J. Microbiol. Biotechnol. (2011), 21(5), 494502 doi: 10.4014/jmb.1012.

12017 First published online 9 April 2011

Coenzyme Q10 Production by Sphingomonas sp. ZUTE03 with Novel Precursors Isolated from Tobacco Waste in a Two-Phase Conversion System
Qiu, Lequan, Weijian Wang, Weihong Zhong*, Li Zhong, Jianjun Fang, Xuanzhen Li, Shijin Wu, and Jianmeng Chen
College of Biological and Environmental Engineering, Zhejiang University of Technology, Hangzhou 310032, China Received: December 13, 2010 / Revised: January 28, 2011 / Accepted: January 31, 2011

Coenzyme Q10 (CoQ10) is a widely used supplement in heart diseases treatment or antioxidative dietary. The microbial production of CoQ10 was enhanced by addition of solanesol and novel precursors recovered from waste tobacco. The novel precursors were separated by silica gel and identified as -linolenic acid (LNA) and butylated hydroxytoluene (BHT) based on the effect on CoQ10 production and GC-MS. The effects of novel precursors on CoQ10 production by Sphingomonas sp. ZUTE03 were further evaluated in a two-phase conversion system. The precursors combination of solanesol (70 mg/l) with BHT (30 mg/l) showed the best effect on the improvement of CoQ10 yield. A maximal CoQ10 productivity (9.5 mg l-1 h-1) was achieved after 8 h conversion, with a molar conversion rate of 92.6% and 92.4% on BHT and solanesol, respectively. The novel precursors, BHT and LNA in crude extracts from waste tobacco leaves, might become potential candidates for application in the industrial production of CoQ10 by microbes. Keywords: Coenzyme Q10, microbial conversion, tobacco waste, precursor, Sphingomonas sp. ZUTE03

Coenzyme Q10 (CoQ10) has been widely used in treating heart diseases for more than 30 years [12]. Additionally, CoQ10 shows a variety of physiological activities indicated for hypertension, brain vascular injury, anemia, muscle dystrophy, and alveolar pyorrhea. Recently, CoQ10 has been used as food supplements owing to its various physiological activities [12, 13]. Extensive attempts have been made to increase the production of CoQ10 to meet the growing demands of the pharmaceutical industry. CoQ10 can be produced by chemical synthesis [17], semi-chemical
*Corresponding author Phone: +86-571-88320739; Fax: +86-571-88320739; E-mail: whzhong@zjut.edu.cn

synthesis [15], and microbial fermentation [28]. However, economical production of CoQ10 by microbes is considered as the most viable approach and has attracted increasing attention of biochemical engineers [3, 4, 11]. The development of strain and the optimization of fermentation strategies and environmental parameters have resulted in yield improvement of CoQ10 [5-9, 21, 24].There also have been an increasing number of reports on the improvement of CoQ10 production by molecular biological methods [2, 14, 18, 19, 22, 23, 29-31]. However, the highest yield of CoQ10 production was achieved by Rhodopseudomonas spheroides with fed-batch process [21], suggesting further studies on metabolic engineering is required to improve CoQ10 biosynthesis. Meanwhile, both the selection of more effective precursors and the optimization of fermentation/ conversion conditions are still necessary to enhance CoQ10 production by microbes. It was widely reported that addition of precursors or regulators, such as para-hydroxybenzoic acid (PHB), solanesol, 4-hydroxybenzoic acid, methionine, L-phenylalanine, and tyrosine, could improve the microbial production of CoQ10 [10, 25]. Some precursors or stimulators are present in the natural plant biomass. For example, carrot juice and tomato juice could be utilized as the natural precursors to enhance the production of CoQ10 by Pseudomonas diminuta NCIM 2865 [1]. Tobacco biomass hydrolysate could also enhance the CoQ10 production by Rhodospirillum rubrum [26]. However, tobacco waste was the widely used material to recover solanesol owing to its high content of this traditional precursor for CoQ10 production [16]. Millions of tons of tobacco waste is produced by the tobacco industry in China per year, which has yet caused environmental problem. Therefore, it is valuable and necessary to study how to recycle such materials. The main strategies to recycle tobacco waste include the reconstitution of tobacco [33], composting the material [20], and the recovery of valuable substances.

