How to Produce CQ10 Ubiquinol by Fermentation?

CQ10, also known as ubiquinone, with a relative molecular weight of 863.4, is a fat-soluble quinone, chemically known as 2'3-dimethoxy-5-methyl-6-decyloisopentenylbenzoquinone.CoQ10 is a yellow or orange-yellow crystalline powder at room temperature. CoQ10 is yellow or orange-yellow crystalline powder at room temperature, with a melting point of 49 ℃, odorless and tasteless, and insoluble in water. The structural formula is as follows.

In 1957, Crane purified CoQ10 from bovine myocardium and measured its chemical structure, confirming that CoQ10 plays an important role as a redox carrier in the respiratory chain of mammals, as a lipid-soluble electron carrier in the respiratory chain, as a cellular energy-generating element, and as a natural activator and antioxidant involved in cellular metabolism. Therefore, CoQ10 has a wide range of applications in clinical and health care, cosmetics and skincare, etc., and has been increasingly emphasized by people [1].

Currently, there are three methods to produce CoQ10: plant and animal tissue extraction, chemical synthesis, and microbial fermentation. Animal and plant tissue extraction method mainly refers to extracting products from animal organs or some plant tissues. Yuan Yi [2] used this method to extract CoQ10 from pig hearts, but the yield of fresh pig hearts was only 75 mg/kg, and due to the limitation of raw material source, the product cost was high and expensive, and the large-scale production was restricted.

Harsh conditions and numerous steps characterize the chemical synthesis method. In contrast, producing CoQ10 by fermentation of microorganisms is a promising method, which is more economical, easy to produce on a large scale, easy to isolate and purify, and easy to be absorbed by the human body.

1. Microbial Strains Producing CoQ10

The content of CoQ10 varies greatly among different strains, and the attached table shows the microbial strains with relatively high content of CoQ10, among which the content of CoQ10 in Rhodobacter sphaericus and Rhodobacter sphaericus is relatively high. These photosynthesizing bacteria belong to the order Rhodobacteria, which is divided into the suborders Rhodobacteria and Green Thiobacteria, and the former is divided into the families Rhodobacteriaceae and Rhodobacteriaceae. The former is divided into Rhodobacter sphaeroides and Rhodobacter sphaeroides. Rhodobacter sphaeroides and Rhodobacter podocarpus belong to the Rhodobacteriaceae family, so Rhodobacter sphaeroides is one of the ideal strains for CoQ10 production.

Table of Coq10-Producing Strains[4]

2.Strain Mutagenesis and Construction of Engineering Bacteria

Generally, wild-type strains have low yield and their production capacity is far from meeting the needs of industrialized production, so it is necessary to genetically modify them by using mutation breeding and genetic engineering techniques.

2.1 Breeding for Metabolic Regulation of Strains

The microbial synthesis pathway of CoQ10 is mainly divided into two routes: synthesis of aromatic ring and biosynthesis of isoprenoid side chain. Therefore, metabolic regulation breeding can be carried out according to the biosynthesis pathway of aromatic ring and isoprenoid side chain.

2.1.1 Breeding of Nutrient Deficient Mutant Strains

According to the principle of metabolic regulation, it is possible to increase CoQ10 production by weakening or cutting off the branching pathways of CoQ10 [5] (such as the metabolic synthesis pathways of aromatic compounds containing benzene rings, such as tyrosine, phenylalanine, and tryptophan, which share precursors with CoQ10 synthesis). Liu Keshan [6] used Agrobacterium tumefaciens as the starting strain and screened a mutant strain with dual defects in tyrosine and aspartic acid through double mutagenesis treatment with nitrosoguanidine and diethyl sulfate. The CoQ10 production of the mutant strain increased by 91.28% compared to the original strain.

