Protective effects of coenzyme Q10 and α-tocopherol against free radical-mediated liver cell injury

In an attempt to provide further confirmation of the antioxidant role of reduced form of coenzyme Q homologue (CoQ_nH₂) and α-tocopherol (α-Toc), we incubated isolated rat hepatocytes with a water-soluble radical initiator, 2,2′-azobis(2-amidinopropane)dihydrochloride (AAPH) in the presence or absence of exogenously added coenzyme Q₁₀ (CoQ₁₀) or α-Toc for 3 h at 37°C under an atmosphere of 95% oxygen and 5% carbon dioxide. In the control experiment without adding AAPH it was confirmed that added CoQ₁₀ and α-Toc were incorporated into the cells and some CoQ₁₀ were converted to CoQ₁₀H₂. Incubation of hepatocytes with 50 mM AAPH resulted in the formation of thiobarbituric acid-reactive substances and the decrease in cell viability and both were inhibited by exogenously added CoQ₁₀ or α-Toc in a dose-dependent manner. The decrease in endogenous CoQ₉H₂ and α-Toc levels was observed by the addition of AAPH. Addition of CoQ₁₀ inhibited the oxidation of CoQ₉H₂ to CoQ₉ dose-dependently while the addition of α-Toc did not. These data suggest that both CoQ_nH₂ and α-Toc act as antioxidants and can inhibit free radical-mediated cell injury.     

Lactate increases coenzyme Q10 production by Agrobacterium tumefaciens

By the optimization of nitrogen source for coenzyme Q₁₀ (ubiquinone, CoQ₁₀) production in Agrobacterium tumefaciens KCCM 10413 culture, the highest CoQ₁₀ production was achieved in medium containing corn steep powder (CSP). Components for a stimulatory effect on the production of CoQ₁₀ in CSP were screened, and lactate was found to increase dry cell weight (DCW) and the specific CoQ₁₀ content. In a fed-batch culture of A. tumefaciens, supplementation with 1.5 g of lactate l⁻¹ further improved DCW, the specific CoQ₁₀ content, and CoQ₁₀ production by 16.0, 5.8, and 22.8%, respectively. It has been reported that lactate stimulates cell growth and acts as an accelerator driving the tricarboxylic acid (TCA) cycle (Roberto et al. 2002, Biotechnol Let 24:427–431; Matsuoka et al. 1996, Biosci Biotechnol Biochem 60:575–579). In this study, lactate supplementation increased DCW and the specific CoQ₁₀ content in A. tumefaciens culture, probably by accelerating TCA cycle and energy production as reported previously, leading to the increase of CoQ₁₀ production.     

Enhanced production of CoQ<Subscript>10</Subscript> by newly isolated <Emphasis Type="Italic">Sphingomonas</Emphasis> sp. ZUTEO3 with a coupled fermentation–extraction process

The use of coenzyme Q₁₀ (CoQ₁₀) as a complementary therapy in heart failure will increase in proportion to the growth of the ageing population and the expansion of statins consumption. Economical production of CoQ₁₀ by microbes will become more important due to the growing demands of the pharmaceutical industry. Process simplification and integration might be one desirable pathway for economic production of CoQ₁₀ by microbial fermentation. In this report, the effect of a coupled fermentation–extraction process on CoQ₁₀ production by newly isolated Sphingomonas sp. ZUTEO3 was evaluated. It was found that the CoQ₁₀ yield of the coupled process was significantly higher than that of the traditional process. As optimal conditions in our experiment, 2% soybean oil was added to the original culture to enhance cell membrane permeability, and 50 mL hexane was added to the 30 h culture as an extracting solvent for the subsequent coupled fermentation–extraction process. The maximal yield of CoQ₁₀ reached 43.2 mg/L and 32.5 mg/g dry cell weight after 38 h of total fermentation period. The coupled process represents one potential pathway for CoQ₁₀ production with even higher yield and lower cost. This is the first report of CoQ₁₀ production by Sphingomonas sp. using a coupled fermentation–extraction process.     

