Coenzyme Q10 Production Research Articles

Precise regulating the specific oxygen consumption rate to strengthen the CoQ10 biosynthesis by Rhodobater sphaeroides

Coenzyme Q10 (CoQ10) is the most consumed dietary supplement and mainly biosynthesized by aerobic fermentation of Rhodobacter sphaeroides (R. sphaeroides). Oxygen supply was identified as a bottleneck for improving CoQ10 yield in R. sphaeroides. In this study, a precise regulation strategy based on dielectric spectroscopy (DS) was applied to further improve CoQ10 biosynthesis by R. sphaeroide. First, a quantitative response model among viable cells, cell morphology, and oxygen uptake rate (OUR) was established. DS could be used to detect viable R. sphaeroides cells, and the relationship among cell morphology, CoQ10 biosynthesis, and OUR was found to be significant. Based on this model, the online specific oxygen consumption rate (QO2) control strategy was successfully applied to the CoQ10 fermentation process. QO2 controlled at 0.07 ± 0.01 × 10− 7mmol/cell/h was most favorable for CoQ10 biosynthesis, resulting in a 28.3% increase in CoQ10 production. Based on the multi-parameters analysis and online QO2 control, a precise online nutrient feeding strategy was established using conductivity detected by DS. CoQ10 production was improved by 35%, reaching 3384 mg/L in 50 L bioreactors. This online control strategy would be effectively applied for improving industrial CoQ10 production, and the precise fermentation control strategy could also be applied to other fermentation process.

Metabolic flux analysis of coenzyme Q10 synthesized by Rhodobacter sphaeroides under the influence of different pH regulators

Coenzyme Q10 (CoQ10) is crucial for human beings, especially in the fields of biology and medicine. The aim of this experiment was to investigate the conditions for increasing CoQ10 production. At present, microbial fermentation is the main production method of CoQ10, and the production process of microbial CoQ10 metabolism control fermentation is very critical. Metabolic flux is one of the most important determinants of cell physiology in metabolic engineering. Metabolic flux analysis (MFA) is used to estimate the intracellular flux in metabolic networks. In this experiment, Rhodobacter sphaeroides was used as the research object to analyze the effects of aqueous ammonia (NH3·H2O) and calcium carbonate (CaCO3) on the metabolic flux of CoQ10. When CaCO3 was used to adjust the pH, the yield of CoQ10 was 274.43 mg·L−1 (8.71 mg·g−1 DCW), which was higher than that of NH3·H2O adjustment. The results indicated that when CaCO3 was used to adjust pH, more glucose-6-phosphate (G6P) entered the pentose phosphate (HMP) pathway and produced more NADPH, which enhanced the synthesis of CoQ10. At the chorismic acid node, more metabolic fluxes were involved in the synthesis of p-hydroxybenzoic acid (pHBA; the synthetic precursor of CoQ10), enhancing the anabolic flow of CoQ10. In addition, Ca2+ produced by the reaction of CaCO3 with organic acids promotes the synthesis of CoQ10. In summary, the use of CaCO3 adjustment is more favorable for the synthesis of CoQ10 by R. sphaeroides than NH3·H2O adjustment. The migration of metabolic flux caused by the perturbation of culture conditions was analyzed to compare the changes in the distribution of intracellular metabolic fluxes for the synthesis of CoQ10. Thus, the main nodes of the metabolic network were identified as G6P and chorismic acid. This provides a theoretical basis for the modification of genes related to the CoQ10 synthesis pathway.

Combining in silico and in vitro approaches to understand the involvement of MEP and shikimate pathway in Agrobacterium tumefaciens for enhanced CoQ10 production.

To carry out integrated comparative metabolic pathway analysis to understand Coenzyme Q10 (CoQ10) production in Agrobacterium tumefaciens. Comparative analysis of the CoQ10 metabolic pathways in 10 organisms using a G2KO program and KEGG database shed light on the completeness of the production pathway in A. tumefaciens. The specific role of the key precursors and the enzymes in the metabolic network was further validated using pathway inhibitors and enhancers. While the use of fosmidomycin and glyphosate displayed 54.54%-99% suppression in CoQ10 production, supplementation of polyprenyl pyrophosphate from the MEP (methylerythritol 4-phosphate) pathway and 4-hydroxybenzoate precursor from the shikimate pathway, enhanced CoQ10 production by 2.3-fold. The present study provides a comprehensive understanding of the CoQ10 biosynthetic pathway in A. tumefaciens, which would assist rational metabolic engineering strategies for boosting CoQ10 biosynthesis.

