Method for producing coenzyme Q.sub.10

Coenzyme Q.sub.10 can be produced extremely quickly in exceedingly high yield in a simple industrial process by cultivating a microorganism of genus Aureobasidium or Trichosporon in a culture medium containing a large quantity of p-hydroxy benzoic acid.

BACKGROUND OF THE INVENTION 
The present invention relates to a method for producing coenzyme Q.sub.10. 
Recently, coenzyme Q.sub.10 has been interested as a raw material for 
medicines for curing various diseases such as heart diseases, 
hypertension, tumor etc. and can be produced by various microorganisms. 
For example, Journal of Fermentation Technology, 47, p 553 (1969) 
discloses a method of improving productivity of coenzyme Q by adding as a 
component of culture medium p-hydroxybenzoic acid (abbreviated POBA) which 
is a precursor in a biosynthetic pathway of coenzyme Q.sub.10. However, 
the method has a defect that the produced coenzyme Q is Q.sub.9 and not 
Q.sub.10. Coenzyme Q.sub.9 is substantially inactive to human body and 
hence it does not exhibit the aforementioned desired pharmaceutical 
effects. 
Japanese patent application publication No. 20,396/72 describes a method 
for producing coenzyme Q by cultivating a yeast, genus Candida capable of 
producing coenzyme Q in a culture medium containing n-alkane and POBA or 
POBA and acetic acid or its salt. However, production of coenzyme Q.sub.10 
is not disclosed and production of Q.sub.9 or Q.sub.7 as disclosed is 
limited due to the feature of using a yeast, genus Candida and to the fact 
that POBA has generally such a strong toxic property e.g. bactericidal 
property to microorganisms that it can be used as a food-preservative so 
that its addition is restricted to 5 mg/l at the most. 
Japanese patent application publication No. 19,034/76 describes a method 
for producing coenzyme Q.sub.10 by cultivating a microorganism of genus 
Alcaligenes, Trichosporon or Aureobasidium in a nutrient culture medium. 
However, the method has deficiencies of necessitating a pretreatment step 
of steam sterilizing the culture medium and thus necessitating a 
complicated production process. 
SUMMARY OF THE INVENTION 
An object of the present invention is to provide a method for producing 
coenzyme Q.sub.10 in high yield in a culture medium containing a large 
quantity of POBA. 
Another object of the present invention is to provide a method for 
producing coenzyme Q.sub.10 quickly in a culture medium containing a large 
quantity of POBA. 
Still another object of the present invention is to provide a method for 
producing coenzyme Q.sub.10 in a simple industrial process in a culture 
medium containing a large quantity of POBA. 
To achieve the above objects, the present invention provides a method for 
producing coenzmye Q.sub.10 wherein coenzyme Q.sub.10 is produced by 
cultivating a microorganism of genus Aureobasidium orTrichosporon in a 
culture medium containing a large quantity of POBA. According to the 
method of the present invention, the culture medium can also contain 
ethanol as a main carbon source in addition to POBA.

DETAILED EXPLANATION OF THE INVENTION 
We have made various experiments and studies on relations between nutrient 
culture media and microorganisms for producing coenzyme Q.sub.10, 
particularly of genera Aureobasidium and Trichosporon, leading to a 
finding that microorganisms of genera Aureobasidium and Trichosporon do 
not die even when POBA is added in such an amount that exceeds far beyond 
generally used. To the contrary, the productivity of coenzyme Q.sub.10 is 
increased. It is quite surprising that microorganisms of genera 
Aureobasidium and Trichosporon can be tolerant to a high concentration of 
POBA, make growth and produce coenzyme Q.sub.10 in exceedingly high yield. 
Accordingly, in an aspect of the present invention, the present invention 
produces coenzyme Q.sub.10 by cultivating a microorganism of genus 
Aureobasidium or Trichosporon in a culture medium wherein a large quantity 
of POBA is added to high concentration. 