Zhang Yanjing [7] used Bulera pseu Doalba as the material to study the effect of additives on yield. It was found that soybean oil, soybean flour, tomato juice, and orange peel juice increased the production of CoQ10 due to their high content of precursors for CoQ10 and carotenoid synthesis; Tobacco leaves and beta carotene block the pathway of synthesizing beta carotene, leading to an increase in the metabolic flux of CoQ10 synthesis and an increase in CoQ10 production. It can be seen that the synthesis of CoQ10 in microorganisms is closely related to the synthesis of carotenoids. Therefore, nutrient deficient strains of carotenoids can be screened as high-yielding strains of CoQ10.

Olson and RunDey [8] developed a carotenoid deficient strain of photosynthetic bacteria to increase the content of CoQ10. YOSHIDA [9] used Ky-4113 as the starting strain of red blood bacteria, with an initial CoQ10 content of 2.4mg/g stem cells, for mutation breeding. High yield mutant strains CL-37, Co-22, and Co-22-11 with carotenoid deficiency were screened, which increased by 88%, 150%, and 263% respectively compared to the starting strain. At present, Japan has used the mutant strain Co-22-11 for fermentation production, and its yield can reach 770mg/L fermentation broth [10].

2.1.2 Selection of Metabolic Deterrent Resistant Mutants

The CoQ10 content can also be increased by removing the feedback effects of metabolic inhibitors on CoQ10 synthesis or its related anabolism. Structural analogs of CoQ10 that are resistant to feedback inhibition of CoQ10 synthesis include L-ethionine (a structural analog of L-methionine, the precursor of CoQ10 synthesis), Zoerythromycin, Vitamin K3, and some aromatic compounds that are structural analogs of CoQ10 or respiratory system inhibitors[9] .

Liu Kesuan[6] used Agrobacterium tume-faciens AGR1. 1416 as the starting strain, and used ultraviolet light (Uv), l-methyl-3-nitro-l-nitrosoguanidine and diethyl sulfate as the mutagen, and screened for mutant strains that were nutrient-deficient in tyrosine, aspartic acid, and were resistant to structural analogs, such as vitamin K3 and ethylthionine, and designed a simple, effective and efficient method to detect the mutation of tyrosine, aspartic acid, and the structural analogs, such as vitamin K3 and ethylthionine.

The mutant strains AGR0619 and AGR0610 were selected by screening tyrosine nutrient-deficient and mutant strains resistant to structural analogues of vitamin K3 and ethionine, and a simple, effective and rapid screening model was designed, and the mutant strains AGR0619 and AGR0610 were obtained, and the yields of CoQ10 reached 29.14 mg/L and 31.42 mg/L, respectively, which were l37.49% and l56.07% higher than that of the starting strains.

YoshiDa used Agrobacteriumtumefaciens Ky-3085 as the starting bacterium and mutated it with nitrosoguanidine to obtain the mutant strains AU-55 and M-37, which were resistant to erythromycin, ethylthionine, and vitamin K3, etc. AU-55 was cultured in a fermentation tank for 58 h, and the maximum yield was 180 mg/L; M-37 was cultured for 72 h before it could be used as the starting bacterial strain. The fermentation time of AU-55 was 58 h in the fermentation tank, and the maximum yield was 180 mg/L. M-37 was cultured for 72 h to reach this level, compared with AU-55, which has a shorter fermentation time, higher yield, and higher tolerance to the high concentration of CoQ10.

Actinomycin D (ActD) is a teratogen and carcinogen, its structure contains a planar phenoxazine ring and two cyclic pentapeptides, which can be inserted into DNA molecules and selectively inhibit transcription and protein synthesis. Therefore, ActD has strong cytotoxicity, and the addition of a certain amount of ActD to the culture medium will produce a stressful and toxic effect on the cells of the bacterium.COQ10 is a physiologically active substance that can enhance the immunity of the organism, and if we can obtain an ActD-resistant mutant strain after mutagenesis, it is possible to increase the amount of intracellular accumulation of physiologically active substances, such as COQ10, in the mutant strain.

Pan Chunmei[11] used RhizObiumradiObacter WSH2601 as the starting strain and used Actinomycin D resistance as the screening model for screening high COQ10-producing mutant strains, and obtained high COQ10-producing strains by combining mutagenesis with UV light and nitrosoguanidine. The COQ10 yield of the strain was optimized by shake flask experiments, and the final COQ10 yield reached 34 mg/L, which was 3.6 times higher than that of the strain before mutagenesis and optimization.