An Overview of Analytical Methods for Quantitative Determination of Coenzyme Q10 in Foods

Food analysts have developed three primary techniques for coenzyme Q10 (CoQ10) production: isolation from animal or plant matrices, chemical synthesis, and microbial fermentation; this literature review is focused on the first method. Choosing the appropriate analytical method for determining CoQ10 in a particular food product is essential, as this analyte is a quality index for healthy foods; various associations of extraction and quantification techniques are available in the literature, each having advantages and disadvantages. Several factors must be considered when selecting an analytical method, such as specificity, linear range, detection limit, quantification limit, recovery rate, operation size, analysis time, equipment availability, and costs. In another train of thought, the food sector produces a significant amount of solid and liquid waste; therefore, waste-considered materials can be a valuable source of CoQ10 that can be recovered and used as a fortifying ingredient or dietary supplement. This review also pursues identifying the richest food sources of CoQ10, and has revealed them to be vegetable oils, fish oil, organs, and meat.

Development of a thin layer chromatographic method for the determination of coenzyme Q10produced byAgrobacterium tumefaciens

AbstractCoenzyme Q10is biosynthesized by living organisms and considered a medically necessary health constituent thereby drawing the attention of the market. Naturally‐producing microbes are commonly used for the commercial production of coenzyme Q10. The detection of extracted coenzyme Q10is usually done by high‐performance liquid chromatography and mass spectrometry methods which require sophisticated instrumentation and trained personnel. However, routine quality assessment is required rapidly at multiple steps during the large‐scale production process which necessitates the development of a rapid and relatively easy method for coenzyme Q10detection. In the present study, a thin‐layer chromatography method is developed for the rapid detection of coenzyme Q10extracted from the natural producerAgrobacterium tumefaciens. Various mobile phases were screened for the thin‐layer chromatography of coenzyme Q10and the optimum separation‐detection was observed with methanol and ethyl acetate (8:2, v/v). The same extracted coenzyme Q10was also analyzed by high‐performance liquid chromatography (the standard method of detection), showing a similar profile as with commercially available standard coenzyme Q10. Furthermore, extract from unstained thin‐layer chromatography spot showed the characteristic spectrum (ultraviolet‐visible) as with standard coenzyme Q10. Conclusively, a simple and inexpensive method for the rapid detection of coenzyme Q10was developed to aid in the commercial production of this medically important coenzyme.

Homozygous COQ7 mutation: a new cause of potentially treatable distal hereditary motor neuropathy.

Distal hereditary motor neuropathy represents a group of motor inherited neuropathies leading to distal weakness. We report a family of two brothers and a sister affected by distal hereditary motor neuropathy in whom a homozygous variant c.3G>T (p.1Met?) was identified in the COQ7 gene. This gene encodes a protein required for coenzyme Q10 biosynthesis, a component of the respiratory chain in mitochondria. Mutations of COQ7 were previously associated with severe multi-organ disorders characterized by early childhood onset and developmental delay. Using patient blood samples and fibroblasts derived from a skin biopsy, we investigated the pathogenicity of the variant of unknown significance c.3G>T (p.1Met?) in the COQ7 gene and the effect of coenzyme Q10 supplementation in vitro. We showed that this variation leads to a severe decrease in COQ7 protein levels in the patient's fibroblasts, resulting in a decrease in coenzyme Q10 production and in the accumulation of 6-demethoxycoenzyme Q10, the COQ7 substrate. Interestingly, such accumulation was also found in the patient's plasma. Normal coenzyme Q10 and 6-demethoxycoenzyme Q10 levels were restored in vitro by using the coenzyme Q10 precursor 2,4-dihydroxybenzoic acid, thus bypassing the COQ7 requirement. Coenzyme Q10 biosynthesis deficiency is known to impair the mitochondrial respiratory chain. Seahorse experiments showed that the patient's cells mainly rely on glycolysis to maintain sufficient ATP production. Consistently, the replacement of glucose by galactose in the culture medium of these cells reduced their proliferation rate. Interestingly, normal proliferation was restored by coenzyme Q10 supplementation of the culture medium, suggesting a therapeutic avenue for these patients. Altogether, we have identified the first example of recessive distal hereditary motor neuropathy caused by a homozygous variation in the COQ7 gene, which should thus be included in the gene panels used to diagnose peripheral inherited neuropathies. Furthermore, 6-demethoxycoenzyme Q10 accumulation in the blood can be used to confirm the pathogenic nature of the mutation. Finally, supplementation with coenzyme Q10 or derivatives should be considered to prevent the progression of COQ7-related peripheral inherited neuropathy in diagnosed patients.

