- Email: [email protected]
Stereoselective synthesis of chaulmoogric acid and related fatty acid from 2-(±)-cyclopentenecarboxylic acid by Bacillus subtilis (ATCC 7059)
Vol. April
99, No. 4,198l 30,
BIOCHEMICAL
AND
BIOPHYSICAL
COMMUNICATIONS
Pages 1226-1229
1981
STEREOSELECTIVE RELATED
FATTY ACID
SYNTHESIS ACID FROM BY BACILLUS
OF
Alberta
March
Research
Council,
CHAULMOOGRIC
ACID
AND
2-(i)-CYCLOPENTENECARBOXYLIC SUBTILIS (ATCC 7059)
Toshi
Received
RESEARCH
Kaneda Edmonton,
Alberta,
Canada
11,1981
Received SUMMARY: 2-(+)-Cyclopentenecarboxylic acid added to the culture medium is incorporated into two new fatty acids by the growing cells of Bacillus subtilis (ATCC 7059). The new fatty acids, amounting to 24% of the total cellular fatty acids, are identified as hydrocarpic [ 11-(2’-cyclopentenvl)-hendecanoic] and chaulmoogric [13-(2’-cvclopentenvI)-tridecanoic] bv gas-liquid chromatography and mass spectrometry. These Cl6 and Cl .fatty acids are optically active, levorotatorv. with the specific rotation of -50.4O as mixture. thus t a e ootical ouritv of aooroximatelv 80%. This ‘indicates that the optical rotation of these bacterial fatty ‘acids are opposite with that ‘of the fatty acids from plant oils. Unusual w-cyclic fatty acids, (+)-chaulmoogric acid [ 13-(2’-cyclopentenyl)-tridecanoic) and related fatty acids, occur as the major fatty acids of chaulmoogra oil (1). This oil is well known as it was believed for centuries to be effective in treating skin disorders such as human leprosy. Recent work by Cramer and Spener (2,3) has indicated that these cyclopentenyl fatty acids may be synthesized in plants from a 2-cyclopentenecarboxylic acid derivative by its elongation in a way similar to de nova palmitic acid synthesis in bacteria, plants and animals (4). A number of carboxylic acids with 3 to 6 carbons have been shown to be incorporated into the related fatty acids with 13 to 19 carbons by de nova fatty acid synthetase of Bacillus subtdis (5-8). The fatty acids synthesized include w-cyclopentanyl fatty acids (9, 10) which are saturated counterparts of w-cyclopentenyl fatty acids. Thus, this bacterial system offers an excellent opportunity of examining the following important points as to whether (a) this system can synthesize chaulmoogric acid and related cyclopentenyl fatty acids and (b) if so these fatty acids are (+)-isomers (1) as those from plant oils. This information is important not only for a better understanding of chaulmoogric acid biosynthesis general.
but
also
tr,,
stereoselectivity
governing
de nova
fatty
acid
synthesis
in nature
in
EXPERIMENTAL Microorganism and culture conditions: Bacillus subtilis (ATCC 7059) was used throughout experiments. The stock culture was maintained on A-K agar slants (Difco) in cold. Glucose (1%)~yeast extract (0.1%) medium (5) was sterilized as usual by a steam sterilizer. A solution of 2-cyclopentenecarboxylic acid, however, was sterilized by a filtration method at room temperature and added to the pre-sterilized cold glucose-yeast extract medium in a flask. A pre-culture was prepared by inoculating a loop of the stock culture into 30 ml of glucose-yeast extract medium in a 125 ml flask and incubating at 35OC for 5 hr on a shaker. Two milliliters of the ore-culture was inoculated into 1 L of glucose-veast extract medium containing 3 mM cyclopentenecarboxylic acid in a 2 L flask and was-incubated at 35OC for 16 hr on a shaker. At this time, the culture attended on the early stationary phase of growth showing 220-230 Klett units by a KlettSummerson calorimeter with a No. 66 filter. The culture of 12 such flasks was harvested and washed once with cold 0.85% NaCl solution yielding 32-36 g of wet-packed cells. 0006-291 X/81/081 226-04$01.00/O Copyrighf G 1981 by Academic Press, Inc. All rights of reproduction in anv form reserved.
