylideneethane and 2Z,4E-alpha-ionylideneethane, along with 2E,4E,6E-allofarnesene and 2Z,4E,6E-allofarnesene, but did not apparently produce carotenoids except for a trace of phytoene. The C15-compounds labeled with 13C were converted to ABA by the fungus, and the incorporation ratio of 2Z,4E-alpha-ionylideneethane was higher than that of 2E,4E-alpha-ionylideneethane. From these results, it was concluded that farnesyl diphosphate was reduced at C-1, desaturated at C-4, and isomerized at C-2 to form 2Z,4E,6E-allofarnesene before being cyclized to 2Z,4E-alpha-ionylideneethane; the ionylideneethane was then oxidized to ABA with molecular oxygen. This direct pathway via ionylideneethane means that the biosynthetic pathway to fungal ABA, not only before but also after isopentenyl diphosphate, differs from that to ABA in plants, since plant ABA is biosynthesized using the non-mevalonate and carotenoid pathways.

Abscisic Acid↗
PubMed1990

[Lipid metabolism in daunomycin-induced nephrotic rats (5)].

Lipid metabolism of intestine, liver and kidney was examined in daunomycin-induced nephrotic rats. 1) Phospholipids, triglycerides and cholesterol contents in chylomicrons were increased in daunomycin-induced nephrotic rats. 2) Cholesterol absorption in intestine (in vivo and situ) was similar in daunomycin-induced nephrotic rats and control rats. 3) Total bile acids levels in bile and portal vein were similar in two groups. 4) There was no difference in incorporation of 14C-mevalonate into cholesterol by liver and kidney slices in two groups. The oxidation of 14CO2 from 14C-mevalonate in kidney slices was decreased in daunomycin-induced nephrotic rats. These results show that the hypercholesterolemia in daunomycin-induced nephrotic rats are not caused by increased absorption of cholesterol in intestine. The increased cholesterol esters in liver are not derived from decreased bile acids catabolism. The increased cholesterogenesis in the liver of daunomycin-induced nephrotic rats may be due to an increased rate at the step prior to mevalonate. The oxidation of 14CO2 from mevalonate by shunt pathway may be decreased in the kidney of daunomycin-induced nephrotic rats.

Animals↗
PubMed2006

Herbicidal effects of statin pharmaceuticals in Lemna gibba.

Statin pharmaceuticals, heavily prescribed in the treatment of hypercholesterolemia, are competitive inhibitors of 3-hydroxy-3-methylglutaryl coenzyme-A reductase (HMGR). In plants, these compounds also inhibit HMGR, which regulates cytosolic isoprenoid biosynthesis in the mevalonic acid (MVA) pathway. Phytotoxicity was evaluated in the higher aquatic plant Lemna gibba exposed to atorvastatin and lovastatin for 7-days by measuring the concentrations of sterols and ubiquinone; products downstream in the MVA pathway. The efficiency of the parallel and unaffected methylerythritol phosphate pathway (MEP) was also evaluated by measuring the end product, plastoquinone. Statin treatment caused an accumulation of plastoquinone, and unexpectedly, ubiquinone, an artifact likely due to metabolite sharing from the plastidial MEP pathway. Statins were, however, highly phytotoxic to L. gibba and HPLC-UV analysis of plant extracts showed significantly decreased concentrations of both stigmasterol and beta-sitosterol, which are critical components of plant membranes and regulate morphogenesis and development. EC10 values for atorvastatin and lovastatin were as small as 26.1 and 32.8 microg/L, respectively. However, hazard quotients indicated that statins present little risk to the model higher aquatic plant Lemna gibba at environmentally relevant concentrations, even though pathway-specific endpoints were 2-3 times more sensitive than traditional gross morphological endpoints typically used in risk assessment.

Atorvastatin↗
PubMed2005

1-Deoxy-D-xylulose 5-phosphate reductoisomerase (IspC) from Mycobacterium tuberculosis: towards understanding mycobacterial resistance to fosmidomycin.

