Process for producing omega-9 highly unsaturated fatty acid and lipid containing the same

The present invention discloses a process for producing lipid containing omega-9 highly unsaturated fatty acid by culturing in a medium a mutant strain obtained by mutation on a microorganism having the ability to produce arachidonic acid belonging to the genus Mortierella and so forth, in which .DELTA.12 desaturation activity is decreased or lost, but at least one of .DELTA.5 desaturation activity, .DELTA.6 desaturation activity and chain length elongation activity is elevated. Moreover, the present invention also discloses a process for producing omega-9 highly unsaturated fatty acid by collecting omega-9 highly unsaturated fatty acid from the culture or lipid described above.

BACKGROUND OF INVENTION 
1. Field of Invention 
The present invention relates to a process for producing omega-9 highly 
unsaturated fatty acid and lipid containing the same by fermentation using 
a mutant strain in which .DELTA.12 desaturation activity has been 
decreased or lost, but at Beast one of .notident.5 desaturation activity, 
.DELTA.6 desaturation activity and chain length elongation activity is 
elevated. 
2. Related Art 
Omega-9 highly unsaturated fatty acids, such as 5,8,11-eicosatrienoic acid 
(referred to as mead acid) and 8,11-eicosadienoic acid, are known to exist 
as one of the constituent fatty acids of animal tissue that has become 
deficient in essential fatty acids. However, it has been extremely 
difficult to isolate and purify them since they are present in extremely 
small amounts. Since it is possible for these highly unsaturated fatty 
acids to become precursors of the leucotriene 3 group in the body, 
considerable expectations have been placed on their physiological 
activity. Their use for anti-inflammatory, anti-allergic and 
anti-rheumatic effects has recently been reported (Japanese Unexamined 
Patent Publication No. 7-41421). 
There is therefore a strong desire to develop a method for producing 
omega-9 highly unsaturated fatty acids in large amounts. A process for 
producing omega-9 highly unsaturated fatty acid and lipid containing the 
same was previously completed by performing mutation on microorganisms 
having the ability to produce arachidonic acid and isolating those 
microorganisms in which A12 desaturation activity has been decreased or 
lost (Japanese Unexamined Patent Publication No. 5-91888). However, 
although it is revolutionary and significant that a process for producing 
omega-9 highly unsaturated fatty acid and lipid containing the same was 
developed since such a process had not existed in the past, there was 
still much room for improvement in yield. Consequently, there has been a 
strong desire to develop a process for efficiently producing a larger 
amount of omega-9 highly unsaturated fatty acids. 
SUMMARY OF INVENTION 
Thus, the present invention is intended to provide a process that makes it 
possible to produce omega-9 highly unsaturated fatty acid or lipid 
containing the same in a large amount using conventional inexpensive 
media. 
As a result of various researches conducted to achieve the above-mentioned 
object, the inventors of the present invention found a mutant in which 
.DELTA.12 desaturation activity has been decreased or lost, but at least 
one of .DELTA.5 desaturation activity, .DELTA.6 desaturation activity and 
chain length elongation activity has been elevated, thereby leading to 
completion of the present invention. 
Thus, the present invention provides a process for producing lipid 
containing omega-9 highly unsaturated fatty acid comprising the steps of: 
culturing in a medium a mutant strain obtained by mutation on a 
microorganism having an ability to produce arachidonic acid belonging to a 
genus selected from the group consisting of the genera Mortierella, 
Conidiobolus, Pythium, Phvto phthora, Penicillium, Cladosporium, Mucor, 
Fusarium, Aspergillus, Rhodotorula, Entomo Ththora, Echinos iorancrium and 
Saprolegnia, in which .DELTA.12 desaturation activity has been decreased 
or lost, but at least one of .DELTA.5 desaturation activity, .DELTA.6 
desaturation activity and chain length elongation activity has been 
elevated; and, 
recovering lipid containing omega-9 highly unsaturated fatty acid from the 
culture. 
Moreover, the present invention provides a process for producing an omega-9 
highly unsaturated fatty acid comprising the step of recovering the 
omega-9 highly unsaturated fatty acid from the culture or lipid obtained 
according to the process described above. 