Effects of dissolved oxygen concentration and DO-stat feeding strategy on CoQ10 production with Rhizobium radiobacter

The cell growth and CoQ₁₀ (coenzyme Q₁₀) formation of Rhizobium radiobacter WSH2601 were investigated in a 7-1 bioreactor under different dissolved oxygen (DO) concentrations. A maximal CoQ₁₀ content (C/B) of 1.91 mg/g dry cell weight (DCW) and CoQ₁₀ concentration of 32.1 mg/l were obtained at the appropriate DO concentration of 40% (of air saturation). High DO concentration was favourable to the cell growth of Rhizobium radiobacter WSH2601. In order to achieve the maximal yield of CoQ₁₀ production, a new DO-stat feeding strategy was proposed, which significantly improved cell growth and CoQ₁₀ formation. With this strategy, the maximal CoQ₁₀ concentration and DCW reached 51.1 mg/l and 23.9 g/l, respectively, which were 67 and 44.8% higher than those obtained in the batch culture with DO concentration controlled.     

A water soluble CoQ10 formulation improves intracellular distribution and promotes mitochondrial respiration in cultured cells

Background

Mitochondria are both the cellular powerhouse and the major source of reactive oxygen species. Coenzyme Q₁₀ plays a key role in mitochondrial energy production and is recognized as a powerful antioxidant. For these reasons it can be argued that higher mitochondrial ubiquinone levels may enhance the energy state and protect from oxidative stress. Despite the large number of clinical studies on the effect of CoQ₁₀ supplementation, there are very few experimental data about the mitochondrial ubiquinone content and the cellular bioenergetic state after supplementation. Controversial clinical and in vitro results are mainly due to the high hydrophobicity of this compound, which reduces its bioavailability.

Principal Findings

We measured the cellular and mitochondrial ubiquinone content in two cell lines (T67 and H9c2) after supplementation with a hydrophilic CoQ₁₀ formulation (Qter®) and native CoQ₁₀. Our results show that the water soluble formulation is more efficient in increasing ubiquinone levels. We have evaluated the bioenergetics effect of ubiquinone treatment, demonstrating that intracellular CoQ₁₀ content after Qter supplementation positively correlates with an improved mitochondrial functionality (increased oxygen consumption rate, transmembrane potential, ATP synthesis) and resistance to oxidative stress.

Conclusions

The improved cellular energy metabolism related to increased CoQ₁₀ content represents a strong rationale for the clinical use of coenzyme Q₁₀ and highlights the biological effects of Qter®, that make it the eligible CoQ₁₀ formulation for the ubiquinone supplementation.     

Redox status and pharmacokinetics of coenzyme Q10 in rat plasma after its single intravenous administration

The pharmacokinetics of the total pool of coenzyme Q₁₀ (CoQ₁₀), its oxidized (ubiquinone) and reduced (ubiquinol, CoQ₁₀H₂) forms have been investigated in rats plasma during 48 h after a single intravenous injection of a solution of solubilized CoQ₁₀ (10 mg/kg) to rats. Plasma levels of CoQ₁₀ were determined by HPLC with spectrophotometric and coulometric detection. In plasma samples taken during the first minutes after the CoQ₁₀ intravenous injection, the total pool of coenzyme Q₁₀ and proportion of CoQ₁₀H₂ remained unchanged during two weeks of storage at ?20°C. The kinetic curve of the total pool of coenzyme Q₁₀ corresponds to a one-compartment model (R ₂ = 0.9932), while the corresponding curve of its oxidized form fits to the two-compartment model. During the first minutes after the injection a significant portion of plasma ubiquinone undergoes reduction, and after 7 h the concentration of ubiquinol predominates. The decrease in total plasma coenzyme Q₁₀ content was accompanied by the gradual increase in plasma ubiquinol, which represented about 90% of total plasma CoQ₁₀ by the end of the first day. The results of this study demonstrate the ability of the organism to transform high concentrations of the oxidized form of CoQ₁₀ into the effective antioxidant (reduced) form and justify prospects of the development of parenteral dosage forms of CoQ₁₀ for the use in the treatment of acute pathological conditions.     

Factors affecting broth viscosity and coenzyme Q10 production by Agrobacterium species

Summary In the production of coenzyme Q₁₀ (CoQ₁₀) by Agrobacterium sp. the culture broth becomes highly viscous. In an attempt to improve the production process, the effects of chemical and physical factors on broth viscosity and CoQ₁₀ production were studied, using Agrobacterium sp. KY-8593. A particular concentration ratio of sugar to ammonium-nitrogen (NH₄–N) in the medium could effectively enhance CoQ₁₀ production without increasing broth viscosity. An increase in culture temperature to between 32°C and 34°C lowered broth viscosity without reducing CoQ₁₀ production. NH₄–N concentration and temperature had a correlative effect on broth viscosity. At a temperature of about 33°C, there was a wide range of NH₄–N concentration which was optimal for both broth viscosity and CoQ₁₀ production. In optimal conditions with 8% sugar the apparent broth viscosity was reduced to less than 10 pseudo-cP and CoQ₁₀ production was increased to more than 80 mg/l.     