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In the present study, we isolated two novel precursors of CoQ10 from waste tobacco leaves by chance. Their effects on CoQ10 production were evaluated in a water-organic solvent, two-phase system, which was designed as the bioconversion system of CoQ10 directly from precursors [34]. The bioconversion system might decrease the cost of downstream purification, because fewer other metabolites would be extracted into the organic phase. Meanwhile, such system is also equal to a kind of conversion-extraction couple process, which might contribute to enhancing the production of CoQ10. To our knowledge, this is the first report on CoQ10 production by Sphingomonas sp. ZUTE03, using novel precursors in a two-phase conversion system. MATERIALS AND METHODS
Chemicals Coenzyme Q (purity above 99%) was purchased from Wako Pure Chemical Industries (Osaka, Japan). Solanesol (purity above 95%) was purchased from Weifang Three Power Group Co. Ltd. (Shandong, China). Alpha-linolenic acid (purity above 80%) was purchased from Beijing Health Science and Technology Co. Ltd. (Beijing, China). Butylated hydroxytoluene (purity above 99.8%) was purchased from Hangzhou Wanjing Novel Material Co. Ltd. (Hangzhou, China). Other chemicals were purchased locally. Strain and Culture Conditions Sphingomonas sp. ZUTE03, which exhibited excellent coenzyme Q production ability, was isolated from the soil in the banks of the Qiantang River, Zhejiang Province, China. The strain has been deposited in the China Center for Type Culture Collection (CCTCC) at Wuhan University, Wuhan, China, under the accession number CCTCC M207084. The seed medium contained 20 g/l glucose, 10 g/l peptone, 10 g/l yeast extract, and 5 g/l NaCl. The fermentation medium contained 15 g/l glucose, 10 g/l (NH ) SO , 1 g/l yeast extract, 0.5 g/l KH PO , 1.5 g/l Na HPO , 0.5 g/l MgSO 7H O. The initial pH of the above medium was adjusted to 7.0 with 2 M NaOH and the medium was then autoclaved at 115 C for 30 min. Culture conditions: One loop of Sphingomonas sp. ZUTE03 from a slant was inoculated into 100 ml of seed medium in a 250 ml flask for 24 h incubation at 180 rpm and 28 C. Then, 5 ml of seed medium was inoculated into 150 ml of fermentation medium in a 500 ml flask. After 12 h cultivation at 180 rpm and 28 C, precursor samples were added into the broth to afford the final concentration 0.75 g/l and started a consequential 24 h conversion before the final biomass and CoQ yield were measured. Analytical Method The extraction of CoQ from cells and the biomass measurement were carried out according to reported methods [32]. The CoQ concentration was analyzed by high-performance liquid chromatography (HPLC) (Type SPD-10AVP; Shimadzu, Japan) equipped with a ZORBAX SB-C18 column (4.6250 mm; Agilent, USA). A mixture of methanol and hexane (83:17 by volume) was used as the mobile phase at a flow rate of 1.0 ml/min. The UV detector wavelength was 275 nm and the sampling quantity was 20 l.
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The concentration of solanesol was analyzed by HPLC (Type SPD-10AVP; Shimadzu, Japan) equipped with a Shim-pack HRCSIL column (4.6150 mm; Shimadzu, Japan). A mixture of hexane and isopropanol (98:2 by volume) was used as the mobile phase at a flow rate of 0.5 ml/min. The UV detector wavelength was 215 nm and the sampling quantity was 20 l. The concentration of butylated hydroxytoluene (BHT) was analyzed by HPLC (Type Agilent 1100, USA) equipped with a ZORBAX SBC18 column (4.6150 mm). Anhydrous ethyl alcohol (chromatographic grade) was used as the mobile phase at a flow rate of 1.0 ml/min, The UV detector wavelength was 215 nm and the sampling quantity was 20 l. Extraction of the Crude Solanesol from the Tobacco Waste Waste tobacco leaves (Hangzhou Liqun Environment Protecting Paper Co. Ltd., Hangzhou, China) were soaked in the distilled water for 10 h before filtration for removing water-soluble impurities. The wet material was dried at 100 C for 2 h and then grinded to pieces of the same size. About 120 g of treated waste tobacco leaves was immersed in 1,500 ml of petroleum ether and extracted for 10 h at 50 C in a 2 l three-neck bottle with stirring paddle. The organic solvent was collected by filtration and concentrated by a vacuum rotatory evaporator to obtain the crude solanesol. Further Separation of the Crude Solanesol and Effective Component Analysis The crude solanesol was saponified by KOH (16 M) at 50 C for 2 h. After dissolving in petroleum ether, the saponified crude solanesol was loaded to a silica gel column (460 cm, height of silica gel about 40 cm), and then slowly eluted by petroleum ether. A greencolor substance