Fermentative Production of Nervonic Acid by Mortierella Capitata

NOTE Fermentative Production of Nervonic Acid by Mortierella capitata RD000969 Hiroki Umemoto1, Keigo Sawada1, Atsushi Kurata1, Shohei Hamaguchi2, Satoshi Tsukahara2, Takashi Ishiguro2 and Noriaki Kishimoto1＊ 1 Department of Applied Biological Chemistry, Kinki University, 3327-204, Nakamachi, Nara City, Nara 631-8505, Japan 2 Miyoshi Oil and Fat Co., Ltd., 4-66-1, Horikiri, Katsushika-ku, Tokyo 124-8510, Japan

Abstract: A high-nervonic acid (cis-15-tetracosenoic acid, C24:1, n-9)-producing filamentous fungus of the Mortierella species was discovered among soil filamentous fungi. The filamentous fungal strain –RD000969– was isolated from soil collected in Kanagawa Prefecture (Japan) and was found to accumulate nervonic acid at a rate of 6.94% of the total cellular fatty acids. The base sequences of 28S rDNA D1/D2 and ITS 5.8S rDNA showed 100% homology with Mortierella capitata CBS 293.96. In addition to nervonic acid, strain

RD000969 produced a large amount of long-chain monounsaturated fatty acids (C20:1, 12.22%; C22:1, 4.07%;

C26:1, 5.91%) and a small amount of ultra-long-chain fatty acids (C28:1, 0.44%; C30:1, 0.06%; C32:1, trace). In the fungal cells, 98.87% of nervonic acid was localized at the sn-1,3 position of triacylglycerol. Nervonic acid production was maximum (186.3 mg・L－1) when the fungus was cultured in potato dextrose (PD) medium

containing yeast extract, CaCl2, and MgSO4・7H2O.

Key words: nervonic acid, Mortierella capitata, very long chain monounsaturated fatty acid, fermentation

1 INTRODUCTION and HIV-1 reverse transcriptase7）.

Nervonic acid（C24:1 n-9）is a major very-long-chain mono- Nervonic acid has been identified in several seed oils, unsaturated fatty acid contained in sphingolipid, compris- such as Lunaria spp., borage, hemp, Acer truncatum ing the white matter and myelin sheath of the human （Purpleblow maple）, Tropaeolum speciosum（Flame brain1）. In demyelinating diseases which cause adrenoleu- flower）, and Cardamine, but only Lunaria annua L. is kodystrophy and multiple sclerosis, the levels of nervonic being studied and grown for future development as a niche acid in cerebral sphingolipid decrease, and the levels of crop8）. However, the use of L. annua to produce nervonic very-long-chain saturated fatty acids, such as C26:0 fatty acid is costly because it is a biennial plant and the seeds acid, increase2）. Oral administration of nervonic acid nor- drop to the ground easily8）. malizes the C26:0 fatty acid level and dietary therapy involv- Thus, we investigated nervonic acid production using ing the ingestion of oil containing nervonic acid has been microorganisms instead of plants. Production of C24:1 fatty shown to be useful for treatment of these diseases2, 3）. acid by some filamentous fungi and bacteria has been re- Moreover, nervonic acid derivatives, which pass through ported. A phytopathogenic filamentous fungus, Macroph- the blood-brain barrier, have been developed for medical omina phaseolina（16.1–48.8％ of total cellular fatty acids treatment, and anti-inflammatory and immunomodulatory ［TFA］）9）, and a zoonotic pathogen, Francisella tularensis 4, 5） 10） activities of these compounds have been reported . （11.2–19.3％ of TFA） , produce C24:1 fatty acid in large Inverse correlations between the plasma nervonic acid quantities, but these are not suitable for industrial produc- level and BMI, leptin, triglyceride, total cholesterol, and tion because of their pathogenicity. Several ruminal fungi11, 12） fasting blood glucose levels have been reported, suggesting have been reported to produce C24:1 fatty acids, but high- that nervonic acid is effective in preventing obesity-associ- level anaerobic culture is necessary. Non-pathogenic fila- ated metabolic diseases6）. In addition, nervonic acid has mentous fungi, Neurospora crassa（1.6％）13）, Phycomyces been shown to inhibit eukaryotic DNA polymerase α and β, blakesleanu（s 2.9％）13）, and Mortierella elongata（4.9％）14）,

