Δ12-Oleate Desaturase-related Enzymes Associated with Formation of Conjugated trans-Δ11, cis-Δ13 Double Bonds

Abstract

Conjugated linolenic acids are present as major seed oils in several plant species. Punicic acid (or trichosanic acid) is a conjugated linolenic acid isomer containingcis-Δ9, trans-Δ11, cis-Δ13 double bonds in the C18 carbon chain. Here we report cDNAs, TkFac and PgFac, isolated from Trichosanthes kirilowii and Punica granatum, that encode a class of conjugases associated with the formation of trans-Δ11,cis-Δ13 double bonds. Expression of TkFac and PgFac inArabidopsis seeds under transcriptional control of the seed-specific napin promoter resulted in accumulation of punicic acid up to ∼10% (w/w) of the total seed oils. In contrast, no punicic acid was found in lipids from leaves even when the conjugases were driven under control of the cauliflower mosaic virus 35S promoter. In yeast cells grown without exogenous fatty acids in the culture medium, TkFac and PgFac expression resulted in punicic acid accumulation accompanied by 16:2Δ9cis, 12cisand 18:2Δ9cis, 12cis production. Thus, TkFac and PgFac are defined as bifunctional enzymes having both conjugase and Δ12-oleate desaturase activity. Furthermore, we demonstrate that 16:2Δ9cis, 12cis and 18:3Δ9cis, 12cis, 15cisas well as 18:2Δ9cis, 12cis are potential substrates for the conjugases to form trans-Δ11 and cis-Δ13 double bonds. Conjugated linolenic acids are present as major seed oils in several plant species. Punicic acid (or trichosanic acid) is a conjugated linolenic acid isomer containingcis-Δ9, trans-Δ11, cis-Δ13 double bonds in the C18 carbon chain. Here we report cDNAs, TkFac and PgFac, isolated from Trichosanthes kirilowii and Punica granatum, that encode a class of conjugases associated with the formation of trans-Δ11,cis-Δ13 double bonds. Expression of TkFac and PgFac inArabidopsis seeds under transcriptional control of the seed-specific napin promoter resulted in accumulation of punicic acid up to ∼10% (w/w) of the total seed oils. In contrast, no punicic acid was found in lipids from leaves even when the conjugases were driven under control of the cauliflower mosaic virus 35S promoter. In yeast cells grown without exogenous fatty acids in the culture medium, TkFac and PgFac expression resulted in punicic acid accumulation accompanied by 16:2Δ9cis, 12cisand 18:2Δ9cis, 12cis production. Thus, TkFac and PgFac are defined as bifunctional enzymes having both conjugase and Δ12-oleate desaturase activity. Furthermore, we demonstrate that 16:2Δ9cis, 12cis and 18:3Δ9cis, 12cis, 15cisas well as 18:2Δ9cis, 12cis are potential substrates for the conjugases to form trans-Δ11 and cis-Δ13 double bonds. A large number of fatty acid species have been found in plant seed oils. Typically, plant seeds contain saturated and unsaturated fatty acids, such as palmitic (16:0), palmitoleic (16:1Δ9cis), stearic (18:0), oleic (18:1Δ9cis), linoleic (18:2Δ9cis, 12cis), and α-linolenic (18:3Δ9cis, 12cis, 15cis) acids. These are typical fatty acids with all other fatty acids regarded as unusual. Typically, polyunsaturation of fatty acids is methylene (-CH2-)-interrupted and occurs incis-configuration as found in linoleic and linolenic acids. In contrast, conjugated (non-methylene-interrupted) fatty acids contain double bonds in cis- or trans-configuration. The conjugated fatty acids occur as diene, triene, and tetraene in which the most common conjugated polyenoic acids are octadecatrienoic acids, termed CLNAs. 1The abbreviations used are: CLNA, conjugated linolenic acid; CaMV, cauliflower mosaic virus; FAD2, Δ12-oleate desaturase; GC, gas chromatography; MS, mass spectrometry; FAME, fatty acid methyl ester; CLA, conjugated linoleic acid 1The abbreviations used are: CLNA, conjugated linolenic acid; CaMV, cauliflower mosaic virus; FAD2, Δ12-oleate desaturase; GC, gas chromatography; MS, mass spectrometry; FAME, fatty acid methyl ester; CLA, conjugated linoleic acid Positional and geometrical isomers of CLNA, three 8,10,12-trienes and four 9,11,13-trienes, have been reported to occur naturally (1Smith Jr., C.R. Prog. Chem. Fats Other Lipids. 