was first eluted and collected. Then subsequent colorless substance was eluted with a mixture of petroleum ether and acetone (9:1 by volume), and collected as 40 ml per tube. The collected eluent was identified by thin-layer chromatography (TLC) on silica gel plates (Type GF254; Qingdao Ocean Chemical Co. Ltd, China) with petroleum ether and ethyl acetate (4:1 by volume) as the developing solvent and colored by iodine spraying method. The fraction of eluent with the same Rf value on the silica gel plates was combined and concentrated. The effects of these concentrates on CoQ fermentation by Sphingomonas sp. ZUTE03 were evaluated. The most effective fractions to improve the CoQ production were further separated by silica gel column and analyzed by TLC, HPLC, and gas chromatography-mass spectrometry (Agilent 5975C GC-MS, USA). Effects of Possible Novel Precursors on CoQ Conversion by Sphingomonas sp. ZUTE03 in a Two-Phase Conversion System The results of the previous research [34] showed that coenzyme Q production of Sphingomonas sp. ZUTE03 in a water-organic solvent, two-phase conversion system was superior to the traditional microbial fermentation. Therefore, the two-phase conversion system was used in the subsequent study for the effects of possible novel precursors on CoQ conversion by Sphingomonas sp. ZUTE03. The two-phase conversion system for CoQ production was a batch-type cultivation and designed as follows. One loopful of strain ZUTE03 from a slant was inoculated into 100 ml of seed medium in a 250 ml flask. After 24 h incubation at 180 rpm and 28 C, cells were harvested by centrifugation at 4,000 rpm and 4 C for 10 min, and then suspended in the 25 ml aqueous phase, acetate buffer (0.2 M, pH=4.6), mixed
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with 25 ml of hexane (as the organic phase) in a 250 ml flask for the consequential bioconversion at 28 C and 180 rpm. The extract solvent was collected from the top layer of the broth as the analytical sample for CoQ yield. Single precursors (100 mg/l, LNA and BHT) and precursor combination (each 100 mg/l, solanesol and PHB, BHT and solanesol, LNA and BHT, PHB and LNA) were dissolved in 25 ml of hexane (as organic phase), respectively. Treatment with no precursors added served as the control. The solution was then mixed with 25 ml of acetate buffer (0.2 M, pH 4.6, as water phase) in a 500 ml flask to form the two-phase conversion system. Then, 170 mg DCW of cells was inoculated and 1 ml of soybean oil was added as a cell membrane permeability accelerant. After 8 h conversion at 28 C and 180 rpm, CoQ in the organic solvent was analyzed by HPLC to determine the most effective treatment with enhanced CoQ production. After the optimal precursor or precursor combination was selected, the effect of precursor concentration on the CoQ production was evaluated in the two-phase conversion system. To optimize the precursor concentration and to test the reciprocal effect between the two different precursors, a double-factor orthogonal experiment using L (3) was performed. Statistical Analysis Methodology Each treatment in the experiment was performed in triplicate. Software Origin 8.0 was used to draw the figures with error bars.
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RESULTS
Effect of Crude Solanesol on CoQ10 Production by Sphingomonas sp. ZUTE03 The purity of the crude solanesol, extracted from waste tobacco leaves was analyzed by HPLC. The results (Fig. 1A and 1B) showed that the solanesol content in the crude extracts was 14.43% (by weight). The crude and the standard solanesols were added to the fermentation medium at the beginning of cultivation to a final solanesol concentration of 100 mg/l, respectively. After 36 h culture, biomass and CoQ10 yield were determined. The results (Fig. 1C) showed that CoQ10 yield with the addition of crude solanesol (3.12 mg/l, 1.81 mg/g-DCW) was higher than that with the addition of standard solanesol (2.03 mg/l, 1.03 mg/g-DCW). Among all the treatments, the CoQ10 yield of control was the lowest (1.59 mg/l, 0.98 mg/g-DCW). The results suggested that something in crude solanesol improved CoQ10 production. Therefore, crude solanesol was separated with silica gel to isolate and identify the possible novel active components. Separation of the Crude Solanesol and Effects of Eluents on CoQ10 Production When the crude solanesol was separated with silica gel, one kind of pigment substance with green color was first eluted and collected. The subsequent colorless eluents were identified by TLC with iodine coloring by spraying method. The results (Fig. 2A) showed that there are no colorable substances in the eluents of tubes from #1 to #6. The Rf