＊Correspondence to: Noriaki Kishimoto, Department of Applied Biological Chemistry, Kinki University, 3327-204, Nakamachi, Nara City, Nara 631-8505, Japan E-mail: [email protected] Accepted March 25, 2014 (received for review February 16 2014) Journal of Oleo Science ISSN 1345-8957 print / ISSN 1347-3352 online http://www.jstage.jst.go.jp/browse/jos/ http://mc.manusriptcentral.com/jjocs

671 H. Umemoto, K. Sawada, A. Kurata et al.

also produce C24:1 fatty acid albeit in small amounts and the was increased from 120 to 180℃ at 30℃/min, then from position and configuration of the double bond in these fatty 180 to 290℃ at 10℃/min, and retained at 290℃ for 5 min. acids has not been identified. Helium was used as the carrier gas at a flow rate of 1 mL/ In this study, we searched for and identified a high-ner- min. The temperatures of the inlet and the detector were vonic acid-producing microorganism and investigated its kept at 250℃, and the split ratio was 10:1. These fatty nervonic acid production ability using it. acids were identified by the mass spectra of GC peaks. The position of the double bond in monounsaturated fatty acids was identified by GC-MS after dimethyl disulfide （DMDS）derivatization. The DMDS derivatives were pre- 2 EXPERIMENTAL pared using the method reported by Shibahara et al.16）. 2.1 Materials The configuration of the double-bond was determined Methanol hydrochloride solution（2 N）was purchased using fourier transform infrared spectroscopy（FTIR）（FT/ from Kokusan Chemica（l Tokyo, Japan）. Methyl tridecano- IR 8200D, Shimadzu）. ate was purchased from Funakosh（i Tokyo）. Sterol esters, sterols, 1,3-diacylglycerol（s 1,3-DAG）, 1,2-diacylglycerols 2.4 Analysis of lipid composition （1,2-DAG）, and monoacylglycerol（s MAG）used for the Total lipids were extracted using the method described identification of lipids were provided by Dr. Toshihiro by Matyash et al.17）. Dried fungal cell（s 100 mg）were vor- Nagao（Osaka Municipal Technical Research Institute, texed with 6 mL of methanol and then added to 20 mL of Osaka, Japan）. All reagents were of analytical grade. methyl-tert-butyl ethe（r MTBE）. The mixture was stirred with a magnetic stirrer for 1 h at room temperature. The 2.2 Strains solution was mixed with 5 mL of distilled water and centri- Filamentous fungi that were non-selectively isolated fuged at 1000×g for 5 min. The upper phase was collected, from soil were tested by primary screening. Soil samples and the lower phase was re-extracted with an 8 mL were collected in Nara Prefectur（e Japan）. The soil samples mixture of MTBE: methanol: water/10: 3: 2.5（v/v/v）. The were suspended in sterile saline and added to potato dex- solvent in the combined upper phase was removed under trose aga（r PDA, Difco）containing rose benga（l 30 mg・L－1） reduced pressure. and chloramphenico（l 100 mg・L－1）for pre-culture at 25℃, The lipid composition was analyzed employing thin-layer and grown filamentous fungal colonies were isolated. In ad- chromatography with flame-ionization detection（TLC-FID, dition, the filamentous fungi provided by the National Insti- Iatroscan MK-5, Mitsubishi Chemical Medience）. Total tute of Technology and Evaluation（NITE, Tokyo）were cul- lipids were dissolved in n-hexane at a concentration of 1％ tured. （vol/vol）. The solution（2 μL）was spotted on the silica gel In secondary screening, filamentous fungi of Mortierella rod, and developed with a mixture of toluene: chloroform: species were selectively isolated. The soil samples were acetic acid/50: 30: 0.7（v/v/v）and then with a mixture of n- suspended with sterile saline and added to Sh3A medium15） hexane: diethyl ether/65: 5（v/v）. The silica gel rod was ana-

（3 g of dried, shrimp flake（s Sergestes lucens）, 15 g of lyzed by TLC-FID. The Rf values of the peaks that appeared agar, and 1 L of distilled water）for pre-culture, and grown were compared with the ones of lipid standard（s sterol Mortierella-like colonies were isolated. In addition, 66 esters, 0.74; TAG, 0.51; free fatty acids, 0.47; sterols, 0.44; strains screened for Mortierella species were provided by 1,3-DAG , 0.29; 1,2-DAG, 0.23; MAG, 0.19; and phospholip- NITE. ids, 0.08）.