DETAILED DESCRIPTION 
In the present invention, the microorganisms used for mutation (to be 
referred to as the "parent strain") are microorganisms that have the 
ability to produce arachidonic acid and belong to the genus Mortierella, 
Conidiobolus, Pythium, Phytophthora, Penicillium, Cladosporium, Mucor, 
Fusarium, Aspergillus, Rhodotorula, Entomophthora, Echinosporancium or 
Saprolegnia. 
These microorganisms convert stearic acid to oleic acid by .DELTA.9 
desaturase, oleic acid to linoleic acid by .DELTA.12 desaturase, linoleic 
acid to .gamma.-linolenic acid by .DELTA.6 desaturase, .gamma.-linolenic 
acid to dihomo-.gamma.-linolenic acid by chain length elongation enzyme, 
and dihomo-.gamma.-linolenic acid to arachidonic acid by .DELTA.5 
desaturase. In addition, these microorganisms biosynthesize 
6,9-octadecadienoic acid from oleic acid by .DELTA.6 desaturase, 
8,11-eicosadienoic acid from 6,9-octadecadienoic acid by chain length 
elongation enzyme, and mead acid from 8,11-eicosadienoic acid by .DELTA.5 
desaturase when .DELTA.12 desaturation activity is inhibited. 
Microorganisms belonging to the subgenus Mortierella in the genus 
Mortierella, which exhibits excellent arachidonic acid productivity, are 
preferable for the parent strain used in the present invention, examples 
of which include the strains Mortierella elongata IFO 8570, Mortierella 
exiqua IFO 8571, Mortierella hygrophila IFO 5941 and Mortierella alpina 
IFO 8568, ATCC 16266, ATCC 32221, ATCC 42430, CBS 219.35, CBS 224.37, CBS 
250.53, CBS 343.66, CBS 527.72, CBS 529.72, CBS 608.70 and CBS 754.68. 
All of these strains are available without restriction from the Institute 
of Fermentation Osaka (IFO) located in Osaka, Japan, the American Type 
Culture Collection (ATCC) located in the USA, or the Centraalbureau voor 
Schimmelcultures (CBS). In addition, the strain Mortierella elongata 
-SAM0219 (FERM P-8703) (FERM BP-1239), which was isolated from the soil by 
the inventors of the present invention, can also be used. Mortierella 
elongata SAM 0219 was deposited as an international deposition under the 
Budapest Treaty as FERM BP-1239 on March 19, 1986 at the Institute of 
Bioscience and Human-Technology Agency of Industrial Science and 
Technology, 1-3 Higashi 1-chome, Tsukuba-shi, Ibaraki-ken, 305, Japan. 
In addition, the parent strain used in the present invention includes 
mutant or recombinant strains of the above-mentioned microorganisms (wild 
strains) having the ability to produce arachidonic acid, namely strains 
intentionally designed so that the content of omega-9 highly unsaturated 
fatty acid, the total lipid content or both is greater than the amount 
produced by the original wild strain when cultured using the same 
substrate. Moreover, said parent strain also includes microorganisms 
designed to produce an amount of omega-9 highly unsaturated fatty acid 
equal to that of the corresponding wild strain by efficiently using a 
substrate having excellent cost benefit. 
In order to obtain a mutant of the present invention having decreased or 
lost .DELTA.12 desaturation activity, but at least one of elevated 
.DELTA.5 desaturation activity, .DELTA.6 desaturation activity and chain 
length elongation activity, mutation is performed on the above-mentioned 
microorganism having the ability to produce arachidonic acid to first 
obtain a mutant having decreased or lost .DELTA.12 desaturation activity. 
Moreover, by then mutation on this mutant strain, a mutant can be obtained 
in which .DELTA.12 desaturation activity has been decreased or lost, but 
at least one of .DELTA.5 desaturation activity, .DELTA.6 desaturation 
activity and chain length elongation activity has been elevated. An 
example of a mutant that can be used having decreased or lost .DELTA.12 
desaturation activity is Mortierella alpina SAM1861 (FERM BP-3590). 