Intracellular cholesterol accumulation and coenzyme Q10 deficiency in Familial Hypercholesterolemia

Familial Hypercholesterolemia (FH) is an autosomal co-dominant genetic disorder characterized by elevated low-density lipoprotein (LDL) cholesterol levels and increased risk for premature cardiovascular disease. Here, we examined FH pathophysiology in skin fibroblasts derived from FH patients harboring heterozygous mutations in the LDL-receptor.Fibroblasts from FH patients showed a reduced LDL-uptake associated with increased intracellular cholesterol levels and coenzyme Q₁₀ (CoQ₁₀) deficiency, suggesting dysregulation of the mevalonate pathway.Secondary CoQ₁₀ deficiency was associated with mitochondrial depolarization and mitophagy activation in FH fibroblasts. Persistent mitophagy altered autophagy flux and induced inflammasome activation accompanied by increased production of cytokines by mutant cells. All the pathological alterations in FH fibroblasts were also reproduced in a human endothelial cell line by LDL-receptor gene silencing.Both increased intracellular cholesterol and mitochondrial dysfunction in FH fibroblasts were partially restored by CoQ₁₀ supplementation. Dysregulated mevalonate pathway in FH, including increased expression of cholesterogenic enzymes and decreased expression of CoQ₁₀ biosynthetic enzymes, was also corrected by CoQ₁₀ treatment.Reduced CoQ₁₀ content and mitochondrial dysfunction may play an important role in the pathophysiology of early atherosclerosis in FH. The diagnosis of CoQ₁₀ deficiency and mitochondrial impairment in FH patients may also be important to establish early treatment with CoQ₁₀.     

Optimization of culture conditions and scale-up to pilot and plant scales for coenzyme Q<Subscript>10</Subscript> production by <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>

This report describes the optimization of culture conditions for coenzyme Q₁₀ (CoQ₁₀) production by Agrobacterium tumefaciens KCCM 10413, an identified high-CoQ₁₀-producing strain (Kim et al., Korean patent. 10-0458818, 2002b). Among the conditions tested, the pH and the dissolved oxygen (DO) levels were the key factors affecting CoQ₁₀ production. When the pH and DO levels were controlled at 7.0 and 0–10%, respectively, a dry cell weight (DCW) of 48.4 g l⁻¹ and a CoQ₁₀ production of 320 mg l⁻¹ were obtained after 96 h of batch culture, corresponding to a specific CoQ₁₀ content of 6.61 mg g-DCW⁻¹. In a fed-batch culture of sucrose, the DCW, specific CoQ₁₀ content, and CoQ₁₀ production increased to 53.6 g l⁻¹, 8.54 mg g-DCW⁻¹, and 458 mg l⁻¹, respectively. CoQ₁₀ production was scaled up from a laboratory scale (5-l fermentor) to a pilot scale (300 l) and a plant scale (5,000 l) using the impeller tip velocity (V _tip) as a scale-up parameter. CoQ₁₀ production at the laboratory scale was similar to those at the pilot and plant scales. This is the first report of pilot- and plant-scale productions of CoQ₁₀ in A. tumefaciens.     

Coenzyme Q concentration and total antioxidant capacity of human milk at different stages of lactation in mothers of preterm and full-term infants

Coenzyme Q₁₀(CoQ₁₀) in human milk at different stages of maturity in mothers of preterm and full-term infants and its relation to the total antioxidant capacity of milk is described for the first time. Thirty healthy breastfeeding women provided colostrum, transition-milk and mature-milk samples. Coenzyme Q, α-, γ- and δ-tocopherol, fatty acids and the total antioxidant capacity of the milk were analyzed. Coenzyme Q₁₀ was found at higher concentrations for colostrum (0.81 ± 0.06 vs. 0.50 ± 0.05 μmol/l) and transition milk (0.75 ± 0.06 vs. 0.45 ± 0.05 μmol/l) in the full-term vs. the preterm group (similar results were found for total antioxidant capacity). Concentrations of α- and γ-tocopherol were higher in the full-term group and decreased with time. In conclusion, CoQ₁₀ is present in breast milk, with higher concentration in mothers of full-term infants. CoQ₁₀ in breast milk decreases through lactation in mothers delivering full-term infants. Also, CoQ₁₀, α- and γ-tocopherol concentration in human milk directly correlates with the antioxidant capacity of the milk.     