Comparative computational study to augment UbiA prenyltransferases inherent in purple photosynthetic bacteria cultured from mangrove microbial mats in Qatar for coenzyme Q10 biosynthesis.

Coenzyme Q10 (CoQ10) is a powerful antioxidant with a myriad of applications in healthcare and cosmetic industries. The most effective route of CoQ10 production is microbial biosynthesis. In this study, four CoQ10 biosynthesizing purple photosynthetic bacteria: Rhodobacter blasticus, Rhodovulum adriaticum, Afifella pfennigii and Rhodovulum marinum, were identified using 16S rRNA sequencing of enriched microbial mat samples obtained from Purple Island mangroves (Qatar). The membrane bound enzyme 4-hydroxybenzoate octaprenyltransferase (UbiA) is pivotal for bacterial biosynthesis of CoQ10. The identified bacteria could be inducted as efficient industrial bio-synthesizers of CoQ10 by engineering their UbiA enzymes. Therefore, the mutation sites and substitution residues for potential functional enhancement were determined by comparative computational study. Two mutation sites were identified within the two conserved Asp-rich motifs, and the effect of proposed mutations in substrate binding affinity of the UbiA enzymes was assessed using multiple ligand simultaneous docking (MLSD) studies, as a groundwork for experimental studies.

Production of Coenzyme Q10 by microbes: an update.

Coenzyme Q10 (CoQ10) is the main CoQ species in human and is used extensively in food, cosmetic and medicine industries because of its antioxidant properties and its benefit in prophylactic medicine and therapy for a variety of diseases. Among various approaches to increase the production of CoQ10, microbial fermentation is the most effective. As knowledge of the biosynthetic enzymes and regulatory mechanisms modulating CoQ10 production increases, opportunities arise for metabolic engineering of CoQ10 in microbial hosts. In this review, we present various strategies used up to date to improve CoQ10 production and focus on metabolic engineering of CoQ10 overproduction in microbes. General strategies of metabolic engineering include providing sufficient precursors for CoQ10, increasing metabolic fluxes, and expanding storage capacity for CoQ10. Based on these strategies, CoQ10 production has been significantly improved in natural CoQ10 producers, as well as in heterologous hosts.

Overexpression of enzymes in glycolysis and energy metabolic pathways to enhance coenzyme Q10 production in Rhodobacter sphaeroides VK-2-3.

We subjected the components of the glycolysis and energy metabolism pathways of Rhodobacter sphaeroides (R. sphaeroides) to metabolic engineering to improve the titer and yield of coenzyme Q10 (CoQ10). Phosphofructokinase (PFK), cyclic adenylate-dependent protein kinase (PKAC), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and adenosine triphosphate hydrolase (KdpC) were overexpressed in R. sphaeroides VK-2-3 (VK-2-3). The strains were labeled R. sphaeroides PFK (RS.PFK), RS.PKAC, RS.PFK–PKAC, RS.KdpC, RS.GAPDH, and RS.KdpC–GAPDH. Results showed that the CoQ10 titers of RS.PFK, RS.PKAC, and RS.PFK–PKAC were 300.96 ± 0.87, 405.94 ± 4.77, and 379.94 ± 0.42 mg/l, respectively. The CoQ10 titers of RS.PFK and VK-2-3 were not significantly different; however, those for RS.PKAC and RS.PFK–PKAC were 13 and 6% higher than that of VK-2-3, respectively. Further, the titers of RS.KdpC, RS.GAPDH, and RS.KdpC–GAPDH were 360.17 ± 0.39, 409.79 ± 0.76, and 359.87 ± 1.14 mg/l, respectively. The titers of RS.KdpC and RS.KdpC–GAPDH were not significantly different from that for VK-2-3, whereas that for RS.GAPDH was 14% higher than that of VK-2-3. Finally, when the cultures of RS.GAPDH and VK-2-3 were scaled up in 5-L fermenters, the CoQ10 titers and RS.GAPDH yields increased by 44.3 and 37.8%, respectively, compared with VK-2-3.To the best of our knowledge, the glycolysis pathway of R. sphaeroides was studied for the first time in this study. We genetically modified the components of the energy metabolism pathway to obtain the strain with high yield of CoQ10 mutant RS.GAPDH. The findings of this study can serve as a basis for future studies involving metabolic engineering of CoQ10-producing strains.