Fig. 1. HPLC analysis of crude extract (A) and solanesol standard (B) and their effects on the CoQ yield by Sphingomonas sp. ZUTE03 in fermentation medium (C).

values of colored substances in tubes #8 to #11 were higher than that of solanesol, and a small amount of solanesol was simultaneously eluted. The main colorable substance in tubes #12 to #20 was solanesol, which gradually decreased in tubes #21 to #25. The Rf values of the main colorable substances in tubes #26 to #43 were lower than that of solanesol. To test the effects of different eluted fractions on CoQ10 fermentation, the eluents in tubes #21 to #25, the eluents in tubes #26 to #43, and pigment substance were combined and concentrated, respectively. Different concentrates were then added to the fermentation medium after 12 h culture. Biomass and CoQ10 yield were determined after 36 h of culture and the results are shown in Fig. 2B. The CoQ10 yield with the concentrate of tubes #26 to #43 as precursors was the highest (11.73 mg/l, 6.19 mg/g-DCW) among all the treatments, which was 6.53 times higher than that of the control, 5.72 times higher than that of

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concentrate from tubes #12 to #25, and 1.56 times higher than that of pigment substance. The above results indicate that more effective components might be present in the eluent in tubes #26 to #43 than saolanesol, which was regarded traditionally as the precursor for CoQ10 production in tobacco waste. Further Separation and Analysis of the Effective Components To further determine the effective component, the concentrate from tubes #26 to #43 was further separated on a silica gel column, identified by TLC, and colored with the iodine spraying method. The results (Fig. 3A) showed that the major colorable components were in tubes #12 to #17. Consequently, the eluents in tubes #12 to #17 were combined, concentrated, and analyzed by HPLC. The HPLC analysis (Fig. 3B) showed that there were two main components in the concentrate, whose retention times were 6.488 min and 9.715 min, respectively. GC-MS analysis showed that the concentrate contained two main kinds of substances (Fig. 3C and 3D), namely LNA, with a structure similar to

solanesol, and BHT, with a benzene ring structure. Based on their special structure, LNA and BHT would be the possible precursors of microbial CoQ10 production, which have never been reported before. Effects of Novel Precursors on CoQ10 Conversion by Sphingomonas sp. ZUTE03 Owing to the advantages like less product loss, lower cost of downstream purification, and higher efficiency [27], over the traditional microbial fermentation in a complex medium and chemical synthesis, a two-phase microbial conversion system was designed to evaluate the effects of novel precursors on the conversion of CoQ10 by Sphingomonas sp. ZUTE03. The results of the previous research [34] showed that the CoQ10 yield with the combination of solanesol and PHB as precursors was much better than with solanesol or PHB alone. In this study, the results (Fig. 4) also showed that CoQ10 yield in precursors combination treatment was superior to that of precursor alone treatment, and among all the treatments, the CoQ10 yield of solanesol-BHT combination treatment was the highest (72.05 mg/l,

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Fig. 2. TLC identification of the eluent of crude extract separated by silicon column (A) and the effects of the different eluents on CoQ yield by Sphingomonas sp. ZUTE03 in fermentation medium (B) (CK: solanesol standard; 1-43: eluents of tubes #1 to #43).

Fig. 4. Effects of precursors on the CoQ production by Sphingomonas sp. ZUTE03 in a two-phase conversion system.