1970; 11: 137-177Crossref Scopus (99) Google Scholar). Five CLNA isomers occur as major seed oils of several plants: α-eleostearic (cis-Δ9, trans-Δ11, trans-Δ13), calendic (trans-Δ8, trans-Δ10,cis-Δ12), punicic (cis-Δ9,trans-Δ11, cis-Δ13), jacaric (cis-Δ8, trans-Δ10, cis-Δ12), and catalpic (trans-Δ9, trans-Δ11,cis-Δ13) acids (1Smith Jr., C.R. Prog. Chem. Fats Other Lipids. 1970; 11: 137-177Crossref Scopus (99) Google Scholar). These isomers have closely related structure; for example, α-eleostearic and jacaric acids are geometrical isomers of punicic and calendic acids, respectively. CLNAs are major seed oils in plants such as tung (Aleurites fordii), karela (Momordica charantia), marigold (Calendula officinalis), and pomegranate (Punica granatum). Tung oil contains high levels of 9,11,13-triene (α-eleostearic acid) and is used mainly in quick-drying enamels and varnishes. There also is growing evidence showing that supplementation with CLNA has cytotoxic effects on tumor cells and that uptake of CLNA has an effect on lipid metabolism (2Igarashi M. Miyazawa T. Cancer Lett. 2000; 148: 173-179Crossref PubMed Scopus (153) Google Scholar, 3Suzuki R. Noguchi R. Ota T. Abe M. Miyashita K. Kawada T. J. Am. Oil Chem. Soc. 2001; 36: 477-482Google Scholar, 4Koba K. Akahoshi A. Yamasaki M. Tanaka K. Yamada K. Iwata T. Kamegai T. Tsutsumi K. Sugano M. J. Am. Oil Chem. Soc. 2002; 37: 343-350Google Scholar). Conjugated eicosapentaenoic and docosahexaenoic acids with conjugated trienoic structure also exhibit cytotoxic effects on tumor cells (5Igarashi M. Miyazawa T. Biochem. Biophys. Res. Commun. 2000; 270: 649-656Crossref PubMed Scopus (70) Google Scholar). It has been further suggested that the biological action of each conjugated fatty acid may not be equivalent (3Suzuki R. Noguchi R. Ota T. Abe M. Miyashita K. Kawada T. J. Am. Oil Chem. Soc. 2001; 36: 477-482Google Scholar). Previous studies have indicated that linoleic acid is the acyl precursor of α-eleostearic acid and linoleoyl phosphatidylcholine is the precursor of α-eleostearoyl phosphatidylcholine (6Liu L. Hammond G. Nikolau B.J. Plant Physiol. 1997; 113: 1343-1349Crossref PubMed Scopus (28) Google Scholar). Recently cDNAs encoding enzymes that catalyze the formation of the conjugated double bonds in CLNA have been identified. These enzymes were termed conjugases and were shown to be divergent forms of Δ12-oleate desaturase (FAD2). Two types of conjugases associated with the formation of conjugated double bonds intrans-configuration have been identified: one catalyzes the conversion of a cis-Δ12 double bond into the conjugatedtrans-Δ11, trans-Δ13 double bonds found in α-eleostearic acid (7Cahoon E.B. Carlson T.J. Ripp K.G. Schweiger B.J. Cook G.A. Hall S.E. Kinney A.J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12935-12940Crossref PubMed Scopus (163) Google Scholar), and the other modifies a cis-Δ9 double bond into the trans-Δ8, trans-Δ10 double bonds of calendic acid (8Cahoon E.B. Ripp K.G. Hall S.E. Kinney A.J. J. Biol. Chem. 2001; 276: 2637-2643Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 9Qiu X. Reed D.W. Hong H. MacKenzie S.L. Covello P.S. Plant Physiol. 2001; 125: 847-855Crossref PubMed Scopus (42) Google Scholar). Very recently a class of conjugase associated with the formation of cis-Δ11,trans-Δ13 double bonds of punicic acid was identified (10Hornung E. Pernstich C. Feussner I. Eur. J. Biochem. 2002; 269: 4852-4859Crossref PubMed Scopus (78) Google Scholar). Although the primary structures of these conjugases are similar, each enzyme specifically catalyzes the formation of α-eleostearic, punicic, or calendic acids. Other classes of conjugases associated with the formation of conjugated double bonds in jacaric and catalpic acids have not been reported. It is known that Trichosanthes kirilowii and P. granatum accumulate punicic acid specifically in seeds up to ∼40 and ∼80% (w/w) of the total seed oil, respectively (11Joh Y.-G. Kim S.-J. Christie W. J. Am. Oil Chem. Soc. 1995; 72: 1037-1042Crossref Scopus (37) Google Scholar, 12Takagi T. Itabashi Y. Lipids. 