2.2 Construction of Genetically Engineered Bacteria

By utilizing molecular biology techniques, the key enzyme genes involved in COQ10 production are introduced into Escherichia coli through gene vectors, increasing in copy number and efficient expression of these genes, thereby enhancing the synthesis ability of COQ10. This is the fundamental approach for constructing fermentation strains for COQ10 production through genetic engineering.

The rate limiting step in the synthesis of COQ10 in microbial cells is the condensation reaction between the precursor of the benzoquinone ring, p-hydroxybenzoic acid (PHB), and the side chain structure, polyisoprene pyrophosphate (PPP). The enzyme catalyzing this reaction is p-hydroxybenzoic acid polyisoprene pyrophosphate transferase [12]. In E. coli, this enzyme is encoded by the ubiA gene and does not require high polymerization length specificity for long-chain polyisoprene pyrophosphate substrates (PPP). It has a relatively broad specificity for substrate recognition [13]; The formation of side chain polyisoprene pyrophosphate is determined by polyisoprene pyrophosphate synthase, which catalyzes the condensation of farnesyl pyrophosphate (FPP) and isoprenoid pyrophosphate (IPP) to form polyisoprene pyrophosphate with a certain degree of polymerization, thereby determining the type of coenzyme Q [14].

In E. coli, this enzyme is encoded by the isPB gene [15] and ultimately produces COQ8. If the isPB gene is inactivated and an exogenous polydecaisoprene pyrophosphate synthase gene is introduced, it is possible to construct a COQ10 biosynthesis system in Escherichia coli.

Zhang An[16] obtained the polydecylenepyrophosphate synthase gene (ddsA) from Gluconobacter oxytoca by PCR amplification and recovered the required fragments by enzymatic digestion. The gene was sequenced and found to be homologous to other isopentenyl pyrophosphate synthase genes (30%~50%), and then cloned into an expression vector, induced expression of the fusion vector, and the corresponding bands appeared on polyacrylamide gel electrophoresis (SDS-PAGE).

Fan Yi [17] selected 10 different E. coli as the receptor for the expression of ddsA, a poly(deca-isopentene pyrophosphate) synthase gene from Gluconobacter oxytoca. The product analysis confirmed that E. coli could express active poly(deca-isoprene pyrophosphate) synthase and then synthesize COQ10, and it was also found that the expression amount of ddsA in one of the strains of E. coli exceeded that of COQ8 in the wild-type, which demonstrated the possibility of using E. coli to produce COQ10 by large-scale fermentation.

Therefore, the selection of a suitable E. coli receptor is important for future research and industrial production. However, the increase of COQ10 production will be accompanied by the production of other coenzyme Q. Therefore, attention should be paid to the study of the enzymatic properties of polyisoprene pyrophosphate synthase and the modification of other related genes (e.g., the inactivation of the isPB gene and the enhancement of the expression of the ubiA gene, etc.), to construct a genetically engineered bacterium for the production of COQ10.

3.Optimization of COQ10 Production Conditions

In addition to the application of metabolic control theory to select high-yielding mutant strains or construct recombinant strains to improve the fermentation yield of COQ10, the optimization of fermentation conditions is also an effective and convenient way to improve the fermentation yield of COQ10. The optimization of fermentation conditions is also an effective and convenient way to improve the fermentation yield, which mainly includes the optimization of culture medium, culture conditions and the addition of prerequisite substances.

Liu Ling[18] extracted COQ10 from Cryptococcus yellows, and after analyzing the conditions of nitrogen source, carbon source, initial pH value, fermentation temperature, etc., the best fermentation conditions were obtained: carbon source of sucrose and glucose 1.25g/L each, nitrogen source of yeast paste and corn syrup 0.3g/L each, pH 6.5, temperature 28 ℃, inoculum volume of 5% and the volume of liquid in 500mL vials 50mL, growth factor of proteins, and growth factors of proteins and proteins, and the inoculum volume was 5%. The inoculum was 5%, and the volume of 500 mL vials was 50 mL, and the growth factor was protein hydrolysate. The fermentation was carried out at 28 ℃, the inoculum volume was 5%, the volume of 500 mL triangular bottle was 50 mL, and the growth factor was protein hydrolysis solution.