2.3 Cultivation and fatty acid analysis 2.5 Regiospecific analysis of fatty acid composition of The isolated ﬁlamentous fungi were inoculated in 5 mL triacylglycerol of potato dextrose（PD）medium in 18 mL test tubes and The lipid classes were fractionated by silica gel column cultured at 28℃ with shaking at 140 rpm for 7 days. The chromatography using a mixture of n-hexane: diisopropyl culture ﬂuid was centrifuged at 14,400×g for 5 min（himac ether: acetic acid/70: 30: 1（v/v/v）. Each lipid was converted CR-G III, Hitachi Koki, Tokyo）. The fungal cells were into its corresponding methyl ester, and the fatty acid washed twice with distilled water and freeze-dried. composition was analyzed using GC. Dried fungal cell（s 30 mg）were mixed with 3 mL of 2 N The fatty acids at the sn-1,3 and sn-2 positions of triac- methanol hydrochloride solution and heated at 100℃ for 2 ylglycero（l TAG）were determined using the method de- h. The reaction solution was mixed with 1 mL of distilled scribed by Watanabe et al.18）. A mixture of 50 mg of TAG water and extracted twice with 3 mL of n-hexane. Using and 500 mg of ethanol was shaken with 22 mg of immobi- methyl tridecanoate as an internal standard, the fatty acid lized Candida antarctica lipase B（Novozym 435）at 30℃ composition was analyzed by GC-MS（JMS-K9, JEOL）using for 3 h, and the fatty acid at the sn-1,3 position was selec- an HP-5 column（Agilent J&W）. The column temperature tively converted to the corresponding ethyl ester. The pro-

672 J. Oleo Sci. 63, (7) 671-679 (2014) Production of nervonic acid by Mortierella capitata RD000969

－1 －1 duced fatty acid ethyl ester and 2-MAG were purified using mg・L ; and KH2PO4, 300 mg・L ）, or MEM Vitamin a silica gel column, and 2-MAG was converted to the corre- Solution（10 mL・L－1）was added to the PD medium. sponding methyl ester. The compositions of the fatty acid Mycelia were inoculated into 200 mL of each medium in ethyl ester derived from the sn-1,3 position and methyl a 500-mL flask, and cultured in a rotary shaker at 140 rpm ester derived from the sn-2 position were analyzed using and 28℃ for 7 days. The fungal cells were freeze-dried and GC. the weight of the dried fungal cells was measured. Fungal cell fatty acids were converted to methyl ester and nervon- 2.6 Identi cation of high-nervonic acid-producing fungus ic acid production was analyzed using GC-MS. The genomic DNA of high-nervonic acid-producing filamentous fungus was extracted according to the method described by Marmur et al.19）. PCR amplification of partial sequences of 28S rDNA D1/D2 and ITS 5.8S rDNA was 3 RESULTS carried out using primers reported in studies20, 21）. The base 3.1 Screening of high-nervonic acid-producing fungi sequences were determined using ABI PRISM 3130xl Fatty acid composition analysis of the 264 filamentous Genetic Analyzer System（Applied Biosystems）. The ana- fungal strains non-selectively isolated in primary screening lyzed base sequences were compared with the GenBank and 51 provided strains revealed that Mortierella elongata 22） database using BLASTN . A phylogenetic tree was con- NBRC 8570 produced C24:1 fatty acid accounting for 1.23％ structed using the Neighbor-joining method in ClustalX23） of the total cellular fatty acids, suggesting that filamentous

with bootstrap values calculated for 1,000 replicates. fungi of Mortierella species have a superior C24:1 fatty acid- Filamentous fungi were cultured in potato dextrose agar, producing ability. Thus, filamentous fungi of Mortierella 2％ Malt aga（r MA）, Bacto Oatmeal Aga（r OA）, and Synthe- species were selected as the target for secondary screen- tischer nährstoffarmer aga（r SNA）24）, and their morphologi- ing. cal characteristics were analyzed by observation under The fatty acid composition of 20 isolates of Mortierella light microscope BX51（Olympus）. species and the 66 provided strains were analyzed. Most of

them produced C24:1 fatty acid accounting for less than 2％ 2.7 Optimization of culture conditions of the total fatty acids, but strain RD000969 isolated from