1981; 16: 546-551Crossref Scopus (100) Google Scholar). The biochemical process resulting in high punicic acid accumulation in the seeds of these plants, however, has not been clear. In this study, for the first step in understanding CLNA accumulation in plant seed oils, we isolated cDNAs that encode enzymes associated with the formation of punicic acid from T. kirilowii andP. granatum and expressed them in Arabidopsisplants. We also analyzed the function of these enzymes in yeast cells. Interestingly the enzymes were demonstrated to possess both Δ12-oleate desaturase and conjugase activities. The finding supports the idea that conjugases have diverged from Δ12-oleate desaturase. The growing number of identified primary structures of fatty acid-modifying enzymes provides valuable information to understand the functional divergence of these enzymes. Total RNA was isolated from maturing seeds of T. kirilowii and P. granatum by the methods of Carpenter et al. (13Carpenter C.D. Simon A. Methods Mol. Biol. 1998; 82: 85-89PubMed Google Scholar). The first strand was with an and from the total RNA and used for were to acid in enzymes. T. a of and were used to the acid and respectively. P. granatum, and were used to the acids and respectively. was with with of and by an step for The were into and a The that types of closely related to were isolated in each kirilowii and and P. granatum and The strand was with a to the double strand cDNAs and and of was to the and were into and cDNAs of four cDNAs were isolated by with a of to the in and The of of and by an step for The was with for The from four for a of were into and as The of TkFac and PgFac were by of the was with a and the to the from the of TkFac with a of PgFac, the was with and the to the from the of PgFac with a The and into were as was with and and of the plant expression with the 35S promoter and and the were from and from with and into the of a C. M. H. J. Plant PubMed Scopus Google in which has been plant expression with a seed-specific promoter of napin M. L. J. Biol. Chem. Full Text PDF PubMed Google and the were into J. M. H. J. Plant Mol. Biol. PubMed Scopus Google in which the been with A. was with the conjugase by methods J. G. Sci. Scholar). Five plants were under with a J. PubMed Scopus Google the conjugase was in yeast and with and the were in and for under and in a The the plants were and to for in a under the the were seeds were and for of were by for in by in and with were for by on and The of TkFac from as was into the yeast expression with the promoter to was with and into the and the PgFac was and into to The of and were by and into as of the was with a and the to the from the of with a of the was with and the to the from the of with a was with and the was into the of The was and into The was from and into The resulting as well as were into S. cells M. J. J. Biochem. 1995; PubMed Scopus Google an S. cells were for on yeast R. K. A. in Scholar). of the cells were grown in culture for with were by in and in culture The was in culture and grown for by for with in the or of or acid cells were by and in and three in The cells were into and under The were with in for the were to the was with The were under and in a of and was used for gas or fatty acid of seeds were with a and and to of in were in of in T. kirilowii and P. granatum, of seeds without seed were used for fatty acid The were to and for the were to of was to the and fatty acid methyl were with of for the was to a and under The were in a of and was used for or acid methyl were analyzed and a gas with a The was to from to a of and for was in a mass to a gas were under as Total fatty acids from maturing seeds of T. P. granatum were analyzed by seeds punicic acid levels of (w/w) and (w/w) of total fatty acids and We RNA from these and isolated four cDNAs from T. kirilowii and and P. granatum and that encode related to These contain three of that are to as to the that have been to form a J. E. PubMed Scopus Google Scholar). of acid of fatty acid-modifying enzymes and and and with Δ12-oleate and that several acids were in Δ12-oleate from a number of plant species. these acids were in and we that these were Δ12-oleate the other the indicated that TkFac and PgFac were a conjugase that were and encode and acids and and acid with the Δ12-oleate the other TkFac and PgFac encode and acids and and acid to Δ12-oleate respectively. PgFac to other conjugases from M. E.B. Carlson T.J. Ripp K.G. Schweiger B.J. Cook G.A. Hall S.E. Kinney A.J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12935-12940Crossref PubMed Scopus (163) Google Scholar), from (7Cahoon E.B. Carlson T.J. Ripp K.G. Schweiger B.J. Cook G.A. Hall S.E. Kinney A.J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12935-12940Crossref PubMed Scopus (163) Google Scholar), and from C. (8Cahoon E.B. Ripp K.G. Hall S.E. Kinney A.J. J. Biol. Chem. 2001; 276: 2637-2643Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 9Qiu X. Reed D.W. Hong H. MacKenzie S.L. Covello P.S. Plant Physiol. 