47NISR Research GRANT
2009 Young Investigator Research Grant

Analysis of Peculiar Fatty Acid Transformation by Anaerobic
Microorganisms and Application to Conjugated Fatty Acid
Production
Shigenobu KISHINO
Graduate School of Agriculture, Kyoto University

Research aims
Various fatty acids with conjugated double bonds occur
in nature. For example, edible fats derived from ruminantanimals contain conjugated linoleic acid (CLA), whichmainly consists of cis-9,trans-11- and trans-10,cis-12-octadecadienoic acid (18 : 2). The occurrence of conjugatedfatty acids has also been reported in plants, for example,cis-9,trans-11,trans-13-octadecatrienoic acid (18 : 3) in Momordica charantia seed oil. The secondary metabolism of
fatty acids by marine algae involves polyunsaturated fatty
acids (PUFAs) containing conjugated olefin systems, forexample, cis-5,trans-7,trans-9,cis-14-eicosatetraenoic acid
(20 : 4) produced from arachidonic acid (cis-5,cis-8,cis11,cis-14-20 : 4). These conjugated fatty acids have attracted much attention as a novel type of biologically beneficial functional lipid1–3). In particular, the unique activities
of CLA have been intensively studied, and there are many
reports that CLA reduces carcinogenesis, atherosclerosis,
and body fat4,5). So, conjugated fatty acids are expected tobe potential materials for pharmaceuticals and dietary supplements.
In this study, I examined whether Clostridium bifermentans, which was previously shown to have the ability to produce CLA from linoleic acid, can transform various PUFAs
into conjugated fatty acids. The results demonstrated that a
Table 1.

variety of conjugated fatty acids could be produced fromPUFAs by the action of C. bifermentans. A putative reaction pathway for the observed fatty acid transformation wasproposed based on the results obtained.
Methods
Clostridium bifermentans was inoculated into GAM
medium and cultured for 3 days. Each transformation reaction was performed at 37°C in a 1.0 ml of reaction mixture
containing 0.6 mg the fatty acid (as the substrate), washedcells and 0.1 M potassium phosphate buffer (pH 6.5). Afterreactions, lipid extraction and methyl esterification werecarried out and fatty acid methyl esters were then separated
by gas chromatography for analysis and by high-performance liquid chromatography for purification. Structures ofpurified methyl esters were identified by NMR and MS
analysis.
Results
In order to examine substrate specificity of the reactions
catalyzed by washed cells of Clostridium bifermentans, anumber of unsaturated fatty acids were subjected as thesubstrate in the reaction. The results were summarized atTable 1. A wide range of C18 and C20 unsaturated fatty acids

Production of conjugated fatty acids by C. bifermentans
C18 FAs

Substrate

Linoleic acida -Linolenic acidg -Linolenic acid
Columbinic acid
Stearidonic acid

(9Z,12Z-18:2)(9Z,12Z,15Z-18:3)(6Z,9Z,12Z-18:3)(5E,9Z,12Z-18:3)(6Z,9Z,12Z,15Z-18:4)

C20 FAs

Substrate

Eicosadienoic acid
Eicosatrienoic acid
Dihomo-g -linolenic acidw -3 Arachidonic acidArachidonic acid
Eicasapentaenoic acidZ, cis configuration; E, trans configuration(11Z,14Z-20:2)(11Z,14Z,17Z-20:3)(8Z,11Z,14Z-20:3)(8Z,11Z,14Z,17Z-20:4)(5Z,8Z,11Z,14Z-20:4)(5Z,8Z,11Z,14Z,17Z-20:4)

Products(9Z,11E-18:2, 9E,11E-18:2)(9Z,11E,15Z-18:3, 9E,11E,15Z-18:3)(6Z,9Z,11E-18:3, 6Z,9E,11E-18:3)(5E,9Z,11E-18:3, 5E,9E,11E-18:3)(6Z,9Z,11E,15Z-18:4, 6Z,9E,11E,15Z-18:4)
Products(11Z,13E-20:2, 11E,13E-20:2)(11Z,13E,17Z-20:3, 11E,13E,17Z-20:3)(8Z,11Z,13E-20:3, 8Z,11E,13E-20:3)(8Z,11Z,13E,17Z-20:4, 8Z,11E,13E,17Z-20:4)(5Z,8Z,11Z,13E-20:4, 5Z,8Z,11E,13E-20:4)(5Z,8Z,11Z,13E,17Z-20:5, 5Z,8Z,11E,13E,17Z-20:5)

48

Fig. 1.

Time course of EPA and AA transformation by C. bifermentans.

Scheme 1.

Putative pathway of polyunsaturated fatty acid transformation by C. bifermentans.

could be reacted with cells of C. bifermentans and wereconverted to their respective conjugated isomers. Polyunsaturated fatty acids that could be substrates for the transformation reaction mediated by C. bifermentans included a linolenic acid, g -linolenic acid, columbinic acid, stearidonicacid, eicosadienoic acid, eicosatrienoic acid, dihomo-g linolenic acid, w 3 arachidonic acid, arachidonic acid (AA),and eicosapentaenoic acid (EPA) with cis double bonds atw 6 and w 9 position.
When cells of C. bifermentans were incubated withlinoleic acid, AA, or EPA for longer periods of time, additional fatty acids were produced concomitantly with thegeneration of conjugated fatty acids. These fatty acids wereidentified as trans-11-octadecenoic acid from linoleic acid,cis-5,cis-8,trans-13-eicosatrienoic acid from AA, and cis5,cis-8,trans-13,cis-17-eicosatetraenoic acid from EPA. The
time courses of EPA and AA conversion were monitored(Fig. 1). EPA and AA were initially converted to conjugated
EPA (CEPAs) and conjugated AA (CAAs), respectively.
When the reactions were allowed to proceed for longer
time, the amounts of CEPAs and CAAs were decreased

with the concomitant increase in the amounts of saturated
EPA and saturated AA, respectively (Fig. 1).
These results suggested that this strain recognizes w 6and w 9 double bonds in the substrate fatty acids and rapidly
converts cis-w 6,cis-w 9 to trans-w 7,cis-w 9 and transw 7,trans-w 9, and finally reduces the double bond at w 9
position in conjugated fatty acid (Scheme 1).
References1)

Nagao, S. and Yanagita, T. (2005) Conjugated fatty acids in food andtheir health benefits. J. Biosci. Bioeng. 100, 152–157.
2) Nagao, S. and Yanagita, T. (2008) Bioactive lipids in metabolic syndrome. Prog. Lipid Res. 47, 127–146.
3) Kishino, S. (2008) Physiologically active of conjugated fatty acids.
Vitamin, 82, 655–657.
4) Pariza, M.W., Park, Y. and Cook, M. (2001) The biologically activity
isomers of conjugated linoleic acid. Prog. Lipid Res. 40, 283–298.
5) Toomey, S., McMonagle, J. and Roche, H. (2006) Conjugatedlinoleic acid: a functional nutrient in the different pathophysiologicalcomponents of the metabolic syndrome? Curr. Opin. Clin. Nutr.
Metab. Care. 9, 740–747.