1-Deoxy-d-xylulose 5-phosphate reductoisomerase (IspC) catalyzes the first committed step in the mevalonate-independent isopentenyl diphosphate biosynthetic pathway and is a potential drug target in some pathogenic bacteria. The antibiotic fosmidomycin has been shown to inhibit IspC in a number of organisms and is active against most gram-negative bacteria but not gram positives, including Mycobacterium tuberculosis, even though the mevalonate-independent pathway is the sole isopentenyl diphosphate biosynthetic pathway in this organism. Therefore, the enzymatic properties of recombinant IspC from M. tuberculosis were characterized. Rv2870c from M. tuberculosis converts 1-deoxy-d-xylulose 5-phosphate to 2-C-methyl-d-erythritol 4-phosphate in the presence of NADPH. The enzymatic activity is dependent on the presence of Mg(2+) ions and exhibits optimal activity between pH 7.5 and 7.9; the K(m) for 1-deoxyxylulose 5-phosphate was calculated to be 47.1 microM, and the K(m) for NADPH was 29.7 microM. The specificity constant of Rv2780c in the forward direction is 1.5 x 10(6) M(-1) min(-1), and the reaction is inhibited by fosmidomycin, with a 50% inhibitory concentration of 310 nM. In addition, Rv2870c complements an inactivated chromosomal copy of IspC in Salmonella enterica, and the complemented strain is sensitive to fosmidomycin. Thus, M. tuberculosis resistance to fosmidomycin is not due to intrinsic properties of Rv2870c, and the enzyme appears to be a valid drug target in this pathogen.

Aldose-Ketose Isomerases↗
PubMed1987

Mevalonic acid products as mediators of cell proliferation in simian virus 40-transformed 3T3 cells.

Effects of treatment with serum-free medium and 25-hydroxycholesterol (25-OH) on the cell cycle of simian virus 40-transformed 3T3 fibroblasts, designated SV-3T3 cells, were studied and compared with simultaneous effects on the activity of 3-hydroxy-3-methylglutaryl (HMG) CoA reductase and incorporation of [3H]mevalonic acid into cholesterol, Coenzyme Q, and dolichol. The data confirm our previous finding (O. Larsson and A. Zetterberg, Cancer Res., 46: 1233-1239, 1986) that 25-OH inhibits the cell cycle traverse of SV-3T3 cells specifically in early G1. In contrast, treatment with serum-free medium had no effect on cell cycle progression. The effect of 25-OH on the cell cycle traverse was correlated to a substantial decrease in the activity of HMG CoA reductase, whereas there was no change in the rate of [3H]mevalonic acid incorporated into cholesterol, Coenzyme Q, and dolichol. When the cells were exposed to serum-free medium, there was no depression of activity of HMG CoA reductase, and the rate of [3H]mevalonic acid incorporated into dolichol and cholesterol was not affected in any appreciable degree. In contrast the rate of Coenzyme Q synthesis was substantially decreased as a result of serum depletion. A similar decrease in Coenzyme Q synthesis was also achieved by treating the cells with cholesterol-poor serum. This indicates that the rate of Coenzyme Q synthesis is dependent on the concentration of cholesterol in the culture medium. In order to analyze whether some of the products in the mevalonic acid biosynthetic pathway may be of importance in the control of G1 traverse and cell proliferation of SV-3T3 cells, cholesterol, Coenzyme Q, and dolichol were added as supplements to cells treated with 25-OH. It was shown that dolichol was capable of overcoming the 25-OH-induced inhibition of G1 traverse efficiently, whereas cholesterol and Coenzyme Q were considerably less effective. Considered together with the fact that the activity of HMG CoA reductase and incorporation of mevalonic acid into dolichol were unaffected following serum-free treatment, the results suggest that maintenance of a certain level of de novo synthesis of dolichol may contribute to the capability of SV-3T3 cells to proliferate in serum-free medium.

Cell Cycle↗
PubMed1988

Isopentenoid synthesis in isolated embryonic Drosophila cells. Farnesol catabolism and omega-oxidation.