2001; 125: 847-855Crossref PubMed Scopus (42) Google TkFac of to to and to PgFac to The TkFac and may that the species to the of from yeast cells TkFac and PgFac that were grown in the of exogenous linoleic from and cells were analyzed by granatum seeds were analyzed for The to methyl of fatty acids are as palmitic acid; palmitoleic acid; acid stearic acid; oleic acid; and linoleic methyl punicic acid; a methyl of which was identified as of from T. kirilowii and P. of acid of from T. and and P. and with of A. of are indicated by acids to in the TkFac are indicated in are indicated of and conjugase the The the to the of were from I. and C. and M. Δ12-oleate C. and A. Δ12-oleate TkFac and PgFac are associated with the formation of punicic the cDNAs encoding TkFac and PgFac were expressed inArabidopsis plants under control of the 35S promoter and or the seed-specific napin promoter and were by and by and fatty acid methyl from leaves of plants were analyzed by gas Although was no in the fatty acid of and plants not a of and which was not present in seeds from was found in seeds of The a gas to that of the methyl of punicic acid kirilowii and from of α-eleostearic and calendic acids from M. and C. respectively not The mass of this was to that of the methyl punicic acid from T. the which was identified as a methyl of an isomer of octadecatrienoic acid and other were not these we that TkFac and PgFac are associated with the formation of the conjugated trans-Δ11,cis-Δ13 double bonds of punicic In to the of methyl punicic an of and which was not found in seeds from plants, was in from The mass of this was by an and other which was identified as a methyl of an isomer of acid a gas from that of the methyl in both and We analyzed of yeast cells with to this fatty acid was as a of TkFac and PgFac to conjugated we not a to the methyl isomer found in not We that a of punicic acid was to form the conjugated 15cis) as seeds possess fatty acid D.W. E. J. I. E. Plant 1995; PubMed Scopus Google Scholar, L. Plant J. 1997; PubMed Scopus Google Scholar). In all plants, punicic acid was in seeds not in not We the fatty acid of seed oils in and plants of punicic acid in seed oils was (w/w) in and (w/w) in not The of punicic acid was in seeds from and the napin and respectively. In contrast, in and were The of the fatty acid identified as in to that of punicic acid and to as as (w/w) of seed oils in the seeds (w/w) punicic acid not Punicic acid accumulation inArabidopsis seeds was accompanied by in of other fatty acids The was in seeds of with cDNAs driven by the napin promoter. of linoleic (18:2Δ9cis, and linolenic acids (18:3Δ9cis, 12cis, which were (w/w) and (w/w) in were in and respectively. In contrast, the of oleic acid was in seeds of plants in of plants acid of seeds from plants and from plants TkFac and 12cis, identified as and are as of the total fatty acids The were from in of seeds from not 35S napin identified as and in a are as of the total fatty acids The were from in of seeds from not 35S napin promoter. further the function of the each was expressed under control of the promoter in yeast cells. each was expressed in yeast cells grown in culture without exogenous fatty acids, and fatty acid were with control yeast cells with In yeast cells and of were that were not present in of control cells and not These were to be methyl of 16:2Δ9cis, 12cis and 18:2Δ9cis, 12cis by gas and mass not We not the to the methyl punicic acid in a large of linoleic acid was present (w/w) of the total fatty acids, and not We that and were Δ12-oleate yeast cells PgFac 16:2Δ9cis, 12cis and 18:2Δ9cis, 12cis which were also found in cells in of (w/w) and respectively and Although TkFac and PgFac were demonstrated to be the enzymes associated with punicic acid formation in plants, these enzymes were shown to possess Δ12-oleate desaturase in to conjugase activity. In to 16:2Δ9cis, 12cisand 12cis, of were found in cells grown in the of exogenous fatty acids. The indicated by was to be methyl punicic acid