Mortierella alpina SAM 1861 was deposited as an international deposition 
under the Budapest Treaty as FERM BP-3590 on September 30, 1991 at the 
Institute of Bioscience and Human-Technology Agency of Industrial Science 
and Technology, 1-3 Higashi 1-chome, Tsukuba-shi, Ibaraki-ken, 305, Japan. 
By using a microorganism having decreased or lost .DELTA.12 desaturation 
activity, and preferably a microorganism in which .DELTA.12 desaturation 
activity is absent, for the parent in the mutant of the present invention, 
whether or not its .DELTA.5 desaturation activity, .DELTA.6 desaturation 
activity or chain length elongation activity is elevated can be easily 
evaluated. 
More specifically, since omega-6 unsaturated fatty acids such as linoleic 
acid, .gamma.-linolenic acid, dihomo-.gamma.-linolenic acid and 
arachidonic acid are inherently either absent or only present in very 
small amounts in microbial cells in the case of a microorganism in which 
.DELTA.12 desaturation activity has been either decreased or lost, 
.gamma.-linolenic acid is formed by .DELTA.6 desaturase if the rest cells 
obtained after culturing are reacted with linoleic acid, arachidonic acid 
is formed by .DELTA.5 desaturase if it is reacted with 
dihomo-.gamma.-linolenic acid, or dihomo-.gamma.-linolenic acid is formed 
by chain length elongation enzyme if it is reacted with .gamma.-linolenic 
acid. Since the activity of each enzyme can be easily assayed, .DELTA.5 
desaturation activity, .DELTA.6 desaturation activity and chain length 
elongation activity of microorganisms obtained by mutation can be 
evaluated by comparing them with the parent strain. 
Although a specific example of a mutant strain of the present invention 
that can be used is Mortierella alpina SAM2086 (FERM P-15766) (which was 
deposited as an international deposition under the Budapest Treaty as FERM 
BP-6032 on Aug. 5, 1996 at the said Institute), a microorganism lack of 
.DELTA.12 desaturation activity and having elevated .DELTA.6 desaturation 
activity that was induced by the inventors of the present invention from 
Mortierella alpina SAM1861, such mutants are not limited to this strain, 
but rather any mutants can be used provided that when the .DELTA.5 
desaturation activity, .DELTA.6 desaturation activity or chain length 
elongation activity of the parent strain in which .DELTA.12 desaturation 
activity is decreased or lost is taken to be expressed "1", at least one 
of these activities exhibits a level of activity that exceeds 1. 
Examples of omega-9 highly unsaturated fatty acids obtained by culturing a 
mutant of the present invention include 6,9-octadecadienoic acid, 
8,11-eicosadienoic acid and 5,8,11-eicosatrienoic acid. 
In the present invention, mead acid can be produced in a large amount by 
using, in particular, a mutant in which .DELTA.12 desaturation activity 
has been absent, and both .DELTA.5 desaturation activity and .DELTA.6 
desaturation activity have been elevated. 
Typical mutation procedures can be performed for inducing mutation, such as 
by irradiating with radiation (X-rays, .gamma.-rays or neutron beam), 
ultraviolet rays or heat treatment, or by suspending the microorganism in 
a suitable buffer, adding a mutagen and incubating for a predetermined 
amount of time followed by suitably diluting and growing on agar medium to 
obtain colonies of the mutant strain. Examples of mutagens include 
alkylating agents such as nitrogen mustard, methylmethane sulfonate (MMS) 
and N-methyl-N-nitro-N-nitrosoguanidine (NTG), base analogs such as 
5-bromouracil, antibiotics such as mitomycin C, base synthesis inhibitors 
such as 6-mercaptopurine, pigments such as proflavin (and other 
derivatives), certain types of carcinogens such as 
4-nitroguinoline-N-oxide, and other compounds such as manganese chloride 
and formaldehyde. In addition, the parent strain may in the form of 
growing cells (mycelium) or spores. 
