coenzyme q10 production by sphingomonas sp
TRANSCRIPT
J. Microbiol. Biotechnol. (2011), 21(5), 494–502doi: 10.4014/jmb.1012.12017First published online 9 April 2011
Coenzyme Q10 Production by Sphingomonas sp. ZUTE03 with NovelPrecursors Isolated from Tobacco Waste in a Two-Phase Conversion System
Qiu, Lequan, Weijian Wang, Weihong Zhong*, Li Zhong, Jianjun Fang, Xuanzhen Li, Shijin Wu, andJianmeng 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 α-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
precursor’s 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
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.
*Corresponding authorPhone: +86-571-88320739; Fax: +86-571-88320739;E-mail: [email protected]
495 Qiu et al.
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 Q10 (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
Q10 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 (NH4)2SO4, 1 g/l yeast extract, 0.5 g/l KH2PO4,
1.5 g/l Na2HPO4, 0.5 g/l MgSO4·7H2O. The initial pH of the above
medium was adjusted to 7.0 with 2 M NaOH and the medium was
then autoclaved at 115oC 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 28oC. 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 28oC, 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 CoQ10 yield were measured.
Analytical Method
The extraction of CoQ10 from cells and the biomass measurement
were carried out according to reported methods [32].
The CoQ10 concentration was analyzed by high-performance liquid
chromatography (HPLC) (Type SPD-10AVP; Shimadzu, Japan)
equipped with a ZORBAX SB-C18 column (4.6×250 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 µl.
The concentration of solanesol was analyzed by HPLC (Type
SPD-10AVP; Shimadzu, Japan) equipped with a Shim-pack HRC-
SIL column (4.6×150 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 µl.
The concentration of butylated hydroxytoluene (BHT) was analyzed
by HPLC (Type Agilent 1100, USA) equipped with a ZORBAX SB-
C18 column (4.6×150 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 µl.
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 100oC 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
50oC 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 50oC for 2 h.
After dissolving in petroleum ether, the saponified crude solanesol
was loaded to a silica gel column (4×60 cm, height of silica gel
about 40 cm), and then slowly eluted by petroleum ether. A green-
color 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 CoQ10 fermentation by Sphingomonas sp. ZUTE03
were evaluated. The most effective fractions to improve the CoQ10
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 CoQ10 Conversion by
Sphingomonas sp. ZUTE03 in a Two-Phase Conversion System
The results of the previous research [34] showed that coenzyme Q10
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 CoQ10 conversion by Sphingomonas sp. ZUTE03. The two-phase
conversion system for CoQ10 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 28oC, cells were harvested by
centrifugation at 4,000 rpm and 4oC for 10 min, and then suspended
in the 25 ml aqueous phase, acetate buffer (0.2 M, pH=4.6), mixed
MICOBIAL PRODUCTION OF COQ10 WITH NOVEL PRECURSORS 496
with 25 ml of hexane (as the organic phase) in a 250 ml flask for
the consequential bioconversion at 28oC and 180 rpm. The extract
solvent was collected from the top layer of the broth as the analytical
sample for CoQ10 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 28oC and 180 rpm,
CoQ10 in the organic solvent was analyzed by HPLC to determine
the most effective treatment with enhanced CoQ10 production.
After the optimal precursor or precursor combination was selected,
the effect of precursor concentration on the CoQ10 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
L9 (3)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.
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
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
Fig. 1. HPLC analysis of crude extract (A) and solanesol standard(B) and their effects on the CoQ10 yield by Sphingomonas sp.ZUTE03 in fermentation medium (C).
497 Qiu et al.
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,
Fig. 2. TLC identification of the eluent of crude extract separated by silicon column (A) and the effects of the different eluents on CoQ10
yield by Sphingomonas sp. ZUTE03 in fermentation medium (B) (CK: solanesol standard; 1-43: eluents of tubes #1 to #43).
MICOBIAL PRODUCTION OF COQ10 WITH NOVEL PRECURSORS 498
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 ofthe eluent with lower Rf than solanesol (in tubes #26 to #43 inFig. 2A) by TLC (A), HPLC (B), and GC-MS (C, D) (CK:solanesol standard; 1-24: further separated eluent tubes).
Fig. 4. Effects of precursors on the CoQ10 production bySphingomonas sp. ZUTE03 in a two-phase conversion system.
499 Qiu et al.
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
Fig. 5. Effects of BHT concentration on the CoQ10 yield (A) andmolar conversion rate of CoQ10 (B) by Sphingomonas sp.ZUTE03 in a two-phase conversion system.
Fig. 6. Effects of solanesol concentration on the CoQ10 yield (A)and molar conversion rate of CoQ10 (B) by Sphingomonas sp.ZUTE03 in a two-phase conversion system.
COENZYME Q
10 P
RODUCTIO
N BY
SPHINGOMONAS S
P. ZU
TE
03 W
ITH
NOVEL P
RECURSORS I
SOLATED
FROM
TOBACCO
WASTE
500
Table 1. Orthogonal test design by L9(3)3 and results.
Sample Solanesol, mg/l (level) BHT, mg/l (level) Solanesol/BHTa (level)CoQ10 yield,
(A) mg/l
Molar conversion
rateb,
CoQ10/BHT, (B) %
Molar conversion
rate,CoQ10/Sol
(C) %
ResidueBHT, (D)mg/l
Residue solanesol,(E) mg/l
1
2
3
4
5
6
7
8
9
65(1)
65(1)
65(1)
70(2)
70(2)
70(2)
75(3)
75(3)
75(3)
25(1)
30(2)
35(3)
25(1)
30(2)
35(3)
25(1)
30(2)
35(3)
(1)
(2)
(3)
(2)
(3)
(1)
(3)
(1)
(2)
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
A B C D E A B C D E A B C D E
K1
K2
K3
k1
k2
k3
R
205.5
221.4
222.1
68.5
73.8
74.0
5.5
267.3
273.6
271.1
89.1
91.2
90.4
2.1
264.3
272.3
269.4
88.1
90.8
89.8
2.7
31.0
29.6
27.2
10.3
9.9
9.1
1.2
24.8
31.5
44.4
8.3
10.5
14.8
6.5
211.1
220.6
217.6
70.4
73.5
72.5
3.1
265.9
273.9
272.2
88.6
91.3
90.7
2.7
263.0
272.5
270.5
87.7
90.8
90.2
3.1
14.9
28.6
44.1
5.0
9.5
14.7
9.7
34.4
32.8
33.5
11.5
10.9
11.2
0.6
216.3
213.6
219.1
72.1
71.2
73.0
1.8
271.2
269.8
271.0
90.4
89.9
90.3
0.5
269.5
267.7
268.8
89.8
89.2
89.6
0.6
28.9
30.5
28.2
9.6
10.2
9.4
0.8
34.4
34.9
31.4
11.5
11.6
10.5
1.1
aReciprocal effect between solanesol and BHT.bMolar conversion rate: CoQ10 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.
501 Qiu et al.
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.
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