by not and by gas of punicic acid in yeast cells grown with linoleic acid were in grown without linoleic acid and the of linoleic acid to the medium, punicic acid was of and (w/w) of the total fatty acids of and respectively and In contrast, no or a of punicic acid was in or cells grown without exogenous linoleic acid indicated by an was also found in yeast cells grown with linoleic acid was by a and other with that of a methyl of acid a gas from 12cis, not The of 16:2Δ9cis, 12cis in cells was in the cells grown with exogenous linoleic acid in grown without linoleic acid of of linoleic acid from the into the lipid of the cells of 16:2Δ9cis, 12cis the 16:2Δ9cis, in cells linoleic the of the isomer was also that the isomer of was by PgFac We a of of the isomer and punicic acid of a large of from cells grown without exogenous fatty acids not we that TkFac and PgFac are in function to each yeast cells TkFac and PgFac were grown with exogenous α-linolenic and of these cells were with of control cells by gas We a of FAME, which was not found in cells grown with linoleic acid not mass the was identified as a methyl of acid which was by a and other not the conjugases were shown to have the to form conjugated trienoic fatty acids of and C18 carbon and the conjugated fatty acid of the C18 carbon chain. conjugated fatty acids were in cells grown without exogenous fatty acid in the or with oleic acid not These that TkFac and PgFac not the cis-Δ9 double bonds of oleic and palmitoleic acids. In this we isolated cDNAs encoding enzymes and in the formation of conjugated trans-Δ11,cis-Δ13 double bonds of punicic Very recently encoding an enzyme to PgFac, was isolated from P. granatum (10Hornung E. Pernstich C. Feussner I. Eur. J. Biochem. 2002; 269: 4852-4859Crossref PubMed Scopus (78) Google Scholar). We have found a of TkFac and have bifunctional activity. cells with TkFac and PgFac 16:2Δ9cis, 12cis and 18:2Δ9cis, 12cis TkFac and PgFac were demonstrated to have both Δ12-oleate desaturase and conjugase activities. supports the idea that a number of fatty acid-modifying enzymes have diverged from fatty acid (7Cahoon E.B. Carlson T.J. Ripp K.G. Schweiger B.J. Cook G.A. Hall S.E. Kinney A.J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12935-12940Crossref PubMed Scopus (163) Google Scholar, P. S. C. Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google Scholar, M. M. A. M. S. M. R. C. A. S. A. S. 1998; PubMed Scopus Google Scholar). of has been shown to be a bifunctional enzyme having both Δ12-oleate and Δ12-oleate desaturase P. S. C. Plant J. 1998; PubMed Scopus Google Scholar). of acids in with in the of the enzyme from to desaturase P. J. E. C. 1998; PubMed Scopus Google Scholar). It is that a number of acids the of Punicic acid and α-eleostearic acid are closely related isomers in which a double bond the is in cis- of acid of the conjugases that several acids in and form conjugated double were in TkFac and PgFac form conjugated trans-Δ11, cis-Δ13 double It is that several acids may be in the divergent of these enzymes. Punicic acid accumulation was in plants punicic acid in seeds not in even when the conjugases were driven under control of the 35S promoter not A has been reported inArabidopsis with Δ12-oleate P. S. C. Plant J. 1998; PubMed Scopus Google Scholar). In seed-specific accumulation of fatty acids from seed-specific expression of enzymes (7Cahoon E.B. Carlson T.J. Ripp K.G. Schweiger B.J. Cook G.A. Hall S.E. Kinney A.J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12935-12940Crossref PubMed Scopus (163) Google Scholar, E.B. Ripp K.G. Hall S.E. Kinney A.J. J. Biol. Chem. 2001; 276: 2637-2643Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 9Qiu X. Reed D.W. Hong H. MacKenzie S.L. Covello P.S. Plant Physiol. 2001; 125: 847-855Crossref PubMed Scopus (42) Google Scholar, P. S. C. Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google Scholar, P. S. C. Plant J. 1998; PubMed Scopus Google Scholar). It is not known conjugated fatty acids are from lipids in of to It has been suggested that fatty acids are a typical fatty acids in P. S. C. Plant J. 1998; PubMed Scopus Google Scholar). accumulation of fatty acids in was accompanied by in and M. L. Plant 1998; 11: Scopus Google Scholar). Expression of the conjugases under control of the or napin promoter to accumulation of punicic acid in seeds The of punicic acid in seeds from from to ∼10% not punicic acid several in fatty acid were acid in to a in seeds with ∼10% (w/w) punicic a in In contrast, linoleic and α-linolenic acid from (w/w) and (w/w) in seeds to (w/w) and (w/w) in seeds with (w/w) punicic respectively. A of Δ12-oleate was reported in plants with enzymes. seeds Δ12-oleate P. S. C. Plant J. 1998; PubMed Scopus Google P. C. Plant Physiol. 