Kc cells divert minimally 40% of their mevalonate carbon to n-fatty acids and unidentified compounds covalently linked to macromolecules (Havel, C., Rector, E. R., II, and Watson, J. A. (1986) J. Biol. Chem. 261, 10150-10156). Furthermore mevalonate carbon diversion appears to occur at the polyprenyl 1-pyrophosphate level. This report summarizes initial efforts to define the mevalonate carbon diversion pathway. We demonstrate that Kc cell extracts readily metabolize [14C]farnesyl 1-pyrophosphate and [14C]farnesol, via common intermediates, to identical 14C-products. Two of the major 14C-products were identified as trans,trans-3,7,11-trimethyl-2,6,10-dodecatrien-1,12-dioic acid and trans-3,7-dimethyl-2,6-decadien-1,10-dioic acid. Similar acids were also synthesized by supplemented rat liver extracts incubated with [14C]farnesol. We conclude that (a) mevalonate carbon diversion at the level of polyprenyl 1-pyrophosphate is a viable metabolic strategy, (b) polyprenols are oxidized to alpha,omega-prenyl dicarboxylic acids which are catabolized from the omega-terminus, and (c) this metabolic process is not limited to insect cells.

Animals↗
PubMed2004

Biochemical characterization of Bacillus subtilis type II isopentenyl diphosphate isomerase, and phylogenetic distribution of isoprenoid biosynthesis pathways.

An open reading frame (Acc. no. P50740) on the Bacillus subtilis chromosome extending from bp 184,997-186,043 with similarity to the idi-2 gene of Streptomyces sp. CL190 specifying type II isopentenyl diphosphate isomerase was expressed in a recombinant Escherichia coli strain. The recombinant protein with a subunit mass of 39 kDa was purified to apparent homogeneity by column chromatography. The protein was shown to catalyse the conversion of dimethylallyl diphosphate into isopentenyl diphosphate and vice versa at rates of 0.23 and 0.63 micromol.mg(-1).min(-1), respectively, as diagnosed by 1H spectroscopy. FMN and divalent cations are required for catalytic activity; the highest rates were found with Ca2+. NADPH is required under aerobic but not under anaerobic assay conditions. The enzyme is related to a widespread family of (S)-alpha-hydroxyacid oxidizing enzymes including flavocytochrome b2 and L-lactate dehydrogenase and was shown to catalyse the formation of [2,3-13C2]lactate from [2,3-13C2]pyruvate, albeit at a low rate of 1 nmol.mg(-1).min(-1). Putative genes specifying type II isopentenyl diphosphate isomerases were found in the genomes of Archaea and of certain eubacteria but not in the genomes of fungi, animals and plants. The analysis of the occurrence of idi-1 and idi-2 genes in conjunction with the mevalonate and nonmevalonate pathway in 283 completed and unfinished prokaryotic genomes revealed 10 different classes. Type II isomerase is essential in some important human pathogens including Staphylococcus aureus and Enterococcus faecalis where it may represent a novel target for anti-infective therapy.

Amino Acid Sequence↗
PubMed1981

The effect of decreased plasma cholesterol concentration on circulatinga mevalonate metabolism in rats.

Circulating mevalonate is metabolized by two mechanisms: the sterol pathways leading to cholesterol and the shunt pathway resulting in CO2 production. The kidney is the chief site of circulating mevalonate metabolism by both pathways. The present study investigated the effect of plasma cholesterol concentration on circulating mevalonate metabolism. 3-Aminopyrazolo(3,4-d)pyrimidine and Triton WR 1339 were utilized to induce "functional hypocholesterolemia". An enhancement of both renal total nonsaponifiable lipid synthesis (36-43%) and cholesterol synthesis (42%) from circulatinga mevalonate was observed when "functional hypocholesterolemia" was induced by either compound. Hepatic total nonsaponifiable lipid synthesis from circulating mevalonate was not enhanced in the Triton-treated animals, but 4-aminopyrazolo(3,4-d)pyrimidine treatment increased accumulation of total labeled nonsaponifiable lipids and cholesterol. No increase in labeled total nonsaponifiable lipids or cholesterol in the carcass was observed after treatment with wither compound. "Functional hypocholesterolemia" reduced the shunt pathway of circulating mevalonate metabolism by approximately 30%. This reduction occurred in both the renal and extrarenal shunt pathways. These data indicate that plasma cholesterol concentration regulates the in vivo metabolism of circulating mevalonate in that hypocholesterolemia reduces the shunt pathway and stimulates sterologenesis, and effect chiefly localized to athe kidneys.