1226
Vol. 99, No. 4,198l
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Isolation of fractionation of bacterial fatty acids: A typical experiment is described below. The total lipids were extracted from the washed wet cells (36 g) with a methanol-chloroform mixture (2: 1 v/v, 520 ml) yielding 379 mg of yellowish oil. The total lipids were saponified with 10% KOH in methanol for 4 hr at room temperature. The total fatty acids were extracted from the saponified sample with n-hexane yielding 182 mg of colorless liquid. The mono-unsaturated fraction was separated from the saturated fraction of the total fatty acids, as methyl ester, by means of AgNO3-silicic acid chromatography (1 1) yielding 43 mg of colorless liquid. Analytical procedure: Fatty acid samples were methylated by reaction with diazomethane for 1 min (12). Two gas-chromatographic columns were used: one, 6 ft by l/8 in O.D. stainless steel tubing packed with 2.5% SE-30 coated on Chromosorb G (loo-120 mesh) (Applied Science Laboratories, State College, PA.), and the other, 50 ft by l/16 in O.D. support-coated open tubular column coated with neopentylglycol adipate olymer (Perkin-Elmer, Norwalk, CT). The SE-30 column was operated isothermally at 220°C with helium as carrier gas at the flow rate of 26 ml per min. The polyester column was also operated isothermally at 165’C with helium at the rate of about 1 ml per min. Equivalent chain length was calculated relative to the methyl esters of normal fatty acids (Applied Science Laboratories) (13). A 5830A Gas Chromatograph (Hewlett Packard, Avondale, PA) was used throughout the present work. Mass spectra were produced by a 270 Gas Chromatograph-Mass Spectrometer system (PerkinElmer) with ionization potential of 70 eV using the SE-30 column at 220°C. Optical rotation was measured in n-hexane by Jasco ORD.CD-5 SS-20-2 modification (Jasco, Easton, MD). Chemicals: 2-(+)--Cyclopentenecarboxylic acid was prepared by Grignard reaction from dicyclopentadiene as starting material (14). The acid was isolated by fractional distillation at 114OC, 17 [M” Hg. The location of unsaturation on cyclopentane ring of the acid as 2-position was confirmed by C-NMR. The detail will be reported elsewhere. Commercial reagent grade n-hexane and petroleum ether were purified by treating with H2SC4 followed by washing with H20 and distillation before used. Other solvents and chemicals were reagent grade and used as received.
RESULTS identification of mono-unsaturated fatty acids, components A and 6: After methylation, the bacterial fatty acids were separated into saturated and mono-unsaturated fractions using an AgN03-silicic acid column. No unsaturated fatty acid with two or more unsaturations was detected. Peak components of the saturated fraction from the same organism have previously been identified as 12-methyltridecanoic, myristic, 13-methyltetradecanoic, 12-methyltetradenoic, 14-methylpentadecanoic, palmitic, 15-methylhexadecanoic, acid by gas-liquid chromatography, melting point and other physical
14-methylhexadecanoic, mass spectrometry, criteria (15).
infrared
16-methylheptadecanoic, spectroscopy, x-ray
and stearic crystallography,
The mono-unsaturated fraction gave two peaks (components A and B) on gas-liquid chromotograms on both SE-30 and polyester columns. These peaks were not detected when the fraction was treated with 82 prior to injection. Equivalent chain length of the two peak components on the two columns is shown in Table 1. The fractions of equivalent chain lengths of components A and B on the two columns are 0.70 and 0.73, respectively, suggesting that both peaks, as expected, are polar compounds. The massspectrum of component A was very similar to that of component B: a fragment peak of component A is m/e 28 less than the corresponding fragment peak of component B with the exception of the base peaks, m/e 67 and m/e 82 (Table 1). Peak B gave the molecular ion of m/e 294, m/e 262 due to the elimination of CH30H from the molecular ion and m/e 213 due to the elimination of the cyclopentene group with an additional methylene group. This is identical to the mass spectrum of methyl chaulmoograte (16). Thus, components A and B were identified as methyl hydrocarpate and methyl chaulmoograte, respectivel!
OPtical Properties of bacterial cyclopentenyl fatty of methyl hydrocarpate and methyl chaulmoograte,
acids: were
1227
The mono-unsaturated fraction, subjected for the measurement
composed of optical
8lOCHEMlCAL
Vol. 99, No. 4,198l
Gas-liquid
AND
chromatographic
BIOPHYSICAL
TABLE 1. and Mass spectrometric components A and B.