Wu Zufang[19] used Rhizobium radiodurans as the COQ10 producing strain, according to the importance of the factors affecting the fermentation effect and their interrelationships, the orthogonal test was carried out on the carbon source (glucose, sucrose, and compound), yeast paste, initial pH value and the amount of liquid loaded, and the final conditions of the fermentation were determined: the carbon source was a mixture of 1.5 g/100mL glucose and 2.5 g/100mL sucrose, yeast paste was 0.8 g/100mL, initial PH value was 0.8 g/100mL, the carbon source was 1.5 g/100mL glucose and 2.5 g/100mL sucrose, and yeast paste was 0.8 g/100mL. The initial pH was 7.0 and the volume of liquid in 500 mL flasks was 50 mL. The growth rate of the bacteria reached 13.8 g/L and the yield of COQ10 reached 22.7 mg/L, which were 34% and 53% higher than that before optimization, respectively, under the shaking flask fermentation conditions.

Yuan Jing[20] optimized the COQ10 medium and culture conditions of photosynthetic bacterium R. caPsulatus, and obtained the following results: yeast paste mass concentration of 3.13 g/L, ammonium sulfate 0.8 g/L, Mg2+0.64 g/L, Fe2+45 2mg/L, Mn2+18mg/L, CO2+16mg/L, initial pH value of 7.0, and the temperature of 30℃. Temperature 30℃. After 4 d of incubation, the mass concentration of COQ10 in the bacterium increased from 15.213 mg/L to 20.365 mg/L, and the yield increased by about 33.87%.

Wang Genhua[21] took RhizObium leguminOsarum as the research object, and studied the culture conditions affecting cell growth and COQ10 synthesis. The optimal growth conditions for the strain were pH 5.0, temperature 30℃, inoculum volume of 2%, 500mL triangular bottle containing 25mL of liquid, and incubation time of 24h.

Liu Ping [22] optimized the fermentation conditions of Saccharomyces cerevisiae and found that the optimal fermentation conditions were 15g/L glucose, 15g/L sucrose, 10g/L protein, 10g/L yeast, 10g/L yeast paste, 41.0g/L MgSO, 41.0g/L K2HPO, 41.0g/L KH2PO, 5.0 PH, 50mL inoculation, and 50mL inoculation in 250mL vials, with 24h incubation time. The inoculum volume was 10%, the incubation temperature was 28℃~30℃, the shaker speed was 200r/min, and the incubation time was 18h. The optimized COQ10 yield reached 26.85mg/L, and the cellular biomass reached 27.56g/L. Based on this study, we investigated the side-chain-supplied precursors (cannabinol) and quinone-ring-supplied precursors (hydroxybenzoic acid and COQ0) for the COQ10 and identified the suitable precursors for the fermentation of fission yeast in corn wine.  

The optimal fermentation conditions for the growth of fission yeast from corn wine and the high conversion of precursors to COQ10 were determined as follows: 28℃, 200r/min, 18h of incubation, 0.5g/L of cannabinol was added to continue the fermentation and incubation for 18h for the conversion of precursors, and the intracellular yield of COQ10 was up to 33.1mg/L, which was 91% higher than that of the control sample.

For the intracellular COQ10 production, the two precursors were added together, which was lower than that of cannabinol alone, while for the extracellular COQ10 production, the different additions of precursors did not have much effect on it. However, from the point of view of COQ10 yield per unit cell, when lycopene and COQ0 were added together, the COQ10 yield per unit cell reached 1.35mg/g, which was 117% higher than that of the blank control.