In order to culture a mutant in the production process of the present 
invention, the spores, mycelia or pre-culture liquid obtained by culturing 
in advance are inoculated into a liquid or solid medium. In the case of 
using a liquid medium, although any typically used substances can be used 
for the carbon source, examples of which include glucose, fructose, 
xylose, saccharose, maltose, soluble starch, molasses, glycerol, mannitol 
and citric acid, glucose, maltose, molasses and glycerol are particularly 
preferable 
In addition, organic nitrogen sources such as yeast. extract, wheat germ 
extract, beef extract, casamino acids, corn steep liquor and urea, or 
inorganic nitrogen sources such as sodium nitrate, ammonium nitrate and 
ammonium sulfate can be used for the nitrogen source. In addition, 
phosphates such as potassium phosphate and potassium dihydrogen phosphate, 
inorganic salts such as ammonium sulfate, sodium sulfate, magnesium 
sulfate, iron sulfate, copper sulfate, magnesium chloride and calcium 
chloride, as well as vitamins can also be used as necessary as trace 
nutrients. 
There are no particular limitations on the concentrations of these medium 
components provided they do not inhibit growth of the microorganism. In 
terms of practicality, the carbon source should typically be used at 0.1 
to 30% by weight, and preferably 1 to 15% by weight, and the nitrogen 
source at 0.01 to 10% by weight, and preferably 0.1 to 5% by weight. 
The culture temperature should be 5 to 40.degree. C. and preferably 20 to 
30 0.degree. C., and after the microorganisms have grown by cultivation at 
20 to 30 0.degree. C., omega-9 highly unsaturated fatty acids can also be 
produced by following cultivation at 5 to 20.degree. C. An amount of 
omega-9 highly unsaturated fatty acids formed in the resulting fatty acids 
can be increased by such a temperature control. The pH value of the medium 
should be 4 to 10, and preferably 5 to 8, and cultivation is performed by 
aerated agitation culture, shaking culture or stationary culture. 
Cultivation is normally performed for 2 to 20 days, preferably for 5 to 20 
days and more preferably for 5 to 15 days. 
In the case of using a solid culture, cultivation is performed for 3 to 14 
days at a temperature of 5 to 40 0C., and preferably 20 to 30 0.degree. 
C., using wheat bran, rice chaff or rice bran containing 50 to 100% by 
weight of water relative to the weight of the solid substances. In this 
case, nitrogen sources, inorganic salts and trace nutrients can be added 
as necessary. In addition, in the present invention, accumulation of 
omega-9 highly unsaturated fatty acids can be promoted by adding a 
precursor of omega-9 highly unsaturated fatty acids to the medium during 
culturing. 
Examples of this precursor include hydrocarbons such as tetradecane, 
hexadecane and octadecane, fatty acids, their salts (e.g., sodium salts or 
potassium salts) or their esters such as tetradecanoic acid, hexadecanoic 
acid and octadecanoic acid, or oils containing fatty acids as their 
constituent ingredients (e.g., olive oil, coconut oil and palm oil). This 
precursor is not limited to these examples, however. The total amount of 
the added substrate is 0.001 to 10% by weight, and preferably 0.5 to 10% 
by weight relative to the amount of medium. In addition, cultivation may 
also be performed using these precursor as the sole carbon source. 
These carbon sources, nitrogen sources, inorganic salts, vitamins or 
substrates may be added before or immediately after inoculation with a 
producer microorganism, or may be added after cultivation has already been 
started. Alternatively, they may be added at either or both times. 
Addition immediately after the start of cultivation may be performed all 
at once or intermittently by dividing over several additions. 
Alternatively, addition may be performed continuously. 
By cultivation in this manner, lipids containing a large amount of omega-9 
highly unsaturated fatty acids will be formed and accumulate 
intracellularly. In the case of liquid culture, lipid containing omega-9 
highly unsaturated fatty acids is recovered from the cultured medium or 
sterilized cultured medium from an intermediate step in the production of 
oil by culturing microorganisms, from the cultured medium or sterilized 
cultured medium at completion of cultivation, or from cultured cells or 
their dried product collected from any of the above cultured media. For 
example, lipid containing omega-9 highly unsaturated fatty acids can be 
recovered from cultured cells and the lipid containing omega-9 highly 
unsaturated fatty acids can be isolated in the manner described below. 