We have also found out that Aureobasidium sp. No. 14 strain which we 
deposited to Fermentation Research Institute (Fermentation Research 
Institute, Agency of Industrial Science and Technology, Ministry of 
International Trade and Industry) on Aug. 23, 1977 with deposit No. 4,197 
is particularly suitable as a microorganism of genus Aureobasidium. With 
regard to this finding, we reported orally in the Annual Meeting of the 
Society of Japanese Agricultural Chemistry held in 1977 that the 
Aureobasidium sp. No. 14 strain exhibits strong tolerance to phenols and 
can utilize POBA as a sole carbon source, and filed a patent application 
as Japanese patent application No. 111,874/77. However, the oral report 
and the Japanese application disclosed merely assimilation or oxidization 
of phenols and growth of the Aureobasidium sp. No. 14 strain and did not 
disclose production of coenzyme Q.sub.10. 
We have found out also that Trichosporon sp. WY 2-2 strain which we 
deposited to Fermentation Research Institute on May 1, 1980 with deposit 
No. 5,500 is particularly suitable as a microorganism of genus 
Trichosporon. 
We have found out also that POBA is preferably added in a concentration of 
about 1,000-4,000 .mu.g/ml to culture medium at a temperature of about 
20.degree.-37.degree. C. when microorganism of genus Aureobasidium is 
used. If the concentration is less than about 1,000 .mu.g/ml, the effect 
of addition can not be exhibited sufficiently to increase the production 
of coenzyme Q.sub.10, while if the concentration is larger than about 
4,000 .mu.g/ml, the bactericidal property of POBA is exhibited so much and 
the growth is suppressed so that the growth rate decreases. A 
concentration of about 1,500-3,500 .mu.g/ml is particularly preferable. As 
for a microorganism of genus Trichosporon, we found out that POBA is 
preferably added in a concentration of about 250-1,000 .mu.g/ml to culture 
medium at a temperature of about 20.degree.-37.degree. C. If the 
concentration is less than about 250 .mu.g/ml, the production of coenzyme 
Q.sub.10 decreases, while if the concentration is larger than about 1,000 
.mu.g/ml, the bactericidal property of POBA is exhibited so much and the 
growth rate decreases. A concentration of about 400-800 .mu.g/ml is 
particularly preferable. 
According to the present invention, any carbon source can be used as far as 
the microorganisms concerning the present invention can assimilate. For 
instance, use is made of carbohydrates such as starch etc.; alcohols such 
as glycerol, propanol etc.; sugars such as glucose, sucrose, molasses 
etc.; hydrocarbons such as aliphatic and aromatic hydrocarbons; or organic 
acids such as palmitic acid, fumalic acid etc. 
We have found out also that coenzyme Q.sub.10 can be produced not only in 
extremely short period of time but also in exceedingly high yield, if 
ethanol is used as a carbon source in addition to POBA. 
Accordingly, in another aspect of the present invention, the method of the 
present invention produces coenzyme Q.sub.10 by cultivating a 
microorganism of genus Aureobasidium or Trichosporon in a culture medium 
containing ethanol as a main carbon source in addition to a large quantity 
of POBA. 
It is quite surprising that, if ethanol is added to the culture medium, the 
growth rate increases remarkably to about twice as much of usual so that 
coenzyme Q.sub.10 can be produced extremely quickly despite the 
bactericidal property of ethanol. 
The concentration of ethanol to be added is about 0.5-4.0% (V/V) to the 
culture medium. If the concentration is less than about 0.5% (V/V), the 
amount of the cells produced becomes less. If the concentration is larger 
than about 4.0% (V/V), the bactericidal property of ethanol is exhibited 
so much and the growth is suppressed to decrease the growth rate. A 
concentration of about 1.0-3.0% (V/V) is particularly preferable. 
In the present invention, the usual nitrogen source, vitamines, inorganic 
salts etc. generally used can be added in usual amounts to the culture 
medium. 
Preferable cultivation conditions are a pH of about 2-8, a temperature of 
about 20.degree.-37.degree. C., the cultivation period of about 20-80 hrs. 
and an aerobic atmosphere. 
If pH value varies noticeably during cultivation to suppress the growth of 
the cells, pH is preferably adjusted by addition of acidic or alkaline 
material. 
DESCRIPTION OF THE PREFERRED EMBODIMENTS 
Hereinafter, the present invention will be explained in more detail with 
reference to preferred embodiments which however should not be construed 
as limitations of the scope of the present invention. 