Therapeutic targeting of oxidative stress with coenzyme Q10 counteracts exaggerated diabetic cardiomyopathy in a mouse model of diabetes with diminished PI3K(p110α) signaling

Diabetes-induced cardiac complications include left ventricular (LV) dysfunction and heart failure. We previously demonstrated that LV phosphoinositide 3-kinase p110α (PI3K) protects the heart against diabetic cardiomyopathy, associated with reduced NADPH oxidase expression and activity. Conversely, in dominant negative PI3K(p110α) transgenic mice (dnPI3K), reduced cardiac PI3K signaling exaggerated diabetes-induced cardiomyopathy, associated with upregulated NADPH oxidase. The goal was to examine whether chronic supplementation with the antioxidant coenzyme Q₁₀ (CoQ₁₀) could attenuate LV superoxide and diabetic cardiomyopathy in a setting of impaired PI3K signaling. Diabetes was induced in 6-week-old nontransgenic and dnPI3K male mice via streptozotocin. After 4 weeks of diabetes, CoQ₁₀ supplementation commenced (10 mg/kg ip, 3 times/week, 8 weeks). At study end (12 weeks of diabetes), markers of LV function, cardiomyocyte hypertrophy, collagen deposition, NADPH oxidase, oxidative stress (3-nitrotyrosine), and concentrations of CoQ₉ and CoQ₁₀ were determined. LV NADPH oxidase (Nox2 gene expression and activity, and lucigenin-enhanced chemiluminescence), as well as oxidative stress, were increased by diabetes, exaggerated in diabetic dnPI3K mice, and attenuated by CoQ₁₀. Diabetes-induced LV diastolic dysfunction (prolonged deceleration time, elevated end-diastolic pressure, impaired E/A ratio), cardiomyocyte hypertrophy and fibrosis, expression of atrial natriuretic peptide, connective tissue growth factor, and β-myosin heavy chain were all attenuated by CoQ₁₀. Chronic CoQ₁₀ supplementation attenuates aspects of diabetic cardiomyopathy, even in a setting of reduced cardiac PI3K protective signaling. Given that CoQ₁₀ supplementation has been suggested to have positive outcomes in heart failure patients, chronic CoQ₁₀ supplementation may be an attractive adjunct therapy for diabetic heart failure.     

Effect of dietary coenzyme Q and fatty acids on the antioxidant status of rat tissues

Summary.  Wistar rats were fed with different diets with or without supplement coenzyme Q₁₀ (CoQ₁₀) and with oil of different sources (sunflower or virgin olive oil) for six or twelve months. Ubiquinone contents (CoQ₉ and CoQ₁₀) were quantified in homogenates of livers and brains from rats fed with the four diets. In the brain, younger rats showed a 3-fold higher amount of ubiquinone than older ones for all diets. In the liver, however, CoQ₁₀ supplementation increased the amount of CoQ₉ and CoQ₁₀ in both total homogenates and plasma membranes. Rats fed with sunflower oil as fat source showed higher amounts of ubiquinone content than those fed with olive oil, in total liver homogenates, but the total ubiquinone content in plasma membranes was similar with both fat sources. Older rats showed a higher amount of ubiquinone after diets supplemented with CoQ₁₀. Two ubiquinone-dependent antioxidant enzyme activities were measured. NADH-ferricyanide reductase activity in hepatocyte plasma membranes was unaltered by ubiquinone accumulation, but this activity increased slightly with age. Both cytosolic and membrane-bound dicumarol-sensitive NAD(P)H:(quinone acceptor) oxidoreductase (DT-diaphorase, EC 1.6.99.2) activities were decreased by diets supplemented with CoQ₁₀. Animals fed with olive oil presented lower DT-diaphorase activity than those fed with sunflower oil, suggesting that the CoQ₁₀ antioxidant protection is strengthened by olive oil as fat source. Received May 22, 2002; accepted September 20, 2002; published online May 21, 2003 RID="*" ID="*" Correspondence and reprints: Departamento de Biología Celular, Fisiología e Inmunología, Universidad de Córdoba Edificio Severo Ochoa, Campus de Rabanales, 14071 Córdoba, Spain.     