Equivalent chain length Mono-unsaturated component
used Polyester
16.40
17.10
B
18.45
19.18
relative
COMMUNICATIONS
characteristics
of
Mass spectrometric
A
a Abundance
rotation. samples
Column SE-30
RESEARCH
Parent (M) 266 (15%)a
peaks,
m/e
Base &
294 (13%)
Others
,10607%,
(M-32) 234 (23%)
(M-81) 185 (22%)
67 (100%)
262 (21%)
213 (15%)
to the base peak intensity.
Both samples contained available for measurement,
these the
esters optical
in the ration of 2:l. rotation was determined
Because of limited
amount of The rotation
as mixtures.
was levoratatory, an average of,-50.4’ (Table 2). This corresponds to the optical the basis of calculated value from the values reported for ethyl esters of plant fatty
purity acids.
of 80%
on
DISCUSSION The present work has shown that the cyclopentenyl fatty acids synthesized from 2-(+I-cyclopentenecarboxylic acid by 6. subtilis are levorotatory, the optical purity of 80%. Because hydrocarpic and chaulmoogric acid from plant oils are both dextrorotatory (11, the following possibilities may be considered for their biosynthetic pathway. In bacteria, one of the isomers of (?I-cyclopentenecarboxylic acid is specifically transformed to the primer precursor, presumable its CoA ester. Then this is elongated to (-)-isomer of cyclopentenyl fatty acids. In plants, Flacourtiaceae, however, of two
the substrate for primer may be a-keto isomers of this a-keto acid is specifically
Optical
Rotation
acid related transformed
TABLE of Cyclopentenyl
to cyclopentenyl to the primer
Code
Concentration Percent
1 3-2
1.4 1.9
Fatty
Acid Methyl
Calculation
hIa
[al a
C16K18
Degree
Degree
-51.8 -49.0
2.0011 1.9611
+62.8 +62.7
d d
b
+65.2
’
Ethyl chaulmoograte
+55.4
tJ
+58.0
’
ester calculated from that of ethyl calculated from values b.
1228
ester.
Optical
Purity
Percent
+61.9
Specific rotation. After reference (1). Specific rotation of methyl Specific rotation of mixture
presumably
Esters
Ethyl hydrocarpate
a b c d
(3). Only
2.
Measurement
Sample
glycine precursor,
82 78
one
Vol. 99, No. 4,198l
cyclopentenecarboxylic Thus, the cyclopentenyl
BIOCHEMICAL
AND
acid CoA ester, which fatty acids synthesized
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
has opposite rotation to that of the bacterial origin. in plants are (+)-isomer. If this is the case, bacterial
de nova fatty acid synthetase should have a stereoselectivity towards the cyclopentenecarboxylic acid primer opposite to that of plant de nova fatty acid synthetase. This appears to contradict the concept of uniformity of biological sytems based on our belief in the evolution of life. Further work is in progress to clarify this apparent contradiction.
ACKNOWLEDGMENTS: I thank Charlotte Stecyk and G.N. Spratt for operation of mass spectrometer.
and Jody
Yu for the excellent
technical
assistance
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.
Markley, K.S. (1960) Fatty Acids pp. 23-249, Interscience Publisher, New York. Cramer, U. and Spener, F. (1976) Biochim. Biophys. Acta. 450, 261-265. Cramer, U. and Spener, F. (1977) Eur. J. Biochem. 74, 495-500. Volpe, J.J. and Vagelos, P.R. (1973) Annu. Rev. Biochem. 42, 21-60. Kaneda, T. (1966) Can. J. Microbial. 12, 501-514. Kaneda, T. (1966) Biochim. Biophys. Acta. 125, 43-54. Kaneda, T. (1971) Biochemistry lU, 340-347. Kaneda, T. (1977) Bacterial. Rev. 41, 391-418. Dreher, R., Poralla, K. and Kong, W.A. (1976) J. Bacterial. 127, 1136-l 140. Kaneda, T. (1977) J. Chromatogr. 136, 323-327. De Vries, B. (1963) J. Amer. Oil Chemists’ Sot. 40, 184-186. Schlenk, M. and Gellerman, J.L. (1960) Anal. Chem. 32, 1412-1414. Ackman, R.G. (1969) Methods in Enzymology,voll4, pp. 329-381, Academic Press, New York. Branner-Jorgensen, S. and Berg, A. (1966) Acta Chem. Stand. 20,2192-2194. Kaneda, T. (1963) J. Biof. Chem. 238, 1222-1228. McCloskey, J.A. (1969) Methods in Enzymology, vol 14 pp. 382-450, Academic Press, New York.