4.Separation and Purification

Since COQ10 is easily oxidized, it is necessary to avoid oxidative destruction of COQ10 during the isolation and extraction process. For example, the antioxidant pyrogallic acid should be added under alkaline conditions. Purification methods for saponification separation and solvent extraction separation are currently used.

4.1 Alcohol and Alkali Saponification Extraction Separation

The prepared fermented bacteria into a round-bottomed flask, add a certain proportion of pyrogallic gallic acid, stirring, and then add a certain amount of sodium hydroxide - ethanol solution, stirring, this time the bacterium into a black paste, and add n-alkane for reflux extraction, quickly cooled to room temperature. Extracted with petroleum ether several times, the extract should be washed with water to neutral, and then anhydrous sodium sulfate to remove water, the purpose is to make COQ10 and hydrocarbon solvents better miscible, to facilitate the subsequent processing.

Concentrate to a small volume, chromatography on silica gel adsorption column, wash with petroleum ether to remove impurities, then elute with ethyl ether-petroleum ether mixture, and distill the eluent under reduced pressure to obtain the yellow oily substance as anhydrous ethanol crystals, and orange-yellow orange crystals as coenzyme COQ10[23-24] . In the presence of ethanol, prolonged saponification will lead to the transposition of the methoxy group on the ring of COQ10 with the ethoxy group of ethanol, resulting in the formation of mono- or di-ethoxy derivatives, which cannot be detected in the product of COQ10, and to avoid the formation of these impurities, KOH can be used instead of NaOH and saponification with methanol [24].

4.2 Alkali Saponification Method of Extraction and Separation

The prepared fermented bacteria into a round-bottomed flask, add a certain amount of sodium hydroxide, heating reflux, rapid cooling, with petroleum ether, ether or ethane, and an isopropanol mixture of extraction. Then water washing, cold precipitation to remove impurities, silica gel column chromatography, the collection of COQ10-containing solution, and then concentrated, anhydrous ethanol crystals, you can get the crystallization of COQ10 [24-25].

4.3 Hydrocarbon Solvent Extraction and Separation from Dried or Freeze-Dried Materials

Direct extraction of COQ10 with petroleum ether, alkane, n-hexane, or n-heptane is more complete. Still, it must be repeated several times or shaken for several hours, and this method must ensure that the material is absolutely dry and finely crushed enough to allow the solvent to penetrate easily into the material. If the freeze-dried material has been frozen, it may absorb water in the open system at room temperature, thus affecting the efficiency of extraction, it is necessary to add 0.5% ~ 1% methanol to hydrocarbon solvents such as petroleum ether beforehand, and subsequent processing is the same as above. Compared with the method of saponification followed by extraction, the amount of extract obtained is less, but it has the advantage of not destroying COQ10 [26-28].

5. Analytical Identification

5.1 Visible Spectrophotometric Method

According to the alkaline conditions, ethyl cyanoacetate can replace the methoxy on the COQ10 molecule to produce the principle of blue, take a small amount of sample and COQ10 standard, respectively, add anhydrous ethanol, ethyl cyanoacetate and potassium hydroxide test solution, shaking well, see there is a blue reaction, the absorbance was measured at 620 nm, the standard curve was plotted COQ10, and the content of COQ10 in the samples was finally calculated. Finally, the content of COQ10 in the sample was calculated [27].

5.2 Ultraviolet Spectrophotometric Method

According to the COQ10 has a stable absorbance at 275nm, take the COQ10 standard sample dissolved in anhydrous ethanol, measure its absorbance at 275nm, make the standard curve of COQ10, and then calculate the content of COQ10 according to the absorbance of the sample. The ultraviolet absorption curve of the sample and the standard curve is generally consistent, but due to the purification of COQ10, repeated use of organic solvents, as well as the leaching of some impurities in the cell, so the observed ultraviolet absorption curve of the sample at 275nm there will be some deviation, there are some small stray peaks, so the determination of the content of COQ10 in this way will bring some errors. In this case, the crude extract can be further purified by thin-layer chromatography and combined with UV detection to determine the content of COQ10 [28].