Following completion of cultivation, the cultured cells are obtained from 
the cultured medium by centrifugation and/or any conventional solid-liquid 
separation technique such as filtration. The cells are preferably washed, 
crushed and dried. Drying can be performed by freeze-drying or air drying. 
The dried cells are preferably extracted with organic solvents in the 
presence of flowing nitrogen gas. Examples of organic solvents that can be 
used include ethyl ether, hexane, methanol, ethanol, chloroform, 
dichloromethane and petroleum ether, while alternating extraction with 
methanol and petroleum ether, and extraction using a single layer solvent 
of chloroform, methanol and water give good results. The organic solvent 
is then distilled off from the extract under reduced pressure to obtain 
lipid containing a high concentration of omega-9 highly unsaturated fatty 
acids. 
In addition, extraction can also be performed using wet cells in place of 
the method described above. In this case, a solvent such as methanol or 
ethanol that is miscible with water, or mixed solvents comprising these 
solvents, water and/or other solvents that are miscible with water, can be 
used. The other parts of the procedure are the same as that described 
above. 
The omega-9 highly unsaturated fatty acids are present in the lipid 
obtained in the above-mentioned manner as a triglyceride, or as a compound 
bonded to phosphatidyl choline, phosphatidyl ethanolamine or phosphatidyl 
inositol. Purification of triglyceride containing omega-9 highly 
unsaturated fatty acids from the lipid containing omega-9 highly 
unsaturated fatty acids recovered from the culture can be performed in 
accordance with routine methods such as hexane extraction followed by 
removal of free acid, decolorization, deodorization, degumming treatment 
or cooling separation. 
In addition, omega-9 highly unsaturated fatty acids are contained in the 
lipid obtained in the manner described above in the form of a lipid 
compound, such as the constituent component of a fat. Although these can 
be separated directly, it is preferable to separate them in the form of an 
ester of a lower alcohol, examples of which include methyl 
8,11-eicosadienoate, methyl 6,9-octadecadienoate and methyl ester of mead 
acid. By converting into esters in this manner, these components can be 
easily separated from other lipid components. In addition, they can also 
be easily separated from other fatty acids formed during cultivation, such 
as palmitic acid and oleic acid (these are also esterified during 
esterification of omega-9 highly unsaturated fatty acids). For example, in 
order to obtain the methyl ester of omega-9 highly unsaturated fatty 
acids, it is preferable to treat the above-mentioned extracted lipid for 1 
to 24 hours at room temperature with 5 to 10% methanolic HCl acid or 10 to 
50% BF3-methanol. 
In order to recover omega-9 highly unsaturated fatty acids from the 
above-mentioned treatment solution, it is preferable to extract with an 
organic solvent such as hexane, ethyl ether or ethyl acetate. Next, by 
drying this extract over anhydrous sodium sulfate and so forth and 
distilling off the organic solvent preferably under reduced pressure, a 
mixture is obtained that consists mainly of fatty acid esters. This 
mixture contains methyl palmitate, methyl stearate, methyl oleate and 
other fatty acid methyl esters in addition to the target omega-9 highly 
unsaturated fatty acid methyl esters. In order to isolate omega-9 highly 
unsaturated fatty acid methyl esters from the mixture of fatty acid methyl 
esters, column chromatography, low-temperature crystallization, urea 
inclusion or liquid-liquid counter-current distribution chromatography and 
so forth can be used alone or in combination. 
In order to obtain omega-9 highly unsaturated fatty acids from the various 
types of omega-9 highly unsaturated fatty acid methyl esters isolated in 
the manner described above, after hydrolysis in the presence of alkali, 
the mixture should be extracted with an organic solvent such as ethyl 
ether or ethyl acetate. 
In addition, in order to recover the omega-9 highly unsaturated fatty acids 
without going through their methyl ester, after hydrolysis of the 
above-mentioned extracted lipid with alkali (by, for example, treating for 
2 to 3 hours at room temperature with 5% sodium hydroxide solution), the 
omega-9 highly unsaturated fatty acids can be extracted and purified from 
the hydrolysate by methods commonly used for extraction and purification 
of fatty acids.