1997; 113: PubMed Scopus Google and S. S. M. S. A. 2001; PubMed Scopus Google and in conjugases (7Cahoon E.B. Carlson T.J. Ripp K.G. Schweiger B.J. Cook G.A. Hall S.E. Kinney A.J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12935-12940Crossref PubMed Scopus (163) Google Scholar), accumulation of fatty acids was accompanied by a in oleic A suggested that expressed enzymes to an in oleic acid by with Δ12-oleate desaturase the or levels S. S. M. S. A. 2001; PubMed Scopus Google Scholar). In a fatty acid that was identified as an isomer of by We analyzed the of yeast cells grown with 12cis not the to the isomer not is that the isomer resulted from conversion of 12cis to conjugated as seeds contain fatty acids in kirilowii and P. granatum which accumulate fatty acids α-linolenic acid and is that the isomer resulted from fatty acid of punicic acid by the of fatty acid D.W. E. J. I. E. Plant 1995; PubMed Scopus Google Scholar, L. Plant J. 1997; PubMed Scopus Google Scholar). The of the conjugases was by expression in yeast cells. 16:2Δ9cis, 12cis in yeast the conjugases and be into the which was to be a conjugated fatty acid containingcis-Δ9, trans-Δ11, cis-Δ13 double bonds and Thus, the conjugases fatty acids with carbon as substrates as well as with C18 carbon In we analyzed the in yeast cells grown with α-linolenic acid (18:3Δ9cis, 12cis, 15cis) and found a a methyl of which was to be of from we also found a of methyl of which a to that of the isomer found in yeast cells not α-linolenic acid ∼10% (w/w) of the total seed oils in contrast, is in seed oils of T. kirilowii and P. In studies yeast cells have demonstrated that α-linolenic acid is a for conjugases associated with the formation of both double bonds and double bonds (7Cahoon E.B. Carlson T.J. Ripp K.G. Schweiger B.J. Cook G.A. Hall S.E. Kinney A.J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12935-12940Crossref PubMed Scopus (163) Google Scholar, E.B. Ripp K.G. Hall S.E. Kinney A.J. J. Biol. Chem. 2001; 276: 2637-2643Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 9Qiu X. Reed D.W. Hong H. MacKenzie S.L. Covello P.S. Plant Physiol. 2001; 125: 847-855Crossref PubMed Scopus (42) Google Scholar). In study, yeast cells large of and however, no fatty acids were found in and cells and that these enzymes a double bond in to which modifies a double bond to conjugated double bonds (8Cahoon E.B. Ripp K.G. Hall S.E. Kinney A.J. J. Biol. Chem. 2001; 276: 2637-2643Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 9Qiu X. Reed D.W. Hong H. MacKenzie S.L. Covello P.S. Plant Physiol. 2001; 125: 847-855Crossref PubMed Scopus (42) Google Scholar). acids with conjugated double bonds have been in and a large number of effects of supplementation with conjugated linoleic acid have been reported. has been shown to the of in the of cells in culture C. Cancer Res. Google Scholar, Cancer Lett. PubMed Scopus Google Scholar, G. Cancer Lett. 1997; PubMed Scopus Google Scholar), and in and Am. J. Physiol. 1998; Google Scholar, Am. J. Physiol. 1999; 276: Google Scholar, P. Lipids. 2000; PubMed Scopus Google Scholar, H. H. E. J. J. 2000; PubMed Scopus Google Scholar). studies have shown that CLNAs are cytotoxic to tumor cells is (2Igarashi M. Miyazawa T. Cancer Lett. 2000; 148: 173-179Crossref PubMed Scopus (153) Google Scholar). In and are in fatty acids in CLNAs K. Akahoshi A. Yamasaki M. Tanaka K. Yamada K. Iwata T. Kamegai T. Tsutsumi K. Sugano M. J. Am. Oil Chem. Soc. 2002; 37: 343-350Google Scholar). CLNAs have been suggested to and metabolism K. Akahoshi A. Yamasaki M. Tanaka K. Yamada K. Iwata T. Kamegai T. Tsutsumi K. Sugano M. J. Am. Oil Chem. Soc. 2002; 37: 343-350Google Scholar), the of the and action of and CLNA In to understanding of fatty acid of conjugases may to the of methods to CLNAs in common oil We for and of We also for the yeast for T. and of for