5.3 High-performance Liquid Chromatography (HPLC) Analysis and Determination

The crude extract of the sample was further purified by thin-layer chromatography and then analyzed by high performance liquid chromatography (HPLC) to identify the purification effect [29]. At this time, the HPLC analysis showed that the impurities were few and did not interfere with the determination of COQ10. To further confirm the results, the standards and samples could be analyzed by HPLC at another wavelength by changing the detection wavelength. At the same time, according to the principle that there is no absorption peak in the reduced state of COQ10, a certain amount of sodium borohydride can be added to the sample to be tested, and if the absorption peaks detected just now disappear, it can be further confirmed that it is COQ10[28] , and the content of COQ10 can be obtained by calculating the area of the absorption peaks.

6.Prospect

At present, the price of COQ10 in the international market is relatively high, and China consumes more than 20t of COQ10 annually, most of which relies on imports, and there is a large gap in the domestic market, which is mainly due to the low isolation and purification yields of strains with low fermentation units. To obtain high-yielding strains, it is necessary to carry out a large number of mutation breeding research, the use of gene mutation and directed mutation to accelerate the search speed and the addition of precursors to promote biosynthesis to improve the content of COQ10, etc.

In recent years, the construction of COQ10-producing strains has been widely recognized by the Chinese government. In recent years, the construction of COQ10-producing recombinant strains has achieved certain results, but due to the complexity of the COQ10 synthesis pathway, the participating genes are many and scattered, to realize large-scale industrial production, further research is needed. In conclusion, to realize the industrial production of COQ10, it is necessary to start from various aspects, including the exploration of in vivo biosynthesis pathway, the selection and breeding of high-yielding strains, the optimization of fermentation conditions, the study of precursors and biosynthesis-promoting substances, the optimization of isolation and purification, and so on.

References:

[1] WU Zufang,et al. Progress of functional studies on coenzyme Q10[J]. Journal of Ningbo University(Science and Technology),2001 , 14(2):85-88.

[2] Yuan Yi. Extraction and purification of coenzyme Q10 from pig heart[J]. Journal of Anhui Agricultural University, 1997,24(2):200-203. [3] Zhao Meifa.

[3] Zhao Meifa. Coenzyme Q10 is an important prodrug[J]. Fine and Specialty Chemicals 2001, (22):35-40.

(22):35-40.

[4] GU Jue-Fen ,CHEN Guang-Ming ,LI Shu-Zhen. Synthesis of coenzyme Q10 by microbial transformation [J]. Pharmaceutical Progress,2001,25(6):339-343.

[5] MEGANATHAN R. Ubiquinone biosynthesis in microorganisms [J]. FEMS Microbiol Lett, 2001,203:l3l-l39.

[6] Liu K-S,Wu W-F,Han S-Q,et al. Selection and development of high-yielding strains of coenzyme Q10[J]. Journal of Shenyang Agricultural University,2005,36(1):89-92.

[7] ZHANG Yanjing , YUAN Qipeng , LIANG Hao. Study on the fermentation conditions of Coenzyme Q10-producing yeast[J]. Microbiology Bulletin,2003,30(2):65-69.

[8] OLSON E O, RUDNEY H. Biosynthesis of ubiquinone [J]. vitam Horm, 1983 ,40:1-43.

[9] YOSHIDA H, KOTANI Y, OcHIAI K, et al. production of u- biquinone-10 using bacteria[J]. J Gen Appl Microbiol,1998,44: 19-26.

[10] TEIZIL U. production of ubiquinone and bacteriochlorophy  Ⅱ a by Rhodobacter sphaeroides and Rhodobacter sulfidophilus [J]. J Fermen Bioeng, 1993 ,76: 191-194.

[11] PAN Chunmei, BUO Guocheng, CHEN Jian. Selection and optimization of fermentation conditions of Rhizobium radiobacter, a coenzyme Q10-producing bacterium[J]. Journal of Process Engineering,2004,4(5):451-456.