EXAMPLES 
The following Examples provide a detailed explanation of the present 
invention. 
Example 1 
Mortierella alpina SAM l86l, a mutant lack of .DELTA.12 desaturation 
activity, was inoculated into Czapek agar medium (0.2% NaN O3, 0.1% 
K.sub.2 HPO.sub.4, 0 .05% MgSO.sub.4, 0.05% KCl, 0.001% FeSO.sub.4, 3% 
sucrose, 2% agar, pH 6.0) to form spores to prepare a spore solution (50 m 
M Tris/malate buffer (pH 7.5), 1.times.10.sup.6 spores/ml). 
0.5 ml of 100 m KM Tris/malate buffer (pH 7.5) were added to 1.0 ml of the 
resulting spore solution followed by the addition of 500 .mu.l of NTG 
solution (5 mg of N-methyl-N-nitro-N-nitrosoguanidine/1ml of deionized 
water) and incubating for 15 minutes at 28.degree. C. to perform mutation 
treatment. 
The NTG-treated spore suspension was diluted to roughly 10.sup.-3 
-10.sup.-4 and applied to a GY agar plate (1% is glucose, 0.5% yeast 
extract, 0.005% Triton X-100, 1.5% agar, pH 6.0). Those colonies that 
appeared during culturing at 28.degree. C. were randomly picked up and 
transferred to a new plate. 
The picked storage colonies were cultured for 2 days at 28 0 0 and 2 days 
at 12.degree. C. on a GY agar plate and then excised while still attached 
to the agar and dried at 100.degree. C. 
The resulting dried cells were placed in a screw-cap test tube (16.5 m min 
diameter) followed by methyl-esterification by treating for 3 hours at 
50.degree. C. by adding 1 ml of methylene chloride and 2 ml of 10% 
methanolic HCl. After adding 4 ml of n-hexane and 1 ml of water, 
extracting two times, and distilling off the solvent from the extract 
using a centrifugal evaporator (40.degree. C., 1 hour), the resulting 
fatty acid methyl esters were analyzed by capillary gas chromatography. As 
a result of screening, Mortierella alpina SAM2086 (FERM P-15766) was 
obtained having higher mead acid productivity than the parent strain, 
Mortierella alpina SAM1861. Mortierella alpina SAM 2086 was deposited as 
FERM P-15766 on Aug. 5, 1996 at the Institute of Bioscience and 
Human-Technology Agency of Industrial Science and Technology, 1-3 Higashi 
1-chome, Tsukuba-shi, Ibaraki-ken, 305, Japan. Moreover, Mortierella 
alpina SAM2104 was obtained by performing similar mutation treatment as 
that described above using SAM2086 for the parent strain. 
Example 2 
Five liters of medium (pH 6.0) containing 4% glucose and 1% yeast extract 
was placed in a 10 liter jar fermentor and sterilized for 30 minutes at 
120.degree. C. The medium was then inoculated with 100 ml of a preculture 
of mutant SAM1861 or SAM2086 of Mortierella alpina followed by aerated 
agitation culture for 8 days with aeration at one volume/volume/min. and 
agitation at 300 rpm. The culture temperature at the start of culturing 
was 28.degree. C. and then lowered to 20.degree. C. on the 2nd day of 
culturing. 1% Glucose was added daily from the 1st to 4th days of 
culturing. Following completion of culturing, the cells were recovered by 
filtration and after adequately washing, the resulting wet cells were 
freeze-dried to obtain 99.7 g and 92.5 g of dried cells for each strain, 
respectively. 
When lipid was extracted from these dried cells according to the extraction 
method of Blight & Dyer using a single layer solvent of chloroform, 
methanol and water, lipids were obtained in the amounts of 48.92 g and 
44.17 g, respectively. In order to confirm the fatty acid composition of 
these lipids, 10 mg of lipid was placed in screw-cap test tubes and 
methyl-esterified by treating for 3 hours at 50.degree. C. by adding 1 ml 
of methylene chloride and 2 ml of 10% methanolic HC1. After adding 4 ml of 
n-hexane and 1 ml of water, extracting two times, and distilling off the 
solvent from the extract using a centrifugal evaporator (40.degree. C., 1 
hour), the resulting fatty acid methyl esters were analyzed by gas 
chromatography. The results are shown in Table 1. 