41.81 mg/g-DCW). The novel precursors combination (i.e., BHT and LNA) was superior to the traditional precursors combination) (i.e., solanesol and PHB). Additionally, the CoQ10 yield of single novel precursor, BHT or LNA, was at a same level as the combination of solanesol and PHB. The results also indicated that both BHT and LNA as precursors were more effective than solanesol or PHB. However, the combination of solanesol and BHT favored CoQ10 production the most. Furthermore, BHT, as an antioxidant capable of preventing the oxidation of CoQ10, is much cheaper than PHB. Therefore, solanesol and BHT were selected as the most effective precursors in the further study. Effects of Concentrations of BHT and Solanesol on CoQ10 Conversion To detect the effects of BHT concentration on CoQ10 production, 20, 30, 40, 50, or 60 mg/l BHT was added with 100 mg/l solanesol, respectively, at the beginning of culture for conversion. The results showed that the CoQ10 yield of 30 mg/l BHT treatment was the highest (74.52 mg/l, Fig. 5A) with the highest conversion ratio of BHT and solanesol (92.15% and 90.31%, respectively, Fig. 5B), suggesting that 30 mg/l was the optimal BHT concentration with a solanesol concentration of 100 mg/l. Based on the above results, 60, 70, 80, 90, or 100 mg/l solanesol with 30 mg/l BHT was added respectively at the beginning of culture to evaluate the effects of BHT concentration on the CoQ10 conversion. The results showed that when 70 mg/l solanesol was added, CoQ10 yield was the highest (75.81 mg/l, Fig. 6A) with the highest conversion ratio of BHT and solanesol (93.19% and 92.28%, respectively, Fig. 6B). It suggested that 70 mg/l solanesol with 30 mg/l BHT might be the optimal concentration for CoQ10 conversion by Sphingomonas sp. ZUTE03 in the two-phase conversion system.

Fig. 3. Further separation by silicon column and identification of the eluent with lower R than solanesol (in tubes #26 to #43 in Fig. 2A) by TLC (A), HPLC (B), and GC-MS (C, D) (CK: solanesol standard; 1-24: further separated eluent tubes).
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In addition to CoQ10 yield and molar conversion rate, the residual precursor should be taken into consideration simultaneously to evaluate the process condition of CoQ10 conversion from precursors. Therefore, the optimization of precursor concentration for CoQ10 production was conducted based on CoQ10 yield, molar conversion rate of precursor, and the residual precursor, simultaneously. Meanwhile, the reciprocal effect between solanesol and BHT should also be evaluated. In the present study, a double-factor orthogonal experiment using L9 (3)3 was designed to optimize precursor concentration and evaluate the effect of the reciprocal effect between solanesol and BHT on the CoQ10 yield. The results (Table 1) indicated that both solanesol and BHT had stronger effects on the CoQ10 yield (larger margin, RA, B, C of solanesol = 5.5, 2.1, 2.7; RA, B, C of BHT = 3.1, 2.7, 3.1) than the reciprocal effect between solanesol and BHT (smaller margin, RA, B, C = 1.8, 0.5, 0.6). Thus, the optimal additional concentrations could be determined by the k

values of solanesol and BHT, which were 70 and 30 mg/l, respectively (Table 1). Under the optimal concentration, the maximal CoQ10 yield reached 76.1 mg/l after 8 h conversion, with a molar conversion rate of 92.6% (on BHT) and 92.4% (on solanesol), respectively. It was also concluded that the maximal volumetric productivity of Sphingomonas sp. ZUTE03 in the two-phase conversion system could reach 9.5 mg l-1 h-1.

DISCUSSION
The exciting finding in the present study was that two novel precursors, identified as LNA and BHT, were isolated from tobacco waste. Both LNA and BHT were more effective than solanesol in improvement of the CoQ10 yield by Sphingomonas sp. ZUTE03. Moreover, the addition of precursors combinations showed more positive effect on

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Fig. 5. Effects of BHT concentration on the CoQ yield (A) and molar conversion rate of CoQ (B) by Sphingomonas sp. ZUTE03 in a two-phase conversion system.
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Fig. 6. Effects of solanesol concentration on the CoQ yield (A) and molar conversion rate of CoQ (B) by Sphingomonas sp. ZUTE03 in a two-phase conversion system.

Table 1. Orthogonal test design by L (3) and results.