To investigate the effects of the components of medium soil in the Kanagawa Prefecture produced C24:1 fatty acid on the growth of ﬁlamentous fungi and their nervonic acid accounting for 6.94％ of the total cellular fatty acids. After

production, 2 types of nitrogen sourc（e yeast extrac（t YE）, conversion to its methyl ester form, C24:1 fatty acid was 5 g・L－1 and casamino acid, 0.44 or 0.88 g・L－1）, 6 minerals subject to DMDS derivatization and then GC-MS analysis. －1 －1 （CuSO4・5H2O, 0.06 mg・L ; FeSO4・7H2O, 0.8 mg・L ; Fragment ion peaks m/z 173 and 301 appeared due to －1 －1 MgSO4・7H2O, 13.5 mg・L ; NaCl, 25 mg・L ; CaCl2, 47.5 cleavage of the double bond at the n-9 position（Fig. 1）.

Fig. 1 Mass spectrum of DMDS derivative of C24:1 fatty acid methyl ester derived from strain RD000969. 673 J. Oleo Sci. 63, (7) 671-679 (2014) H. Umemoto, K. Sawada, A. Kurata et al.

FT-IR analysis of C24:1 fatty acid methyl ester revealed that bered long-chain fatty acid（s C17:1, C19:1, C21:1）possessed a the trans absorbance band did not appear at 966 cm－1; double bond at the n-8 position. however, the cis absorbance band appeared at 3,005 cm－1. Thus, strain RD000969 was selected as a high-nervonic

On the basis of these ﬁndings, the C24:1 fatty acid produced acid-producing fungus and was examined in further by strain RD000969 was identiﬁed as nervonic acid. studies. The fatty acid composition of strain RD000969 and In addition to nervonic acid, strain RD000969 produced GC chromatogram are shown in Table 1 and Fig. 2. a large amount of long-chain monounsaturated fatty acids

（C20:1, C22:1, C26:1）and a small amount of ultra-long-chain 3.2 Analysis of lipid composition and TAG

fatty acid（s C28:1, C30:1, C32:1）. Odd-numbered chain monoun- Total lipids were extracted from strain RD000969 cul-

saturated fatty acid（s C17:1 to C27:1）were also produced. tured in PD medium, and the lipid composition was ana-

Even-numbered chain fatty acids except C16:1 and odd- lyzed using TLC-FID. Total lipids were comprised of: TAG,

numbered very-long-chain fatty acid（s C23:1, C25:1, C27:1）pos- 70.7％; total phospholipids, 21.3％; sterol esters, 2.58％;

sessed a double-bond at the n-9 position, C16:1 fatty acid 1,3-DAG, 2.58％; 1,2-DAG, 0.74％; free fatty acids, 1.46％; possessed a double bond at the n-7 position, and odd-num- and other lipids, 0.64％. The lipid classes were fractionated

Table 1 Fatty acid composition of strain RD000969. Molecular ion peak of Fragment ion peaks of Fatty acids Content (% of TFA)a methy ester (m/z) DMDS adducts (m/z)

C16:0 13.68±1.33 270

C16:1 (n-7) 0.28±0.12 268 145 (n-7), 217 (Δ9)

C17:1 (n-8) 0.32±0.06 282 159 (n-8), 217 (Δ9)

C18:0 5.19±0.56 298

C18:1 (n-9) 17.37±1.11 296 173 (n-9), 217 (Δ9)

C18:2 6.18±1.08 294

C18:3 2.18±0.44 292

C19:1 (n-8) 0.55±0.13 310 159 (n-8), 245 (Δ11)

C20:0 0.64±0.06 326

C20:1 (n-9) 12.22±1.44 324 173 (n-9), 245 (Δ11)

C20:2 + C20:3 3.98±1.01 322, 320

C20:4 9.37±0.60 318

C21:1 (n-8) 0.03±0.01 338 159 (n-8), 273 (Δ13)

C22:0 1.37±0.16 354

C22:1 (n-9) 4.07±0.33 352 173 (n-9), 273 (Δ13)

C23:1 (n-9) 0.05±0.03 366 173 (n-9), 287 (Δ14)

C24:0 4.46±0.67 382

C24:1 (n-9) 6.94±0.06 380 173 (n-9), 301 (Δ15)

C25:1 (n-9) 0.08±0.05 394 173 (n-9), 315 (Δ16)

C26:0 1.76±0.67 410

C26:1 (n-9) 5.91±0.38 408 173 (n-9), 329 (Δ17)

C27:1 (n-9) trace 422 173 (n-9), 343 (Δ18)