Activating SIRT-1 Signalling with the Mitochondrial-CoQ10 Activator Solanesol Improves Neurobehavioral and Neurochemical Defects in Ouabain-Induced Experimental Model of Bipolar Disorder.

Bipolar disorder (BD) is a chronic mental illness characterized by mood fluctuations that range from depressive lows to manic highs. Several studies have linked the downregulation of SIRT-1 (silent mating type information regulation-2 homologs) signaling to the onset of BD and other neurological dysfunctions. This research aimed to look into the neuroprotective potential of Solanesol (SNL) in rats given ICV-Ouabain injections, focusing on its effect on SIRT-1 signaling activation in the brain. Ouabain, found in hypothalamic and medullary neurons, is an endogenous inhibitor of brain Na+/K+ ATPase. The inhibition of brain Na+/K+ ATPase by Ouabain may also result in changes in neurotransmission within the central nervous system. SNL is a Solanaceae family active phytoconstituent produced from the plant Nicotiana tabacum. SNL is used as a precursor for the production of CoQ10 (Coenzyme Q10), a powerful antioxidant and neuroprotective compound. In the current study, lithium (Li), an important mood stabilizer drug, was used as a control. This study looked at the neuroprotective potential of SNL at dosages of 40 and 80 mg/kg in ICV-OUA injections that caused BD-like neurobehavioral and neurochemical defects in Wistar rats. Wistar rats were placed into eight groups (n = 6) and administered 1 mM/0.5 µL ICV-OUA injections for three days. Neurochemical assessments were done in rat brain homogenates, CSF, and blood plasma samples at the end of the experiment protocol schedule. Long-term SNL and lithium administration have been shown to decrease the number of rearing and crossings and reduce time spent in the center, locomotor activities, and immobility time. Solansesol treatment gradually raises the amount of Na+/K+ ATPase, limiting the severity of behavioural symptoms. These findings also revealed that SNL increases the levels of SIRT-1 in CSF, blood plasma, and brain homogenate samples. Moreover, in rat brain homogenates and blood plasma samples, SNL modulates apoptotic markers such as Caspase-3, Bax (pro-apoptotic), and Bcl-2 (anti-apoptotic). Mitochondrial-ETC complex enzymes, including complex-I, II, IV, V, and CoQ10, were also restored following long-term SNL treatment. Furthermore, SNL lowered inflammatory cytokines (TNF-α, IL-1β) levels while restoring neurotransmitter levels (serotonin, dopamine, glutamate, and acetylcholine) and decreasing oxidative stress markers. Histological examinations also validated Solanesol’s protective effect. As a result, our findings suggest that SNL, as a SIRT-1 signalling activator, may be a promising therapeutic approach for BD-like neurological dysfunctions.

Rational Engineering of Non-Ubiquinone Containing Corynebacterium glutamicum for Enhanced Coenzyme Q10 Production.

Coenzyme Q10 (CoQ10) is a lipid-soluble compound with important physiological functions and is sought after in the food and cosmetic industries owing to its antioxidant properties. In our previous proof of concept, we engineered for CoQ10 biosynthesis the industrially relevant Corynebacterium glutamicum, which does not naturally synthesize any CoQ. Here, liquid chromatography–mass spectrometry (LC–MS) analysis identified two metabolic bottlenecks in the CoQ10 production, i.e., low conversion of the intermediate 10-prenylphenol (10P-Ph) to CoQ10 and the accumulation of isoprenologs with prenyl chain lengths of not only 10, but also 8 to 11 isopentenyl units. To overcome these limitations, the strain was engineered for expression of the Ubi complex accessory factors UbiJ and UbiK from Escherichia coli to increase flux towards CoQ10, and by replacement of the native polyprenyl diphosphate synthase IspB with a decaprenyl diphosphate synthase (DdsA) to select for prenyl chains with 10 isopentenyl units. The best strain UBI6-Rs showed a seven-fold increased CoQ10 content and eight-fold increased CoQ10 titer compared to the initial strain UBI4-Pd, while the abundance of CoQ8, CoQ9, and CoQ11 was significantly reduced. This study demonstrates the application of the recent insight into CoQ biosynthesis to improve metabolic engineering of a heterologous CoQ10 production strain.