Adenine↗
PubMed2005

Comparative actions of clomazone on beta-carotene levels and growth in rice (Oryza sativa) and watergrasses (Echinochloa spp).

Seedlings of rice, early watergrass (thiobencarb-resistant and thiobencarb-susceptible biotypes, R and S, respectively), and late watergrass (thiobencarb-resistant and thiobencarb-susceptible biotypes, R and S, respectively) were hydroponically exposed to clomazone at concentrations ranging from 0.08 to 7.9 microM. Whole-plant growth (mg fresh wt) and beta-carotene concentrations (microg g(-1) fresh wt) were measured after a 7-day exposure period. For growth, the no observed effect concentrations (NOECs) were 7.9, 0.21, 0.21, 0.46 and 0.46 microM clomazone for rice, early watergrass (R), early watergrass (S), late watergrass (R) and late watergrass (S), respectively, while the concentrations causing 25% inhibition in response (IC25) were 5.6 (+/-1.6), 0.46 (+/-0.06), 0.42 (+/-0.08), 0.92 (+/-0.45) and 0.79 (+/-0.08) microM clomazone, respectively. Clomazone inhibits beta-carotene synthesis via inhibition of the non-mevalonate isoprenoid synthetic pathway. For assessment of clomazone effects, beta-carotene levels proved to be a more sensitive toxicological endpoint than growth. For rice, early watergrass (R), early watergrass (S), late watergrass (R) and late watergrass (S), the beta-carotene NOECs were 0.21, <0.08, <0.08, 0.08 and 0.46 microM clomazone respectively, while IC25 values were 0.42 (+/-0.26), 0.08 (+/-0.02), 0.08 (+/-0.02), 0.33 (+/-0.09) and 0.54 (+/-0.15) microM, respectively. No evidence was found that the thiobencarb-resistance mechanisms present in early and late watergrasses impart resistance to clomazone. Due to similar sensitivity between rice and late watergrass, use of clomazone in rice culture will require the use of a safening technique.

Dose-Response Relationship, Drug↗
PubMed2000

Low-copy plasmids can perform as well as or better than high-copy plasmids for metabolic engineering of bacteria.

Multicopy plasmids are often chosen for the expression of recombinant genes in Escherichia coli. The high copy number is generally desired for maximum gene expression; however, the metabolic burden effects that usually result from multiple plasmid copies could prove to be detrimental for maximum productivity in certain metabolic engineering applications. In this study, low-copy mini-F plasmids were compared to high-copy pMB1-based plasmids for production of two metabolites in E. coli: polyphosphate (polyP) and lycopene derived from isopentenyl diphosphate (IPP). The stationary-phase accumulation of polyP on a per cell basis was enhanced approximately 80% when either high- or low-copy plasmids were used, from 120 micromol/g DCW without augmented polyP kinase (PPK) activity to approximately 220 micromol/g DCW. The cell density of the high-copy plasmid-containing culture at stationary phase was approximately 24% lower than the low-copy culture and 30% lower than the control culture. This difference in cell density is likely a metabolic burden effect and resulted in a lower overall product concentration for the high-copy culture (approximately 130 micromol/L culture) relative to the low-copy culture (approximately 160 micromol/L culture). When the gene for DXP (1-deoxy-D-xylulose 5-phosphate) synthase, the first enzyme in the IPP mevalonate-independent biosynthetic pathway, was expressed from the tac promoter on multicopy and low-copy plasmids, lycopene production was enhanced two- to threefold over that found in cells expressing the chromosomal copy only. Cell growth and lycopene production decreased substantially when isopropyl beta-D-thiogalactosidase (IPTG) was added to the high-copy plasmid-containing culture, suggesting that overexpression of DXP synthase was a significant metabolic burden. In the low-copy plasmid-containing culture, no differences in cell growth or lycopene production were observed with any IPTG concentrations. When dxs was placed under the control of the arabinose-inducible promoter (P(BAD)) on the low-copy plasmid, the amount of lycopene produced was proportional to the arabinose concentration and no significant changes in cell growth resulted. These results suggest that low-copy plasmids may be useful in metabolic engineering applications, particularly when one or more of the substrates used in the recombinant pathway are required for normal cellular metabolism.

Acid Anhydride Hydrolases↗