3 9

1 2 3 4 5 6 7 8 9 A B

65(1) 65(1) 65(1) 70(2) 70(2) 70(2) 75(3) 75(3) 75(3) C D E A B

25(1) 30(2) 35(3) 25(1) 30(2) 35(3) 25(1) 30(2) 35(3) C D E A B

(1) (2) (3) (2) (3) (1) (3) (1) (2) C D E

66.8 69.6 69.1 70.4 76.1 74.9 73.9 74.6 73.6

87.9 89.8 89.6 89.2 92.6 91.8 88.8 91.5 90.8

86.8 88.9 88.6 88.4 92.4 91.5 87.8 91.2 90.4

5.4 10.2 15.4 5.9 9.2 14.3 3.6 9.2 14.4

9.0 7.9 7.9 11.6 9.7 10.2 13.8 15.2 15.4

K1 205.5 267.3 264.3 31.0 24.8 211.1 265.9 263.0 14.9 34.4 216.3 271.2 269.5 28.9 34.4 K2 221.4 273.6 272.3 29.6 31.5 220.6 273.9 272.5 28.6 32.8 213.6 269.8 267.7 30.5 34.9 K3 222.1 271.1 269.4 27.2 44.4 217.6 272.2 270.5 44.1 33.5 219.1 271.0 268.8 28.2 31.4 k1 k2 k3 R
b a

68.5 73.8 74.0 5.5

89.1 91.2 90.4 2.1

88.1 10.3 90.8 89.8 2.7

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70.4 73.5 72.5 3.1

88.6 91.3 90.7 2.7

87.7 90.8

5.0 11.5 9.5 10.9

72.1 71.2 73.0 1.8

90.4 89.9 90.3 0.5

89.8

9.6 11.5

9.9 10.5 9.1 14.8 1.2 6.5

89.2 10.2 11.6 89.6 0.6 9.4 10.5 0.8 1.1

90.2 14.7 11.2 3.1 9.7 0.6

Reciprocal effect between solanesol and BHT. Molar conversion rate: CoQ quantity (calculated as molar number) / reduced quantity of precursor (calculated as molar number). K, Sum A, B, C, D, E of different factors at the same level. k, The average of K. R, The difference between the maximum and minimum k values.

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Residue Residue BHT, solanesol, (D) (E) mg/l mg/l

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CoQ10 production than single precursor, and the combinational addition of solanesol and BHT improved the CoQ10 yield most significantly. Based on the special structure of LNA and BHT (i.e., a structure similar to solanesol, and a benzene ring structure similar to PHB), LNA and BHT would be the potential precursors of microbial CoQ10 production, which have never been reported before. Usually, it could be proved by radioactive labeling experimental data that they are exactly the precursor. However, in the two-phase system, BHT and LNA were the only chemicals with benzene-ring and the side chain similar to the structure of CoQ10, respectively. Thus, the most possible usage of BHT or LNA is as a precursor of CoQ10. Specifically, we found that the maximal CoQ10 yield reached 76.1 mg/l after 8 h conversion, with a nearly equal molar conversion rate of 92.6% (on BHT) and 92.4% (on solanesol), respectively. It suggested that they possibly incorporated in the CoQ10 structure with a proportion of 1:1. However, not all the used compounds were incorporated into CoQ10, some of which (about 8%) might be utilized as substrates for cell basic metabolism or other necessary reaction to support the conversion of BHT and solanesol to CoQ10. So far, several precursors such as solanesol, PHB, methionine, L-phenylalanine, and tyrosine have been used in CoQ10 synthesis. However, most attention was paid on solanesol and PHB, because the chemical structure of solanesol was similar to the CoQ10 side chain (isoprene pyrophosphate), and PHB could be used as a precursor of the quinone ring of CoQ10. Solanesol is usually regarded as the main precursor recovered from waste tobacco leaves owing to its high content in tobacco leaves [16]. However, we found that there were at least another two novel precursors in crude extracts from waste tobacco leaves, namely BHT and LNA, which might provide the benzene ring structure and side chain similar to the solanesol, respectively, for CoQ10 synthesis. The CoQ10 yield in the present study seemed to be higher than that in previous report about precursor effect on CoQ10 production, probably either due to the high efficiency of novel precursors or the two-phase process [1, 16, 26]. Moreover, the maximal volumetric productivity of Sphingomonas sp. ZUTE03 in the two-phase conversion system (9.5 mg l-1 h-1) was higher than that of R. spheroides with fed-batch process (5.1 mg l-1 h-1), although the latter showed the highest CoQ10 yield (770 mg/l) among all the previous literature [4, 21]. Therefore, the novel precursors, BHT and LNA in crude extracts from waste tobacco leaves, might become the potential candidates for application in industrial production of CoQ10 by microbes. However, it is still necessary to increase the CoQ10 yield and decrease the residual precursor concentration. We will design a repeat or continuous bioreactor for CoQ10 bioconversion in a two-phase system in our future study,