Accurate and online quantification of viable Rhodobacter sphaeroides cells using a flow cytometry-dielectric spectroscopy (FCM-DS) method

Accurate and online quantification of viable cells is one of the necessary requirements during the microbial fermentation process for high productivity. The flow cytometry (FCM)-based method accurately quantifies viable cells, but this offline method cannot reflect the counts constantly. The dielectric spectroscopy (DS) sensor is widely utilized to monitor viable cells online; however, accurately converting the capacitance value of the DS sensor to the viable bacterial cell counts has barely been tried. We have developed a method by coupling the principles and techniques of FCM and the DS sensor to quantify viable Rhodobacter sphaeroides cells. Using specific fluorescent antibodies and propidium iodide (PI), viable R. sphaeroides cells were accurately quantified within 30 min by FCM. The DS sensor was combined with the FCM to create a direct capacitance-viable cell count quantification system. The LOD (limit of detection) of the FCM-DS method was 8 × 108 CFU/mL, RSD (relative standard deviation) < 5%, along with good reproducibility of the results. Finally, the viable cell count, obtained from the FCM-DS method, was applied to regulate the specific oxygen uptake rate (QO2) that increased the production of coenzyme Q10 by 8.1%. Together, our results strongly suggest that viable cells can be accurately quantified online by the integrated FCM-DS method, which would help to devise precise fermentation control strategies.

Enhanced production and characterization of coenzyme Q10 from Rhodobacter sphaeroides using a potential fermentation strategy

BackgroundThe demand for Coenzyme Q10 (CoQ10) to the human body is increasing due to its potential health function. However, the production of CoQ10 in yields remains a big problem for commercialization. MethodsThis study attempts to develop a useful fermentation strategy for enhancing the production of CoQ10 from the Rhodobacter sphaeroides. Significant FindingsIt was observed that the cell growth, the concentration and the content of CoQ10 were further enhanced to 11.44 g/L, 60.02 mg/L and 5.25 mg/g by the assimilation of 2 g/L of vitamin B1. This fed-batch culture using vitamin B1 as a feeding substrate resulted into the cell growth, the CoQ10 production and the CoQ10 content of 16.5 g/L, 88.7 mg/L and 5.38 mg/g respectively. Moreover, the fed-batch culture with 100% medium replacement of 12 h-, 24 h- and 48 h-cultivations were also performed. The maximal cell growth, CoQ10 production and CoQ10 content were found to be 37.65 g/L, 138.24 mg/L and 3.67 mg/g, respectively. Eventually, this study also identified the CoQ10 produced by R. sphaeroides using FAB-MS and 1HNMR . It was suggested that by optimizing the environmental factors, the CoQ10 production was enhanced by 3.42 folds for fed-batch culture when compared with the batch cultivation.

Coenzyme Q10 to manage chronic heart failure with a reduced ejection fraction: a systematic review and economic evaluation.