Attenuation of Cocaine and Methamphetamine Neurotoxicity by Coenzyme Q10

The neurotoxic effects of cocaine and methamphetamine (METH) were studied in mice brain with a primary objective to determine the neuroprotective potential of coenzyme Q₁₀ (CoQ₁₀) in drug addiction. Repeated treatment of cocaine or METH induced significant reduction in the striatal dopamine and CoQ₁₀ in mice. Cocaine or METH-treated mice exhibited increased thiobarbituric acid reactive substances (TBARs) in the striatum and cerebral cortex without any significant change in the cerebellum. Complex I immunoreactivity was inhibited in both cocaine and METH-treated mice, whereas tyrosine hydroxylase (TH) immunoreactivity was decreased in METH-treated mice and increased in cocaine-treated mice. Neither cocaine nor METH could induce significant change in α-synuclein expression at the doses and duration we have used in the present study. CoQ₁₀ treatment attenuated cocaine and METH-induced inhibition in the striatal ¹⁸F-DOPA uptake as determined by high-resolution microPET neuroimaging. Hence exogenous administration of CoQ₁₀ may provide neuroprotection in drug addiction.     

Effects of dietary L-carnitine and coenzyme Q10 supplementation on performance and ascites mortality of broilers

The study was conducted to determine the effects of dietary L-carnitine and coenzyme Q₁₀ (CoQ₁₀) supplementation on growth performance and ascites mortality of broilers. A 3 × 3 factorial arrangement was employed with three levels (0, 75 and 150 mg/kg) of L-carnitine and three levels of CoQ₁₀ (0, 20 and 40 mg/kg) supplementation during the experiment. Five hundred and forty one-day-old Arbor Acre male broiler chicks were randomly allocated into nine groups with six replicates each. All birds were fed with the basal diets from day 1 to 7 and changed to the experimental diets from day 8. During day 15 to 21 all the birds were exposed to low ambient temperature (15 - 18°C) to induce ascites. The results showed that under this condition, growth performance of broilers were not significantly affected by CoQ₁₀ or L-carnitine + CoQ₁₀ supplementation during week 0 - 3 and 0 - 6, but body weight gain (BWG) of broilers was significantly reduced by 150 mg/kg L-carnitine during week 0 - 6. Packed cell volume (PCV) of broilers was significantly decreased by L-carnitine and L-carnitine + CoQ₁₀ supplementation (P < 0.05). Erythrocyte osmotic fragility (EOF), ascites heart index (AHI) and ascites mortality of broilers were significantly decreased by L-carnitine, CoQ₁₀ and L-carnitine + CoQ₁₀ supplementation. Though no significant changes were observed in total antioxidative capability (T-AOC), total superoxide dismutase (T-SOD) was increased by L-carnitine, CoQ₁₀ and L-carnitine + CoQ₁₀ supplementation (P < 0.05). Malonaldehyde (MDA) content was significantly decreased by CoQ₁₀ and L-carnitine + CoQ₁₀ supplementation. The results indicate that dietary L-carnitine and CoQ₁₀ supplementation reduce ascites mortality of broilers; the reason may be partially associated with their antioxidative effects.     

Analysis of coenzyme Q in human blood and tissues

The major coenzyme Q species in humans is the decaprenyl quinoid derivative coenzyme Q₁₀ (CoQ₁₀), and its measurement is somewhat challenging owing to its hydrophobicity and tendency to be oxidized. There are three major methods which are suited for analysis of CoQ₁₀: HPLC-coupled UV or electrochemical detection, and tandem mass spectrometry. The techniques are discussed, and results of these applications to determine CoQ₁₀ concentrations in various human fluids and tissues are summarized.     

Treatment of CoQ10 Deficient Fibroblasts with Ubiquinone,CoQ Analogs,and Vitamin C: Time- and Compound-Dependent Effects

Background

Coenzyme Q₁₀ (CoQ₁₀) and its analogs are used therapeutically by virtue of their functions as electron carriers, antioxidant compounds, or both. However, published studies suggest that different ubiquinone analogs may produce divergent effects on oxidative phosphorylation and oxidative stress.