aimed at recycling of residual precursors. In addition, the process optimization of economical preparation of novel precursors from waste tobacco is also necessary and would be favorable to its potential application in the more economical microbial production of CoQ10.

Acknowledgment
This study was supported by the Science and Technology Department of Zhejiang Province of P.R. China under Grant No. 2007C23035, for which the authors are grateful. REFERENCES
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12. Katagiri, T. 1996. Appraisal of coenzyme Q as a drug for the management of cardiac failure. Kiso To Rinshyo 30: 1559-1567. 13. Ken, S., W. Masanori, S. Yoshito, I. Akihiro, and N. Napavarn. 2005. Applications of photosynthetic bacteria for medical fields. J. Biosci. Bioeng. 100: 481-488. 14. Kim, S. J., M. D. Kim, J. H. Choi, S. Y. Kim, Y. W. Ryu, and J. H. Seo. 2006. Amplification of 1-deoxy-D-xyluose 5-phosphate (DXP) synthase level increases coenzyme Q production in recombinant Escherichia coli. Appl. Microbiol. Biotechnol. 72: 982-985. 15. Lipshutz, B. H., P. Mollard, S. S. Pfeiffer, and W. Chrisman. 2002. A short, highly efficient synthesis of coenzyme Q . J. Am. Chem. Soc. 124: 14282-14283. 16. Machado, P. A., H. Fu, R. J. Kratochvil, Y. H. Yuan, T. S. Hahm, C. M. Sabliov, C. I. Wei, and Y. M. Lo. 2010. Recovery of solanesol from tobacco as a value-added byproduct for alternative applications. Bioresour. Technol. 101: 1091-1096. 17. Negishi, E., S. Y. Lou, C. Xu, and S. Huo. 2002. A novel, highly selective, and general methodology for the synthesis of 1,5-diene-containing oligoisoprenoids of all possible geometrical combinations exemplified by an iterative and convergent synthesis of coenzyme Q . Org. Lett. 4: 261-264. 18. Okada, K., T. Kainou, K. Tanaka, T. Nakagawa, H. Matsuda, and M. Kawamukai. 1998. Molecular cloning and mutational analysis of the ddsA gene encoding decaprenyl diphosphate synthase from Gluconobacter suboxydans. Eur. J. Biochem. 255: 52-59. 19. Park, Y. C., S. J. Kim, J. H. Choi, W. H. Lee, K. M. Park, K. Mokoto, Y. W. Ryu, and J. H. Seo. 2005. Batch and fed-batch production of coenzyme Q in recombinant Escherichia coli containing the decaprenyl diphosphate synthase gene from Gluconobacter suboxydans. Appl. Microbiol. Biotechnol. 67: 192-196. 20. Piotrowska-Cyplik, A., A. P. Olejnik, J. D. Cyplik, and Z. Czarnecki. 2009. The kinetics of nicotine degradation, enzyme activities and genotoxic potential in the characterization of tobacco waste composting. Bioresour. Technol. 100: 5037-5044. 21. Sakato, K., H. Tanaka, S. Shibata, and Y. Kuratsu. 1992. Agitation aeration studies on coenzyme-Q10 production using Rhodopseudomonas sphaeroides. Biotechnol. Appl. Biochem. 16: 19-28. 22. Seo, M. J., E. M. Im, J. Y. Nam, and S. O. Kim. 2007. Increase of CoQ10 production level by the coexpression of decaprenyl diphosphate synthase and 1-deoxy-D-xylulose 5-phosphate synthase isolated from Rhizobium radiobacter ATCC 4718 in recombinant Escherichia coli. J. Microbiol. Biotechnol. 17: 1045-1048.
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01

MICOBIAL PRODUCTION OF COQ

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NOVEL PRECURSORS

502