Chronic heart failure is a debilitating condition that accounts for an annual NHS spend of £2.3B. Low levels of endogenous coenzyme Q10 may exacerbate chronic heart failure. Coenzyme Q10 supplements might improve symptoms and slow progression. As statins are thought to block the production of coenzyme Q10, supplementation might be particularly beneficial for patients taking statins. To assess the clinical effectiveness and cost-effectiveness of coenzyme Q10 in managing chronic heart failure with a reduced ejection fraction. A systematic review that included randomised trials comparing coenzyme Q10 plus standard care with standard care alone in chronic heart failure. Trials restricted to chronic heart failure with a preserved ejection fraction were excluded. Databases including MEDLINE, EMBASE and CENTRAL were searched up to March 2020. Risk of bias was assessed using the Cochrane Risk of Bias tool (version 5.2). A planned individual participant data meta-analysis was not possible and meta-analyses were mostly based on aggregate data from publications. Potential effect modification was examined using meta-regression. A Markov model used treatment effects from the meta-analysis and baseline mortality and hospitalisation from an observational UK cohort. Costs were evaluated from an NHS and Personal Social Services perspective and expressed in Great British pounds at a 2019/20 price base. Outcomes were expressed in quality-adjusted life-years. Both costs and outcomes were discounted at a 3.5% annual rate. A total of 26 trials, comprising 2250 participants, were included in the systematic review. Many trials were reported poorly and were rated as having a high or unclear risk of bias in at least one domain. Meta-analysis suggested a possible benefit of coenzyme Q10 on all-cause mortality (seven trials, 1371 participants; relative risk 0.68, 95% confidence interval 0.45 to 1.03). The results for short-term functional outcomes were more modest or unclear. There was no indication of increased adverse events with coenzyme Q10. Meta-regression found no evidence of treatment interaction with statins. The base-case cost-effectiveness analysis produced incremental costs of £4878, incremental quality-adjusted life-years of 1.34 and an incremental cost-effectiveness ratio of £3650. Probabilistic sensitivity analyses showed that at thresholds of £20,000 and £30,000 per quality-adjusted life-year coenzyme Q10 had a high probability (95.2% and 95.8%, respectively) of being more cost-effective than standard care alone. Scenario analyses in which the population and other model assumptions were varied all found coenzyme Q10 to be cost-effective. The expected value of perfect information suggested that a new trial could be valuable. For most outcomes, data were available from few trials and different trials contributed to different outcomes. There were concerns about risk of bias and whether or not the results from included trials were applicable to a typical UK population. A lack of individual participant data meant that planned detailed analyses of effect modifiers were not possible. Available evidence suggested that, if prescribed, coenzyme Q10 has the potential to be clinically effective and cost-effective for heart failure with a reduced ejection fraction. However, given important concerns about risk of bias, plausibility of effect sizes and applicability of the evidence base, establishing whether or not coenzyme Q10 is genuinely effective in a typical UK population is important, particularly as coenzyme Q10 has not been subject to the scrutiny of drug-licensing processes. Stronger evidence is needed before considering its prescription in the NHS. A new independent, well-designed clinical trial of coenzyme Q10 in a typical UK heart failure with a reduced ejection fraction population may be warranted. This study is registered as PROSPERO CRD42018106189. This project was funded by the National Institute for Health Research (NIHR) Health Technology Assessment programme and will be published in full in Health Technology Assessment; Vol. 26, No. 4. See the NIHR Journals Library website for further project information.

Iterative mutagenesis induced by atmospheric and room temperature plasma treatment under multiple selection pressures for the improvement of coenzyme Q10 production by Rhodobacter sphaeroides.

CoQ10, which has been widely applied in medicine by dietary supplement, possesses important functions in antioxidant process and bioenergy generation. Iterative mutagenesis introduced by atmospheric and room temperature plasma (ARTP) treatment was studied to improve the coenzyme Q10 (CoQ10) production of Rhodobacter sphaeroides (R. sphaeroides), and multiple selection pressures including vitamin K3 (VK3), Na2S and benzoic acid (BA) were adopted for the first time. After two rounds of mutation and screening, a mutant strain R.S 17 was obtained, and the product titer was increased by 80.37%. The CoQ10 titer and cell density reached 236.7mg L-1 and 57.09g L-1, respectively, in the fed-batch fermentation, and the CoQ10 content was 22.1% higher than that of the parent strain. In addition, the spectral scanning results indicated the metabolic flux improvement contributing to the CoQ10 production in R.S 17, and the genetic stability was validated. Based on the iterative mutagenesis introduced by ARTP under multiple selection pressures, the promotion of CoQ10 production by R. sphaeroides was achieved. The significant improvement in fermentation performances and the good genetic stability of R.S 17 indicate a potential way for the efficient biosynthesis of CoQ10.

Optimization of Coenzyme Q10 Production by Gluconobacter japonicus FM10 Using Response Surface Methodology

Introduction: Coenzyme Q10 is one of the antioxidants with a worldwide market. Nowadays the coenzymeQ10 production has been considered by fermentation using microorganisms. In this study, the Response Surface Methodology was used to optimize culture composition for coenzyme Q10 production by a previously isolated bacterium, Gluconobacter japonicus FM10. Materials and methods: A central composite design was employed to optimize the culture composition including sorbitol, yeast extract, peptone, KH2PO4, and MgSO4 for coenzyme Q10 production. The dry cell weight and coenzymeQ10 concentration were monitored as response variables and the desirability function approach was applied to obtain the optimum level for each factor. Results: Results showed that an average, 3 mg/L of coenzyme Q10 was obtained when the optimized culture composition was employed (110 g/L of sorbitol, 25 g/L of yeast extract, 35 g/L of peptone, 0.5 g/L of KH2PO4, and 0.55 g/L of MgSO4). In addition, the expected dry cell weight reached 6 g/L in the presence of 90 g/L of sorbitol, 17.5 g/L of yeast extract, 35 g/L of peptone, 0 g/L of KH2PO4, and 1.7 g/L of MgSO4. Conclusions: The results of regression analysis revealed that the concentrations of peptone and sorbitol were the most effective factors in producing coenzyme Q10 and dry cell weight, respectively.