Methodology/Principal Findings

To test these concepts, we have evaluated the effects of CoQ₁₀, coenzyme Q₂ (CoQ₂), idebenone, and vitamin C on bioenergetics and oxidative stress in human skin fibroblasts with primary CoQ₁₀ deficiency. A final concentration of 5 µM of each compound was chosen to approximate the plasma concentration of CoQ₁₀ of patients treated with oral ubiquinone. CoQ₁₀ supplementation for one week but not for 24 hours doubled ATP levels and ATP/ADP ratio in CoQ₁₀ deficient fibroblasts therein normalizing the bioenergetics status of the cells. Other compounds did not affect cellular bioenergetics. In COQ2 mutant fibroblasts, increased superoxide anion production and oxidative stress-induced cell death were normalized by all supplements.

Conclusions/Significance

These results indicate that: 1) pharmacokinetics of CoQ₁₀ in reaching the mitochondrial respiratory chain is delayed; 2) short-tail ubiquinone analogs cannot replace CoQ₁₀ in the mitochondrial respiratory chain under conditions of CoQ₁₀ deficiency; and 3) oxidative stress and cell death can be counteracted by administration of lipophilic or hydrophilic antioxidants. The results of our in vitro experiments suggest that primary CoQ₁₀ deficiencies should be treated with CoQ₁₀ supplementation but not with short-tail ubiquinone analogs, such as idebenone or CoQ₂. Complementary administration of antioxidants with high bioavailability should be considered if oxidative stress is present.     

Suppression of coenzyme Q10 levels and the induction of multiple PDSS and COQ genes in human cells following oligomycin treatment

Abstract

Endogenous coenzyme Q₁₀ (CoQ₁₀) is a lipid-soluble antioxidant and essential for the electron transport chain. We previously demonstrated that hydrogen peroxide enhanced CoQ₁₀ levels, whereas disruption of mitochondrial membrane potential by a chemical uncoupler suppressed CoQ₁₀ levels, in human 143B cells. In this study, we investigated how CoQ₁₀ levels and expression of two PDSS and eight COQ genes were affected by oligomycin, which inhibited ATP synthesis at Complex V without uncoupling the mitochondria. We confirmed that oligomycin increased the production of reactive oxygen species (ROS) and decreased mitochondria-dependent ATP production in 143B cells. We also demonstrated that CoQ₁₀ levels were decreased by oligomycin after 42 or 48 h of treatment, but not at earlier time points. Expression of PDSS2 and COQ2–COQ9 were up-regulated after 18-hour oligomycin treatment, and the expression of PPARGC1A (PGC1-1α) elevated concurrently. Knockdown of PPARGC1A down-regulated the basal mRNA levels of PDSS2 and five COQ genes and suppressed the induction of COQ8 and COQ9 genes by oligomycin, but did not affect CoQ₁₀ levels under these conditions. N-acetylcysteine suppressed the augmentation of ROS levels and the enhanced expression of COQ2, COQ4, COQ7, and COQ9 induced by oligomycin, but did not modulate the changes in CoQ₁₀ levels. These results suggested that the condition of mitochondrial dysfunction induced by oligomycin decreased CoQ₁₀ levels independent of oxidative stress. Up-regulation of PDSS2 and several COQ genes by oligomycin might be regulated by multiple mechanisms, including the signaling pathways mediated by PGC-1α and ROS, but it would not restore CoQ₁₀ levels.     

Disruption of the human COQ5-containing protein complex is associated with diminished coenzyme Q10 levels under two different conditions of mitochondrial energy deficiency