SAM2086, induced by mutation from SAM1861, was clearly shown to demonstrate 
both excellent mead acid productivity and containing ratio. 
TABLE 1 
__________________________________________________________________________ 
Growth 
.omega.9 PUFA Production (g/l)** 
Strain (g/l)* 
18:2 (.omega.9) 
20:2 (.omega.9) 
20:3 (.omega.9) 
__________________________________________________________________________ 
SAM1861 19.94 1.27 0.32 1.61 
SAM2086 18.49 1.10 0.31 1.84 
__________________________________________________________________________ 
Fatty Acid Composition (%).sup.+ 
18:2 20:2 
20:3 
16:0 18:0 18:1 LA (.omega.9) GLA 20:1 (.omega.9) (.omega.9) DGLA Ara 
EPA 24:0 Other 
__________________________________________________________________________ 
SAM 6.61 7.74 41.23 0 12.94 0 2.26 3.29 16.43 0 0 0 4.58 4.92 
1861 
SAM 6.96 6.88 38.51 0 12.41 0 2.19 3.53 20.87 0 0 0 3.80 4.85 
2086 
__________________________________________________________________________ 
.sup.+ LA: linoleic acid, 18:2 (.omega.9): 6,9octadecadienoic acid, GLA: 
linolenic acid, 20:2 (.omega.9): 8,11eicosadienoic acid, 20:3 (.omega.9): 
mead acid, DGLA: dihomolinolenic acid, Ara: arachidonic acid, EPA: 
eicosapentaenoic acid 
*Dry cell weight per liter of medium 
**Weight of omega9 unsaturated fatty acids per liter of medium 
Example 3 
1 ml of 0.1 M phosphate buffer (pH 7.4), 30 mg of wet cells obtained in 
Example 2 of mutant SAM1861 or SAM2086 of Mortierella alpina and 100 .mu.l 
of BSA suspended substrate solution (prepared by mixing 20 mg of linoleic 
acid, .gamma.-linolenic acid or dihomo-.gamma.-linolenic acid in 2 ml of 
5% bovine serum albumin (fatty acid-free BSA, Sigma) and suspending by 
sonication for approximately 20 minutes) were added to screw-cap test 
tubes (16.5 mm in diameter) after which the test tubes were capped with a 
silicone stopper and shaken at 28.degree. C. and 120 rpm. The reaction was 
stopped after 0, 2, 6 or 20 hours by adding 4 ml of ethanol. 
After drying with a centrifugal evaporator (40.degree. C., 1 hour), 
methyl-esterification was performed in the same manner as Example 2, and 
the resulting fatty acid methyl esters (substrates and reaction products) 
were analyzed by capillary gas chromatography. In this analysis, the same 
amount of 5% BSA solution was used as control. Thus, if 
dihomo-.gamma.-linolenic acid is used for the substrate, .DELTA.5 
desaturation activity is determined from the amount of the reaction 
product, i.e., arachidonic acid; if .gamma.-linolenic acid is used for the 
substrate, chain length elongation activity is determined from the amount 
of the reaction product, i.e., dihomo-.gamma.-linolenic acid, and .DELTA.5 
desaturation activity is determined from the amount of arachidonic acid; 
and if linoleic acid is used for the substrate, .DELTA.6 desaturation 
activity is determined from the amount of the reaction product, i.e., 
.gamma.-linolenic acid, chain length elongation activity is determined 
from the amount of dihomo-.gamma.-linolenic acid, and .DELTA.5 
desaturation activity is determined from the amount of arachidonic acid. 
Those results are shown in Table 2. 
In the case of taking the activity of SAM1861 to be 1 for the .DELTA.5 
desaturation activity using dihomo-.gamma.-linolenic acid for the 
substrate, the activity of SAM2086 was 1.74. In the case of taking the 
activity of SAM1861 to be 1 for the .DELTA.6 desaturation activity using 
linoleic acid for the substrate, the activity of SAM2086 was 1.42. The 
increases in mead acid productivity and ratio of SAM2086 induced by 
mutation from SAM1861 of Example 2 were clearly the result of increased 
.DELTA.5 desaturation activity and .DELTA.6 desaturation activity. 