Co-production of polyhydroxybutyrate (PHB) and coenzyme Q10 (CoQ10) via no-sugar fermentation\u2014a case by Methylobacterium sp. XJLW

PurposeTo explore a competitive PHB-producing fermentation process, this study evaluated the potential for Methylobacterium sp. XJLW to produce simultaneously PHB and coenzyme Q10 (CoQ10) using methanol as sole carbon and energy source.MethodsThe metabolic pathways of PHB and CoQ10 biosynthesis in Methylobacterium sp. XJLW were first mined based on the genomic and comparative transcriptomics information. Then, real-time fluorescence quantitative PCR (RT-qPCR) was employed for comparing the expression level of important genes involved in PHB and CoQ10 synthesis pathways’ response to methanol and glucose. Transmission electron microscope (TEM), gas chromatography/mass spectrometry (GC-MS), nuclear magnetic resonance (NMR), Fourier transformation infrared spectrum (FT-IR), and liquid chromatography/mass spectrometry (LC-MS) methods were used to elucidate the yield and structure of PHB and CoQ10, respectively. PHB and CoQ10 productivity of Methylobacterium sp. XJLW were evaluated in Erlenmeyer flask for medium optimization, and in a 5-L bioreactor for methanol fed-batch strategy according to dissolved oxygen (DO) and pH control.ResultsComparative genomics analysis showed that the PHB and CoQ10 biosynthesis pathways co-exist in Methylobacterium sp. XJLW. Transcriptomics analysis showed that the transcription level of key genes in both pathways responding to methanol was significantly higher than that responding to glucose. Correspondingly, strain Methylobacterium sp. XJLW can produce PHB and CoQ10 simultaneously with higher yield using cheap and abundant methanol than using glucose as sole carbon and energy source. The isolated products showed the structure characteristics same to that of standard PHB and CoQ10. The optimal medium and cultural conditions for PHB and CoQ10 co-production by Methylobacterium sp. XJLW was in M3 medium containing 7.918 g L-1 methanol, 0.5 g L-1 of ammonium sulfate, 0.1% (v/v) of Tween 80, and 1.0 g L-1 of sodium chloride, under 30 °C and pH 7.0. In a 5-L bioreactor coupled with methanol fed-batch process, a maximum DCW value (46.31 g L-1) with the highest yields of PHB and CoQ10, reaching 6.94 g L-1 and 22.28 mg L-1, respectively.ConclusionMethylobacterium sp. XJLW is potential for efficiently co-producing PHB and CoQ10 employing methanol as sole carbon and energy source. However, it is still necessary to further optimize fermentation process, and genetically modify strain pathway, for enhanced production of PHB and CoQ10 simultaneously by Methylobacterium sp. XJLW. It also suggests a potential strategy to develop efficiently co-producing other high-value metabolites using methanol-based bioprocess.

Increasing coenzyme Q10 yield from Rhodopseudomonas palustris by expressing rate-limiting enzymes and blocking carotenoid and hopanoid pathways.

Coenzyme Q10 (CoQ10 ), a strong antioxidant, is used extensively in food, cosmetic and medicine industries. A natural producer, Rhodopseudomonas palustris, was engineered to overproduce CoQ10 . For increasing the CoQ10 content, crtB gene was deleted to block the carotenoid pathway. crtB gene deletion led to 33% improvement of CoQ10 content over the wild type strain. However, it was found that the yield of hopanoids was also increased by competing for the precursors from carotenoid pathway with CoQ10 pathway. To further increase the CoQ10 content, hopanoid pathway was blocked by deleting shc gene, resulting in R. palustris [Δshc, ΔcrtB] to produce 4·7mgg-1 DCW CoQ10 , which was 1·2 times higher than the CoQ10 content in the wild type strain. The common strategy of co-expression of rate-limiting enzymes (DXS, DPS and UbiA) was combined with the pathway blocking method resulted in 8·2mgg-1 DCW of CoQ10 , which was 2·9 times higher than that of wild type strain. The results suggested a synergistic effect among different metabolic engineering strategies. This study demonstrates the potential of R. palustris for CoQ10 production and provides viable strategies to increase CoQ10 titer.