C28:0 0.13±0.01 438

C28:1 (n-9) 0.44±0.09 436 173 (n-9), 357 (Δ19)

C30:1 (n-9) 0.06±0.03 464 173 (n-9), 385 (Δ21)

C32:1 (n-9) trace 492 173 (n-9), 413 (Δ23) othersb 2.74 a Mean ± SD (n=3) b C14:0, C15:0, C17:0, C17:2, C17:3, C19:0, C19:2, C19:3, C19:4, C21:0, C23:0, C25:0, C27:0, C29:0 and C30:0 were included. 674 J. Oleo Sci. 63, (7) 671-679 (2014) Production of nervonic acid by Mortierella capitata RD000969

Fig. 2 GC chromatogram of fatty acid methyl ester derived from strain RD000969. The region from r.t. 12.5 min to

16.5 min was zoomed. Peak 1: C13:0 (internal standard), 2: C14:0, 3: C15:0, 4: C16:1, 5: C16:0, 6: C17:2+C17:3, 7: C17:1, 8:

C17:0, 9: C18:3, 10: C18:2, 11: C18:1, 12: C18:0, 13: C19:2+C19:3+C19:4, 14: C19:1, 15: C19:0, 16: C20:4, 17: C20:2+C20:3, 18:

C20:1, 19: C20:0, 20: C21:1, 21: C21:0, 22: C22:1, 23: C22:0, 24: C23:1, 25: C23:0, 26: C24:1, 27: C24:0, 28: C25:1, 29: C25:0, 30:

C26:1, 31: C26:0, 32: C27:1, 33: C28:1, 34: C28:0, 35: C30:1, 36: C32:1.

Table 2 Localization of the fatty acids in TAG produced by the strain RD000969. Fatty acids Localization at sn-1,3 position (%)

C16:0 95.97

C18:0 96.27

C18:1 (n-9) 27.71

C18:2 11.92

C18:3 28.22

C20:0 100

C20:1 (n-9) 85.33

C20:3 18.67

C20:4 28.44

C22:0 100

C22:1 (n-9) 94.92

C24:0 99.20

C24:1 (n-9) 98.87

C26:0 100

C26:1 (n-9) 100 and the fatty acid composition of each fraction was investi- position regardless of the level of unsaturation. gated. Nervonic acid was detected only in TAG. The sn-1,3 position of TAG was selectively converted to 3.3 Identi cation of strain RD000969 ethyl ester, and the fatty acids were analyzed speciﬁcally The partial sequences of 28S rDNA D1/D2 and ITS 5.8S for their localization at the sn-1,3 position（Table 2）. Ner- rDNA of strain RD000969 were determined and deposited vonic acid was mostl（y 98.87％）localized at the sn-1,3 posi- in the GenBank databank（Accession No. AB908051, tion, and minor amounts were found at the sn-2 position. AB908052）. Partial sequences of 28S rDNA D1/D2 and ITS

Many polyunsaturated fatty acid（s PUFA）, such as C18:3 and 5.8S rDNA showed 100％ homology with the sequences of

C20:4, presented at the sn-2 position, and C22 or longer very- Mortierella capitata CBS 293.96（Accession No. long-chain fatty acids tended to be localized at the sn-1,3 KC018334, JX976123）. In the phylogenetic tree prepared

675 J. Oleo Sci. 63, (7) 671-679 (2014) H. Umemoto, K. Sawada, A. Kurata et al.

Fig. 3 Phylogenetic tree derived from 28S rDNA D1/D2 sequences. The tree was constructed employing the neighbor- joining method. Bootstrap values for 1,000 replicates are given at branch points.

3.4 Effects of mineral salts and yeast extract concentration on nervonic acid production Strain RD000969 showed slow rate of growth in PD medium, and only a small number of fungal cells grew. Thus, nitrogen sources, minerals, and vitamins were added to PD medium, and their effects on growth and nervonic acid production were investigated. The addition of casami-

no acid, CuSO4・5H2O, FeSO4・7H2O, NaCl, KH2PO4, and MEM Vitamin Solution to the PD medium did not increase growth and nervonic acid production, but the addition of