TABLE 2 
______________________________________ 
Reaction Rates of SAM1861 and SAM2086 
(nmol/30 mg wet cells/hour) 
Substrate Substrate 
Substrate 
DGLA GLA LA 
Product Product Product 
Ara DGLA GLA 
Strain (.DELTA.5 DS) (EL) (.DELTA.6 DS) 
______________________________________ 
SAM1861 0.85 7.50 11.01 
SAM2086 1.48 6.60 15.6 
______________________________________ 
LA: linoleic acid, GLA: linolenic acid, 
DGLA: dihomolinolenic acid, Ara: arachidonic acid 
.DELTA.5 DS: .DELTA.5 desaturation activity 
.DELTA.6 DS: .DELTA.6 desaturation activity 
EL: chain length elongation activity 
Example 4 
2 ml of medium (pH 6.0) containing 2% glucose, 1% yeast extract and 0.5% of 
each of the precursors of the omega-9 highly unsaturated fatty acids 
indicated in Table 3, or oils containing the same, was placed in 10 ml 
Erlenmeyer flasks and sterilized for 20 minutes at 120.degree. C. The 
flasks were each inoculated with a piece of cells of mutant SAM2086 of 
Mortierella alpina followed by culturing for 8 days at 28.degree. C. using 
a reciprocating shaker (110 rpm). The results are shown in Table 3. 
TABLE 3 
______________________________________ 
Amount of Omega-9 Highly 
Unsaturated Fatty Acids Produced 
(g/l) 
Added Substance 
18:2 20:2 20:3 
______________________________________ 
No addition 0.23 0.04 0.27 
Hexadecane 0.32 0.06 0.39 
Octadecane 0.38 0.06 0.48 
Palmitic acid 0.40 0.07 0.51 
Stearic acid 0.47 0.07 0.58 
Oleic acid 0.57 0.11 0.70 
Sodium palmitate 0.35 0.08 0.44 
Sodium stearate 0.37 0.08 0.46 
Sodium oleate 0.49 0.09 0.60 
Methyl palmitate 0.45 0.10 0.57 
Methyl stearate 0.52 0.11 0.64 
Methyl oleate 0.66 0.16 0.81 
Ethyl oleate 0.67 0.15 0.82 
Palm oil 0.45 0.08 0.56 
Olive oil 0.48 0.12 0.58 
Coconut oil 0.36 0.07 0.41 
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18:2; 6,9octadecadienoic acid 
20:2; 8,11eicosadienoic acid 
20:3; 5,8,11eicosatrienoic acid (mead acid) 
Example 5 
Five liters of medium (pH 6.0) containing 2% glucose, 1% yeast extract, 
0.1% olive oil and 0.01% Adecanol (defoaming agent; Trademark) was placed 
in a 10 liter jar fermentor followed by sterilization for 30 minutes at 
120.degree. C. 100 ml of a preculture of Mortierella alpina SAM2104 was 
inoculated. Cultivation was carried out for 8 days with aeration at 1 
volume/volume/min. and agitation at 300 rpm. 
The culture temperature was 28.degree. C. at the start of culturing and 
then lowered to 20.degree. C. starting on the 2nd day of culturing. 1.5% 
glucose was added on the 2nd and 3rd days of culturing. Following 
completion of culturing, 15.80 g of dried cells was obtained per liter of 
medium by following the same procedure as that of Example 2. The lipid was 
extracted in the same manner as Example 2, said lipids were 
methyl-esterified, the resulting fatty acid methyl esters were analyzed by 
gas chromatography. The amounts produced and percentages of mead acid, 
8,11-eicosadienoic acid and 6,9-octadecadienoic acid relative to the total 
amount of fatty acids were 1.76 g/liter and 23.76% for mead acid, 0.35 g 
liter and 4.75% for 8,11-eicosadienoic acid, and 0.84 g /liter and 11.35% 
for 6,9-octadecadienoic acid, respectively.