99, No. 4,198l 30,
BIOCHEMICAL
AND
BIOPHYSICAL
COMMUNICATIONS
Pages 1226-1229
1981
STEREOSELECTIVE RELATED
FATTY ACID
SYNTHESIS ACID FROM BY BACILLUS
OF
Alberta
March
Research
Council,
CHAULMOOGRIC
ACID
AND
2-(i)-CYCLOPENTENECARBOXYLIC SUBTILIS (ATCC 7059)
Toshi
Received
RESEARCH
Kaneda Edmonton,
Alberta,
Canada
11,1981
Received SUMMARY: 2-(+)-Cyclopentenecarboxylic acid added to the culture medium is incorporated into two new fatty acids by the growing cells of Bacillus subtilis (ATCC 7059). The new fatty acids, amounting to 24% of the total cellular fatty acids, are identified as hydrocarpic [ 11-(2’-cyclopentenvl)-hendecanoic] and chaulmoogric [13-(2’-cvclopentenvI)-tridecanoic] bv gas-liquid chromatography and mass spectrometry. These Cl6 and Cl .fatty acids are optically active, levorotatorv. with the specific rotation of -50.4O as mixture. thus t a e ootical ouritv of aooroximatelv 80%. This ‘indicates that the optical rotation of these bacterial fatty ‘acids are opposite with that ‘of the fatty acids from plant oils. Unusual w-cyclic fatty acids, (+)-chaulmoogric acid [ 13-(2’-cyclopentenyl)-tridecanoic) and related fatty acids, occur as the major fatty acids of chaulmoogra oil (1). This oil is well known as it was believed for centuries to be effective in treating skin disorders such as human leprosy. Recent work by Cramer and Spener (2,3) has indicated that these cyclopentenyl fatty acids may be synthesized in plants from a 2-cyclopentenecarboxylic acid derivative by its elongation in a way similar to de nova palmitic acid synthesis in bacteria, plants and animals (4). A number of carboxylic acids with 3 to 6 carbons have been shown to be incorporated into the related fatty acids with 13 to 19 carbons by de nova fatty acid synthetase of Bacillus subtdis (5-8). The fatty acids synthesized include w-cyclopentanyl fatty acids (9, 10) which are saturated counterparts of w-cyclopentenyl fatty acids. Thus, this bacterial system offers an excellent opportunity of examining the following important points as to whether (a) this system can synthesize chaulmoogric acid and related cyclopentenyl fatty acids and (b) if so these fatty acids are (+)-isomers (1) as those from plant oils. This information is important not only for a better understanding of chaulmoogric acid biosynthesis general.
but
also
tr,,
stereoselectivity
governing
de nova
fatty
acid
synthesis
in nature
in
EXPERIMENTAL Microorganism and culture conditions: Bacillus subtilis (ATCC 7059) was used throughout experiments. The stock culture was maintained on A-K agar slants (Difco) in cold. Glucose (1%)~yeast extract (0.1%) medium (5) was sterilized as usual by a steam sterilizer. A solution of 2-cyclopentenecarboxylic acid, however, was sterilized by a filtration method at room temperature and added to the pre-sterilized cold glucose-yeast extract medium in a flask. A pre-culture was prepared by inoculating a loop of the stock culture into 30 ml of glucose-yeast extract medium in a 125 ml flask and incubating at 35OC for 5 hr on a shaker. Two milliliters of the ore-culture was inoculated into 1 L of glucose-veast extract medium containing 3 mM cyclopentenecarboxylic acid in a 2 L flask and was-incubated at 35OC for 16 hr on a shaker. At this time, the culture attended on the early stationary phase of growth showing 220-230 Klett units by a KlettSummerson calorimeter with a No. 66 filter. The culture of 12 such flasks was harvested and washed once with cold 0.85% NaCl solution yielding 32-36 g of wet-packed cells. 0006-291 X/81/081 226-04$01.00/O Copyrighf G 1981 by Academic Press, Inc. All rights of reproduction in anv form reserved.