Coenzyme Q10 Biosynthesis Established in the Non-Ubiquinone Containing Corynebacterium glutamicum by Metabolic Engineering.

Coenzyme Q10 (CoQ10) serves as an electron carrier in aerobic respiration and has become an interesting target for biotechnological production due to its antioxidative effect and benefits in supplementation to patients with various diseases. For the microbial production, so far only bacteria have been used that naturally synthesize CoQ10 or a related CoQ species. Since the whole pathway involves many enzymatic steps and has not been fully elucidated yet, the set of genes required for transfer of CoQ10 synthesis to a bacterium not naturally synthesizing CoQ species remained unknown. Here, we established CoQ10 biosynthesis in the non-ubiquinone-containing Gram-positive Corynebacterium glutamicum by metabolic engineering. CoQ10 biosynthesis involves prenylation and, thus, requires farnesyl diphosphate as precursor. A carotenoid-deficient strain was engineered to synthesize an increased supply of the precursor molecule farnesyl diphosphate. Increased farnesyl diphosphate supply was demonstrated indirectly by increased conversion to amorpha-4,11-diene. To provide the first CoQ10 precursor decaprenyl diphosphate (DPP) from farnesyl diphosphate, DPP synthase gene ddsA from Paracoccus denitrificans was expressed. Improved supply of the second CoQ10 precursor, para-hydroxybenzoate (pHBA), resulted from metabolic engineering of the shikimate pathway. Prenylation of pHBA with DPP and subsequent decarboxylation, hydroxylation, and methylation reactions to yield CoQ10 was achieved by expression of ubi genes from Escherichia coli. CoQ10 biosynthesis was demonstrated in shake-flask cultivation and verified by liquid chromatography mass spectrometry analysis. To the best of our knowledge, this is the first report of CoQ10 production in a non-ubiquinone-containing bacterium.

Production of Coenzyme Q10 in Recombinant Schizosaccharomyces Pombe through Metabolic Engineering Approaches

Coenzyme Q10 is an important cofactor in the electron transport chain, which powers oxidative phosphorylation. Similar to other cofactors such as vitamin B12, deficiency in coenzyme Q10 (CoQ) manifests as clinical symptoms. Hence, coenzyme Q10 can be used as a health supplement. But, difficulty in extracting it from natural sources and the high cost involved explains continuing interest in improving its production titer in various cellular hosts. A metabolic cofactor, and thus naturally of low cellular abundance, various genetic engineering approaches are explored for improving its concentration in heterologous hosts ranging from Escherichia coli to Schizosaccharomyces pombe. While the native pathway for CoQ biosynthesis feeds into that of cholesterol, and is conserved across the three domains of life, slight variation in pathway architecture exists. Specifically, some segments of the pathway are truncated for improving overall metabolic efficiency in hosts. Hence, choice of organisms as production host for CoQ10 is crucial and these typically exhibit high growth rates with ease for genetic manipulation such as heterologous expression of missing pathway genes. Whole cell metabolomics profiled by high performance liquid chromatography tandem mass spectrometry (HPLC- MS/MS) coupled with metabolic flux analysis (MFA) and metabolic flux control (MCA), points to specific metabolic choke points where enhanced expression of enzyme increase the cellular concentration of important precursors and the final coenzyme product. One such choke point is the rate limiting enzyme, HMG-CoA reductase, whose over-expression improves CoQ titer. Systematic exploration of how fermentation parameters affect production yield is another important approach given myriad problems and challenges affecting the industrial use of recombinant CoQ production hosts. But CoQ production by recombinant routes usually results in CoQ of different side chain lengths; thus, efforts are directed towards tunable control of CoQ side chain moiety from the metabolic engineering perspective, which improves product quality and pharmaceutical properties. Another interesting basic science angle involves the conjugation of different cell localization signals for probing subcellular localization of CoQ. Finally, RNA interference (RNAi) mediated modulation of CoQ expression is a useful tool for indirectly assessing cellular energy state and redox balance, which opens up its use in studies examining how energy status affect physiological response to various nutrition and environment stressors. Collectively, much effort has focused on improving CoQ yield through metabolic engineering, and effecting tunable control of side chain length. However, important questions remain on understanding the subcellular localization of CoQ and examining the utility of tuning CoQ expression level for affecting cellular energy level, and its use as a physiology probe.