[12] SIBERT M, et al. Ubiquinone biosynthesis: cloning of the genes coding  for  chorismate  pyruvatelyase  and 4-hydroxybenzoate octaprenyl transferase from Escherichia coli   [J].  FEBS  Lett, 1992 , 307:347.

[13]  El  Hachimi  Z ,  SAMUEL  O ,  et al.  Biochemical  study  on  ubiquinone biosynthesis in Escherichia coli.1.Specificity of para- hydrabenzoate polyprenyl-transferase [J]. Biochimie, 1974,56: 1239-1247.

[14] OKADA K, et al. polyprenyl diphosphate synthase essentiallydefines the length of the side chain of ubiquinone [J]. Biochem Biophys Acta, 1996 ,1302: 217-223.

[15] ASAI KI, et al. The identification of Escherichia coli ispB(cel) gene encoding the octaprenyl diphosphate synthase [J]. Biochem Bioph Res co, 1994,202:340-345.

[16] ZHANG An, WU Haizhen, ZHOU Xuefeng, et al. Cloning of ddsA gene from Gluconobacter oxytoca and its expression in Escherichia coli [J]. Journal of East China University of Science and Technology,2002, (3):321-325

[17] FAN Yi, LIU Xinyi, CHEN Guohao. Expression of the ddsA gene of Gluconobacter oxytocin in different host bacteria of Escherichia coli[J]. Industrial Microbiology,2002,32(4):39-41.

[18] LIU Ling, LI Ge. Selection of optimal conditions for the production of coenzyme Q10 by fermentation of Cryptococcus yellows[J]. Journal of Dalian Fisheries Academy,2004, 19(3): 199-203.

[19] WU Zufang, JU Guocheng, CHEN Jian. Shake flask fermentation of Rhizobium radiodurans WSH2601 for the production of coenzyme Q10[J]. Journal of Wuxi University of Light Industry,2003,1(1):65-69.

[20] YUAN Jing, WEI Hong. Study on the fermentation conditions of coenzyme Q10 produced by photosynthetic bacteria[J]. Amino acids and bioresources,2003,25(2): 24-26.

[21] WANG Gen-Hua, QIAN He. Effects of fermentation conditions on cell growth and coenzyme Q10 synthesis in Rhizobium pea[J]. Journal of Wuxi University of Light Industry,2003, (3): 101-104.

[22] LIU Ping , QIU Weihua , ZHENG Ya'an , et al. Effects of precursor substances on coenzyme Q10 biosynthesis[J]. Food and Fermentation Industry,2005, (4): 1-5.

[23] WANG Chunlin. Extraction, isolation, and characterization of coenzyme Q10 from Chinese soybean[J]. Chinese Journal of Pharmaceutical Industry, 1996,27(3): 102-104.

[24] QIU Wei-Hua, LIU Ping, ZHONG Gui-Fang, et al. Extraction and determination of coenzyme Q10 in fission yeast from corn wine[J]. Food and Fermentation Industry,2004,30(11):31-35.

[25] Yunnan Zoological Research Institute, Jiangsu Taizhou Biopharmaceutical Factory. Isolation of coenzyme Q10 from pig heart residue for the preparation of cytochrome C[J]. Pharmaceutical Industry,1976,2: 22-25.

[26] Ouyang Pingkai, Hu Yonghong. Production and application of coenzyme Q10[J]. Progress of chemical industry,1994, (4:9-11).

(4):9-11.

[27] Wang Chunlin. Extraction, isolation, and characterization of coenzyme Q10 from Chinese soybeans[J]. Chinese Journal of Pharmaceutical Industry, 1996,27(3):102-104.

[28] WU Zufang, JU Guocheng, CHEN Jian. Separation, purification and quantitative analysis of coenzyme Q10 in fermentation broth[J]. Journal of Wuxi University of Light Industry, 2002,21 (4):420-423.

[29] YAMADA M K. The ubiquinone system in Hasegawaea japon- ica(Yukawet Maki)Yamada et Banno:A new method for identi- fyingubiquinone homologs from yeast cells [J]. IFORes comm, 1999 , 19:41-46.