In the succeeding examples, all quantities of materials are shown by gram 
unit, unless otherwise specified. 
EXAMPLE 1 
In this Example, Aureobasidium sp. No. 14 strain is used for producing 
coenzyme Q.sub.10. 
Inoculum cells are cultivated by the MPY medium which has the following 
composition. 
Composition of the MPY medium: 
Malt extract broth: 30 
Peptone: 5 
Yeast extract: 0.1 
Tap water: 1 l 
The culture medium used for pre-culture and main culture has the following 
composition. 
Composition of culture medium used for pre-culture and main culture: 
NH.sub.4 NO.sub.3 : 5.0 
KH.sub.2 PO.sub.4 : 2.5 
MgSO.sub.4.7H.sub.2 O: 1.0 
NaCl: 0.1 
Yeast extract: 0.1 
CaCl.sub.2.2H.sub.2 O: 0.01 
FeCl.sub.3.6H.sub.2 O: 0.01 
C.sub.2 H.sub.5 OH: 20 ml 
POBA: 
0 (pre-culture) 
0-2,000 .mu.g/ml (main culture) 
Tap water: 980 ml 
pH: 5.0 
Cultivations are effected as follows. 
A loopful cells from the young slant culture is inoculated to the MPY 
medium 10 ml, and cultivated at 30.degree. C. for 7 days to obtain 
inoculum cells. 
A drop of the inoculum cell suspension is inoculated to 200 ml of the 
above-mentioned pre-culture medium not containing POBA and subsequently 
subjected to shaking cultivation at 30.degree. C. for 48 hrs. to yield a 
pre-culture broth. 
The pre-culture broth is added to 800 ml of the aforementioned main culture 
medium containing POBA in a concentration of 0-2,000 .mu.g/ml in a volume 
ratio of 1:4 to a total volume of 1 l and subjected again to shaking 
cultivation at 30.degree. C. for 48 hrs. 
The growth cells are separated from supernatant solution by centrifugal 
precipitation of the culture broth and washed three times with distilled 
water. Suspension of wet cells thus obtained are treated in the following 
procedure to give a raw sample for extracting coenzyme Q.sub.10. 
The mixture of 50 ml of the suspension of the wet cells, 150 ml of 
methanol, 5 g of pyrogallol and 20 g of sodium hydroxide are subjected to 
saponification by treating under a reflux condenser at 90.degree. C. for 1 
hr. and then extracted twice with each 100 ml of n-hexane. The n-hexane 
layer of total 200 ml is washed three times with distilled water, then 
dehydrated and dried overnight with anhydrous sodium sulfate, and 
thereafter concentrated under reduced pressure. The sample thus obtained 
is used for the quantitative analysis of coenzyme Q.sub.10 in the 
following way. 
The concentrate is dissolved in 7 ml of ethanol to give a sample solution. 
2 ml of the sample solution, 0.5 ml of ethylcyanoacetate (ECA) and 0.5 ml 
of 0.2N-KOH aqueous solution are mixed. After exactly 10 min., Optional 
Density at 625 nm (OD.sub.625) is determined. As a control, OD.sub.625 is 
determined for a similar solution wherein instead of ECA an equal amount 
of ethanol is used and reacted in the same way. The control value is 
subtracted from the above determined value. The results are shown in Table 
1. 
TABLE 1 
______________________________________ 
(Analytical results) 
Amount* 
of produced 
POBA coenzyme 
concen- 
Q.sub.10 (.mu.g) 
Dry cell 
tration 
1 l weight (g) Q.sub.10 production (.mu.g) 
(.mu.g/ml) 
culture broth 
1 l culture broth 
1 g dry cell weight 
______________________________________ 
0 770 3.5 220 
100 980 3.6 270 
500 980 3.6 270 
1,000 1,190 3.7 320 
2,000 1,820 2.9 630 
______________________________________ 
*Amount of coenzyme Q.sub.10 obtained from 1 l culture broth. 
The above results are also shown as characteristic graphs in the attached 
FIGS. 1A and 1B. 