1226
Vol. 99, No. 4,198l
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Isolation of fractionation of bacterial fatty acids: A typical experiment is described below. The total lipids were extracted from the washed wet cells (36 g) with a methanol-chloroform mixture (2: 1 v/v, 520 ml) yielding 379 mg of yellowish oil. The total lipids were saponified with 10% KOH in methanol for 4 hr at room temperature. The total fatty acids were extracted from the saponified sample with n-hexane yielding 182 mg of colorless liquid. The mono-unsaturated fraction was separated from the saturated fraction of the total fatty acids, as methyl ester, by means of AgNO3-silicic acid chromatography (1 1) yielding 43 mg of colorless liquid. Analytical procedure: Fatty acid samples were methylated by reaction with diazomethane for 1 min (12). Two gas-chromatographic columns were used: one, 6 ft by l/8 in O.D. stainless steel tubing packed with 2.5% SE-30 coated on Chromosorb G (loo-120 mesh) (Applied Science Laboratories, State College, PA.), and the other, 50 ft by l/16 in O.D. support-coated open tubular column coated with neopentylglycol adipate olymer (Perkin-Elmer, Norwalk, CT). The SE-30 column was operated isothermally at 220°C with helium as carrier gas at the flow rate of 26 ml per min. The polyester column was also operated isothermally at 165’C with helium at the rate of about 1 ml per min. Equivalent chain length was calculated relative to the methyl esters of normal fatty acids (Applied Science Laboratories) (13). A 5830A Gas Chromatograph (Hewlett Packard, Avondale, PA) was used throughout the present work. Mass spectra were produced by a 270 Gas Chromatograph-Mass Spectrometer system (PerkinElmer) with ionization potential of 70 eV using the SE-30 column at 220°C. Optical rotation was measured in n-hexane by Jasco ORD.CD-5 SS-20-2 modification (Jasco, Easton, MD). Chemicals: 2-(+)--Cyclopentenecarboxylic acid was prepared by Grignard reaction from dicyclopentadiene as starting material (14). The acid was isolated by fractional distillation at 114OC, 17 [M” Hg. The location of unsaturation on cyclopentane ring of the acid as 2-position was confirmed by C-NMR. The detail will be reported elsewhere. Commercial reagent grade n-hexane and petroleum ether were purified by treating with H2SC4 followed by washing with H20 and distillation before used. Other solvents and chemicals were reagent grade and used as received.
RESULTS identification of mono-unsaturated fatty acids, components A and 6: After methylation, the bacterial fatty acids were separated into saturated and mono-unsaturated fractions using an AgN03-silicic acid column. No unsaturated fatty acid with two or more unsaturations was detected. Peak components of the saturated fraction from the same organism have previously been identified as 12-methyltridecanoic, myristic, 13-methyltetradecanoic, 12-methyltetradenoic, 14-methylpentadecanoic, palmitic, 15-methylhexadecanoic, acid by gas-liquid chromatography, melting point and other physical
14-methylhexadecanoic, mass spectrometry, criteria (15).
infrared
16-methylheptadecanoic, spectroscopy, x-ray
and stearic crystallography,
The mono-unsaturated fraction gave two peaks (components A and B) on gas-liquid chromotograms on both SE-30 and polyester columns. These peaks were not detected when the fraction was treated with 82 prior to injection. Equivalent chain length of the two peak components on the two columns is shown in Table 1. The fractions of equivalent chain lengths of components A and B on the two columns are 0.70 and 0.73, respectively, suggesting that both peaks, as expected, are polar compounds. The massspectrum of component A was very similar to that of component B: a fragment peak of component A is m/e 28 less than the corresponding fragment peak of component B with the exception of the base peaks, m/e 67 and m/e 82 (Table 1). Peak B gave the molecular ion of m/e 294, m/e 262 due to the elimination of CH30H from the molecular ion and m/e 213 due to the elimination of the cyclopentene group with an additional methylene group. This is identical to the mass spectrum of methyl chaulmoograte (16). Thus, components A and B were identified as methyl hydrocarpate and methyl chaulmoograte, respectivel!
OPtical Properties of bacterial cyclopentenyl fatty of methyl hydrocarpate and methyl chaulmoograte,
acids: were
1227
The mono-unsaturated fraction, subjected for the measurement
composed of optical
8lOCHEMlCAL
Vol. 99, No. 4,198l
Gas-liquid
AND
chromatographic
BIOPHYSICAL
TABLE 1. and Mass spectrometric components A and B.