BackgroundThe Coq protein complex assembled from several Coq proteins is critical for coenzyme Q₆ (CoQ₆) biosynthesis in yeast. Secondary CoQ₁₀ deficiency is associated with mitochondrial DNA (mtDNA) mutations in patients. We previously demonstrated that carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) suppressed CoQ₁₀ levels and COQ5 protein maturation in human 143B cells.MethodsThis study explored the putative COQ protein complex in human cells through two-dimensional blue native-polyacrylamide gel electrophoresis and Western blotting to investigate its status in 143B cells after FCCP treatment and in cybrids harboring the mtDNA mutation that caused myoclonic epilepsy with ragged-red fibers (MERRF) syndrome. Ubiquinol-10 and ubiquinone-10 levels were detected by high-performance liquid chromatography. Mitochondrial energy status, mRNA levels of various PDSS and COQ genes, and protein levels of COQ5 and COQ9 in cybrids were examined.ResultsA high-molecular-weight protein complex containing COQ5, but not COQ9, in the mitochondria was identified and its level was suppressed by FCCP and in cybrids with MERRF mutation. That was associated with decreased mitochondrial membrane potential and mitochondrial ATP production. Total CoQ₁₀ levels were decreased under both conditions, but the ubiquinol-10:ubiquinone-10 ratio was increased in mutant cybrids. The expression of COQ5 was increased but COQ5 protein maturation was suppressed in the mutant cybrids.ConclusionsA novel COQ5-containing protein complex was discovered in human cells. Its destabilization was associated with reduced CoQ₁₀ levels and mitochondrial energy deficiency in human cells treated with FCCP or exhibiting MERRF mutation.General significanceThe findings elucidate a possible mechanism for mitochondrial dysfunction-induced CoQ₁₀ deficiency in human cells.     

Identification of bottlenecks in Escherichia coli engineered for the production of CoQ(10)

In this work, Escherichia coli was engineered to produce a medically valuable cofactor, coenzyme Q₁₀ (CoQ₁₀), by removing the endogenous octaprenyl diphosphate synthase gene and functionally replacing it with a decaprenyl diphosphate synthase gene from Sphingomonas baekryungensis. In addition, by over-expressing genes coding for rate-limiting enzymes of the aromatic pathway, biosynthesis of the CoQ₁₀ precursor para-hydroxybenzoate (PHB) was increased. The production of isoprenoid precursors of CoQ₁₀ was also improved by the heterologous expression of a synthetic mevalonate operon, which permits the conversion of exogenously supplied mevalonate to farnesyl diphosphate. The over-expression of these precursors in the CoQ₁₀-producing E. coli strain resulted in an increase in CoQ₁₀ content, as well as in the accumulation of an intermediate of the ubiquinone pathway, decaprenylphenol (10P-Ph). In addition, the over-expression of a PHB decaprenyl transferase (UbiA) encoded by a gene from Erythrobacter sp. NAP1 was introduced to direct the flux of DPP and PHB towards the ubiquinone pathway. This further increased CoQ₁₀ content in engineered E. coli, but decreased the accumulation of 10P-Ph. Finally, we report that the combined over-production of isoprenoid precursors and over-expression of UbiA results in the decaprenylation of para-aminobenzoate, a biosynthetic precursor of folate, which is structurally similar to PHB.     

Controlling the sucrose concentration increases Coenzyme Q10 production in fed-batch culture of <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>

The production yield of Coenzyme Q₁₀ (CoQ₁₀) from the sucrose consumed by Agrobacterium tumefaciens KCCM 10413 decreased, and high levels of exopolysaccharide (EPS) accumulated after switching from batch culture to fed-batch culture. Therefore, we examined the effect of sucrose concentration on the fermentation profile by A. tumefaciens. In the continuous fed-batch culture with the sucrose concentration maintained constantly at 10, 20, 30, and 40 g l⁻¹, the dry cell weight (DCW), specific CoQ₁₀ content, CoQ₁₀ production, and the production yield of CoQ₁₀ from the sucrose consumed increased, whereas EPS production decreased as maintained sucrose concentration decreased. The pH-stat fed-batch culture system was adapted for CoQ₁₀ production to minimize the concentration of the carbon source and osmotic stress from sucrose. Using the pH-stat fed-batch culture system, the DCW, specific CoQ₁₀ content, CoQ₁₀ production, and the product yield of CoQ₁₀ from the sucrose consumed increased by 22.6, 13.7, 39.3, and 39.3%, respectively, whereas EPS production decreased by 30.7% compared to those of fed-batch culture in the previous report (Ha SJ, Kim SY, Seo JH, Oh DK, Lee JK, Appl Microbiol Biotechnol, 74:974–980, 2007). The pH-stat fed-batch culture system was scaled up to a pilot scale (300 l), and the CoQ₁₀ production results obtained (626.5 mg l⁻¹ of CoQ₁₀ and 9.25 mg g DCW⁻¹ of specific CoQ₁₀ content) were similar to those obtained at the laboratory scale. Thus, an efficient and highly competitive process for microbial CoQ₁₀ production is available.