EXAMPLE 2 
In this Example, the same microorganism and cultivation conditions as in 
Example 1 are used, and the same treatments as in Example 1 are effected, 
except that main culture is not adjusted to the pH value of 5.0 and 
conducted in an acidic condition of a pH value of 2.5-4. 
A loopful cells from the young slant culture is inoculated to the MPY 
medium 10 ml, and cultivated at 30.degree. C. for 7 days to obtain 
inoculum cells. 
A drop of the inoculum cell suspension is inoculated to 200 ml of the 
pre-culture medium not containing POBA and subsequently subjected to 
shaking cultivation at 30.degree. C. for 48 hrs. to yield a pre-culture 
broth. 
The pre-culture broth is added to 800 ml of the main culture medium 
containing POBA in a concentration of 0-2,000 .mu.g/ml and a pH value of 
2.5-4.0 depending on POBA concentration in a volume ratio of 1:4 to a 
total volume of 1 l and subjected again to shaking cultivation at 
30.degree. C. for 48 hrs. 
The cells are separated from supernatant solution by centrifugal 
precipitation of the culture broth and washed three times with distilled 
water. Suspension of wet cells thus obtained are treated in the following 
prcedure to give a raw sample for extracting coenzyme Q.sub.10. 
The mixture of 50 ml of the suspension of the wet cells, 150 ml of 
methanol, 5 g of pyrogallol and 20 g of sodium hydroxide are subjected to 
saponification by treating under a reflux condenser at 90.degree. C. for 1 
hr. and then extracted twice with each 100 ml of n-hexane. The n-hexane 
layer of total 200 ml is washed three times with distilled water, then 
dehydrated and dried overnight with anhydrous sodium sulfate, and 
thereafter concentrated under reduced pressure. The sample thus obtained 
is used for quantitative analysis of coenzyme Q.sub.10 in the same way as 
in Example 1. The results are shown in Table 2. 
TABLE 2 
______________________________________ 
(Analytical results) 
Amount* 
of produced 
POBA coenzyme 
concen- 
Q.sub.10 (.mu.g) 
Dry cell 
tration 
1 l weight (g) Q.sub.10 production (.mu.g) 
(.mu.g/ml) 
culture broth 
1 l culture broth 
1 g dry cell weight 
______________________________________ 
0 700 2.5 280 
100 840 2.6 320 
500 1,120 2.1 530 
1,000 1,190 2.1 570 
2,000 not grown -- -- 
______________________________________ 
*Amount of coenzyme Q.sub.10 obtained from 1 l culture broth. 
The above results are also shown as characteristic graphs in the attached 
FIGS. 2A and 2B. 
EXAMPLE 3 
The same experiment as in Example 1 is repeated, except that a loopful 
cells from the young slant culture is inoculated to 10 ml of the 
pre-culture medium not containing POBA instead of the MPY medium and 
cultivated at 30.degree. C. for 10 days to obtain inoculum cells, and that 
POBA concentrations in the main culture medium is raised up. 
A drop of the inoculum cell suspension is inoculated to 200 ml of the 
pre-culture medium not containing POBA and subsequently subjected to 
shaking cultivation at 30.degree. C. for 48 hrs. to yield a pre-culture 
broth. 
The pre-culture broth is added to 800 ml of the main culture medium 
containing POBA in a concentration of 1,000-4,000 .mu.g/ml in a volume 
ratio of 1:4 to a total volume of 1 l and subjected again to shaking 
cultivation at 30.degree. C. for 48 hrs. 
The green cells are separated from supernatant solution by centrifugal 
precipitation of the culture broth and washed three times with distilled 
water. Suspension of wet cells thus obtained are treated in the following 
procedure to give a raw sample for extracting coenzyme Q.sub.10. 
The mixture of 50 ml of the suspension of the wet cells, 150 ml of 
methanol, 5 g of pyrogallol and 20 g of sodium hydroxide are subjected to 
saponification by treating under a reflux condenser at 90.degree. C. for 1 
hr. and then extracted twice with each 100 ml of n-hexane. The n-hexane 
layer of total 200 ml is washed three times with distilled water, then 
dehydrated and dried overnight with anhydrous sodium sulfate, and 
thereafter concentrated under reduced pressure. The sample thus obtained 
is used for quantitative analysis of coenzyme Q.sub.10 in the same way as 
in Example 1. 