Equivalent chain length Mono-unsaturated component
used Polyester
16.40
17.10
B
18.45
19.18
relative
COMMUNICATIONS
characteristics
of
Mass spectrometric
A
a Abundance
rotation. samples
Column SE-30
RESEARCH
Parent (M) 266 (15%)a
peaks,
m/e
Base &
294 (13%)
Others
,10607%,
(M-32) 234 (23%)
(M-81) 185 (22%)
67 (100%)
262 (21%)
213 (15%)
to the base peak intensity.
Both samples contained available for measurement,
these the
esters optical
in the ration of 2:l. rotation was determined
Because of limited
amount of The rotation
as mixtures.
was levoratatory, an average of,-50.4’ (Table 2). This corresponds to the optical the basis of calculated value from the values reported for ethyl esters of plant fatty
purity acids.
of 80%
on
DISCUSSION The present work has shown that the cyclopentenyl fatty acids synthesized from 2-(+I-cyclopentenecarboxylic acid by 6. subtilis are levorotatory, the optical purity of 80%. Because hydrocarpic and chaulmoogric acid from plant oils are both dextrorotatory (11, the following possibilities may be considered for their biosynthetic pathway. In bacteria, one of the isomers of (?I-cyclopentenecarboxylic acid is specifically transformed to the primer precursor, presumable its CoA ester. Then this is elongated to (-)-isomer of cyclopentenyl fatty acids. In plants, Flacourtiaceae, however, of two
the substrate for primer may be a-keto isomers of this a-keto acid is specifically
Optical
Rotation
acid related transformed
TABLE of Cyclopentenyl
to cyclopentenyl to the primer
Code
Concentration Percent
1 3-2
1.4 1.9
Fatty
Acid Methyl
Calculation
hIa
[al a
C16K18
Degree
Degree
-51.8 -49.0
2.0011 1.9611
+62.8 +62.7
d d
b
+65.2
’
Ethyl chaulmoograte
+55.4
tJ
+58.0
’
ester calculated from that of ethyl calculated from values b.
1228
ester.
Optical
Purity
Percent
+61.9
Specific rotation. After reference (1). Specific rotation of methyl Specific rotation of mixture
presumably
Esters
Ethyl hydrocarpate
a b c d
(3). Only
2.
Measurement
Sample
glycine precursor,
82 78
one
Vol. 99, No. 4,198l
cyclopentenecarboxylic Thus, the cyclopentenyl
BIOCHEMICAL
AND
acid CoA ester, which fatty acids synthesized
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
has opposite rotation to that of the bacterial origin. in plants are (+)-isomer. If this is the case, bacterial
de nova fatty acid synthetase should have a stereoselectivity towards the cyclopentenecarboxylic acid primer opposite to that of plant de nova fatty acid synthetase. This appears to contradict the concept of uniformity of biological sytems based on our belief in the evolution of life. Further work is in progress to clarify this apparent contradiction.
ACKNOWLEDGMENTS: I thank Charlotte Stecyk and G.N. Spratt for operation of mass spectrometer.
and Jody
Yu for the excellent
technical
assistance
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.
Markley, K.S. (1960) Fatty Acids pp. 23-249, Interscience Publisher, New York. Cramer, U. and Spener, F. (1976) Biochim. Biophys. Acta. 450, 261-265. Cramer, U. and Spener, F. (1977) Eur. J. Biochem. 74, 495-500. Volpe, J.J. and Vagelos, P.R. (1973) Annu. Rev. Biochem. 42, 21-60. Kaneda, T. (1966) Can. J. Microbial. 12, 501-514. Kaneda, T. (1966) Biochim. Biophys. Acta. 125, 43-54. Kaneda, T. (1971) Biochemistry lU, 340-347. Kaneda, T. (1977) Bacterial. Rev. 41, 391-418. Dreher, R., Poralla, K. and Kong, W.A. (1976) J. Bacterial. 127, 1136-l 140. Kaneda, T. (1977) J. Chromatogr. 136, 323-327. De Vries, B. (1963) J. Amer. Oil Chemists’ Sot. 40, 184-186. Schlenk, M. and Gellerman, J.L. (1960) Anal. Chem. 32, 1412-1414. Ackman, R.G. (1969) Methods in Enzymology,voll4, pp. 329-381, Academic Press, New York. Branner-Jorgensen, S. and Berg, A. (1966) Acta Chem. Stand. 20,2192-2194. Kaneda, T. (1963) J. Biof. Chem. 238, 1222-1228. McCloskey, J.A. (1969) Methods in Enzymology, vol 14 pp. 382-450, Academic Press, New York.