The results are shown in Table 3. 
TABLE 3 
______________________________________ 
(Analytical results) 
Amount* 
of produced 
POBA coenzyme 
concen- 
Q.sub.10 (.mu.g) 
Dry cell 
tration 
1 l weight (g) Q.sub.10 production (.mu.g) 
(.mu.g/ml) 
culture broth 
1 l culture broth 
1 g dry cell weight 
______________________________________ 
1,000 2,310 7.1 330 
2,000 3,920 5.5 710 
3,300 3,220 5.8 560 
4,000 2,940 7.3 400 
______________________________________ 
*Amount of coenzyme Q.sub.10 obtained from 1 l culture broth. 
The above results are also shown as characteristic graphs in the attached 
FIGS. 3A and 3B. 
EXAMPLE 4 
The same experiment as in Example 3 is repeated, except that a loopful 
cells from the young slant culture is cultivated for 7 days instead of 10 
days and that a temperature of 37.degree. C. is used for the main 
cultivation instead of 30.degree. C. 
The results are shown in Table 4. 
TABLE 4 
______________________________________ 
(Analytical results) 
Amount* 
of produced 
POBA coenzyme 
concen- 
Q.sub.10 (.mu.g) 
Dry cell 
tration 
1 l weight (g) Q.sub.10 production (.mu.g) 
(.mu.g/ml) 
culture broth 
1 l culture broth 
1 g dry cell weight 
______________________________________ 
1,000 1,120 8.4 130 
2,000 2,520 10.4 240 
2,700 3,220 9.2 350 
3,300 3,080 7.2 430 
4,000 280 2.4 120 
______________________________________ 
*Amount of coenzyme Q.sub.10 obtained from 1 l culture broth. 
The above results are also shown as characteristic graphs in the attached 
FIGS. 4A and 4B. 
EXAMPLE 5 
In this Example, coenzyme Q.sub.10 is produced using Trichosporon sp. WY 
2-2 strain instead of Aureobasidium sp. No. 14 strain. 
Cultivations are effected as follows. 
A loopful cells from the young slant culture is inoculated to the 
aforementioned MPY medium 10 ml and cultivation at 30.degree. C. for 7 
days to obtain inoculum cells. 
A drop of the inoculum cell suspension is inoculated to 200 ml of the 
above-mentioned pre-culture medium not containing POBA and subsequently 
subjected to shaking cultivation at 30.degree. C. for 48 hrs. to yield a 
pre-culture broth. 
The pre-culture broth is added to 800 ml of the aforementioned main culture 
medium containing POBA in a concentration of 0-2,000 .mu.g/ml in a volume 
ratio of 1:4 to a total volume of 1 l and subjected again to shaking 
cultivation at 30.degree. C. for 48 hrs. 
The cells are separated from supernatant solution by centrifugal 
precipitation of the culture broth and washed three times with distilled 
water. Suspension of wet cells thus obtained is treated in the following 
procedure to give a raw sample for extracting coenzyme Q.sub.10. 
The mixture of 50 ml of the suspension of the wet cells, 150 ml of 
methanol, 5 g of pyrogallol and 20 g of sodium hydroxide are subjected to 
saponification by treating under a reflux condensor at 90.degree. C. for 1 
hr. and then extracted twice with each 100 ml of n-hexane. The n-hexane 
layer of total of 200 ml is washed three times with distilled water, then 
dehydrated and dried overnight with anhydrous sodium sulfate, and 
thereafter concentrated under reduced pressure. The sample thus obtained 
is used for quantitative analysis of coenzyme Q.sub.10 in the following 
way. 
The concentration is dissolved in 2 ml of acetone to give a sample 
solution. Ten .mu.l of the sample solution is used for the quantitative 
analysis by high performance liquid chromatography. The quantitative value 
is determined by the standard curve obtained from the authentic specimen. 
The results are shown in Table 5. 
TABLE 5 
______________________________________ 
(Analytical results) 
Amount* 
of produced 
POBA coenzyme 
concen- 
Q.sub.10 (.mu.g) 
Dry cell 
tration 
1 l weight (g) Q.sub.10 production (.mu.g) 
(.mu.g/ml) 
culture broth 
1 l culture broth 
1 g dry cell weight 
______________________________________ 
0 6.03 0.053 114.0 
100 9.50 0.098 97.0 
500 17.80 0.173 103.0 
1,000 13.20 0.190 69.4 
2,000 8.52 0.181 47.0 
______________________________________ 
*Amount of coenzyme Q.sub.10 obtained from 1 l culture broth. 
The above results are also shown as characteristic graphs in the attached 
FIGS. 5A and 5B. 
REFERENTIAL EXAMPLE 
Growth characteristics of Aureobasidium sp. No. 14 strain is studied on 
various utilizable main carbon sources other than ethanol. The main carbon 
sources used are glycerol, glucose, n-paraffin mixture (Carbon number 
C.sub.11 .about.C.sub.14), n-propanol, acetone, ethyl acetate, 
cyclohexanol, n-dodecane or palmitic acid. 
Cultivations are effected as follows. 
A drop of the inoculum cell suspension obtained in the same procedure as in 
Example 1 is inoculated to 10 ml of a culture medium which has the same 
composition employed in Example 1 for pre-culture, except that as a main 
carbon source 20 ml of ethanol is replaced by 20 g or 20 ml of the above 
mentioned materials. 
When the main carbon source used is insoluble in water, 0.5 g surfactant, 
Tween 80 is added per 1 l medium. 
The inoculated culture medium is subjected to shaking cultivation at 
30.degree. C. and cell growth expressed in OD.sub.660 unit and the 
cultivation time are observed. 
The results are shown in Table 6. 
TABLE 6 
______________________________________ 
(Growth characteristics 
on various main carbon sources) 
Growth Cultivation time 
Main carbon source 
(OD.sub.660) 
(hrs.) 
______________________________________ 
glycerol 4.14 54 
glucose 4.46 54 
n-paraffin (C.sub.11 .about.C.sub.14) 
2.86 54 
n-propanol 2.50 248 
acetone 1.02 248 
ethylacetate 2.29 96 
cyclohexanol 0.17 224 
n-dodecane 3.54 54 
palmitic acid 3.64 73 
______________________________________ 
EXAMPLE 6 
In this Example, coenzyme Q.sub.10 is produced by Aureobasidium sp. No. 14 
strain grown in the media containing various main carbon sources and a 
large quantity of POBA, 2,000 .mu.g/ml. The main carbon sources used are 
glycerol, glucose or n-paraffin (C.sub.11 .about.C.sub.14). 
Cultivation is effected in the same way as in Referential Example to obtain 
100 ml of a pre-culture broth. 
The pre-culture broth thus obtained is added to 400 ml of the culture 
medium described in Referential Example containing POBA in the 
concentration of 2,000 .mu.g/ml in a volume ratio of 1:4 to a total volume 
of 500 ml and subjected again to shaking cultivation at 30.degree. C. for 
a given period. 
The grown cells are separated from supernatant solution by centrifugal 
precipitation of the culture broth and washed three times with distilled 
water. Suspension of wet cells thus obtained are treated in the same 
procedure as in Example 1 to give a raw sample for extracting and 
quantitative analysis of coenzyme Q.sub.10. 
The results are shown in Table 7. 
TABLE 7 
______________________________________ 
(Production of Q.sub.10 
on various main carbon sources) 
Main Wet cell 
Pre-culture 
culture weight Q.sub.10 
Main carbon source 
(hrs.) (hrs.) (g) (.mu.g/l) 
______________________________________ 
glycerol 42 52 14.2 2,900 
glucose 47 47 10.0 1,900 
n-paraffin (C.sub.11 .about.C.sub.14) 
47 70 1.4 100 
______________________________________ 
As clearly indicated from the above quantitative analytical results, the 
present invention is exceedingly superior to conventional methods. 
Although the present invention has been explained in detail with specific 
values and embodiments, it will of course be apparent to those skilled in 
the art that many variations and modifications are possible without 
departing from the broad aspect and scope of the present invention as 
defines in the appended claims.