YE, MgSO4・7H2O, and CaCl2 promoted growth and nervonic acid production. When YE（5 g・L－1）was added to PD medium, the dried fungal cell weight increased by 4.3-fold, but the nervonic acid production was only 5.9-fold higher －1 （Fig. 5）. In contrast, when CaCl（2 47.5 mg・L ）and MgSO4・－1 7H2O（13.5 mg・L ）were added to PD medium in combina- tion, the dried fungal cell weight increased by a maximum of 2-fold, but nervonic acid production increased 7.7-fold. When YE（1 g・L－1）was added to PD medium containing Fig. 4 Strain RD000969 colonies grown in PDA. CaCl2 and MgSO4・7H2O, nervonic acid production reached maximum concentration（186.3±20.6 mg・L－1）（Fig. 6）. from the partial 28S rDNA D1/D2 sequences, strain RD000969 belonged to the clade of M. capitata including M. capitata CBS 293.96（Fig. 3）. Strain RD000969 colonies grown in PDA were white with 4 DISCUSSION a rosette-like surface（Fig. 4）and had a garlic-like odor, The genus Mortierella has two subgenera, Mortierella 25） which is consistent with the characteristics of Mortierella and Micromucor . The subgen. Mortierella produced C20 25） subgen. Mortierella , but strain RD000969 grown in PDA, PUFA, such as arachidonic acid（C20:4, n-6）, whereas the

MA, OA, and SNA did not form a sporangium or sporagio- subgen. Micromucor produced C18 PUFA but did not 26） spore. produce C20 PUFA . Strain RD000969 accumulated C20:4 fatty acid at a rate of 9.37％ of the total cellular fatty acids,

676 J. Oleo Sci. 63, (7) 671-679 (2014) Production of nervonic acid by Mortierella capitata RD000969

Fig. 5 Effects of the CaCl2 concentration on the growth of and nervonic acid production by strain RD000969. Lane 1: －1 －1 PD medium, lane 2: PD + YE (0.5 g L ), lane 3: PD + CaCl2 (47.5 mg L ), lanes 4-10: PD + MgSO4・7H2O －1 －1 (13.5 mg L ) + CaCl2 (0-760 mg L ) Mean ± SD.

Fig. 6 Effects of the YE concentration on the growth of and nervonic acid production by strain RD000969. Lane 1: PD －1 －1 －1 medium, lane 2: PD + YE (0.5 g L ), lanes 3-8: PD + MgSO4・7H2O (13.5 mg L ) + CaCl2 (47.5 mg L ) + YE (0-5 g L－1) Mean ± SD. suggesting that it belongs to the subgen. Mortierella. Mortierellales proposed by Wagner et al.27）, M. capitata The partial sequences of 28S rDNA D1/D2 and ITS 5.8S belongs to Group 5（strangulata and wolfii）. Since this rDNA of strain RD000969 showed 100％ homology with group contains only a few species, all of which could be the base sequences of M. capitata CBS 293.96. M. capitata identiﬁed by molecular data, we identiﬁed strain RD000969 belongs to the subgen. Mortierella25）. The characteristic of as M. capitata RD000969 based on the base sequence ho- M. capitata is expansion of the lower region of the termi- mology. nal sporangium located at the end of numerous narrow Odd-numbered long-chain monounsaturated fatty acids 25） branches forming a globular sporangiospore . However, （C17:1, C19:1 and C21:1 fatty acids）produced by strain strain RD000969 did not form a sporangium or sporangio- RD000969 possess a double bond at the n-8 position（Table spore in the media tested. Based on the classification of 1）. The synthesis of these fatty acids is initiated by Δ9 de-

677 J. Oleo Sci. 63, (7) 671-679 (2014) H. Umemoto, K. Sawada, A. Kurata et al.

saturase acting on C17:0 fatty acid and producing C17（:1 n-8） Res. 6（4）, 545-551（1965）. fatty acid, and the chain extension enzyme acts on this 2） Sargent, J. R.; Coupland, K.; Wilson, R. Nervonic acid fatty acid and synthesizes C19（:1 n-8）and C21（:1 n-8）fatty and demyelinating disease. Med. Hypotheses 42（4）, acids. On the other hand, odd-numbered very-long-chain 237-242（1994）. monounsaturated fatty acid（s C23:1, C25:1 and C27:1 fatty 3） Tanaka, K.; Shimizu, T.; Ohtsuka, Y.; Yamashiro, Y.; Os- acids）possess a double bond at the n-9 position, suggesting hida, K. Early dietary treatments with Lorenzo’s oil that C23:1 fatty acid was not synthesized from C21:1 fatty acid and docosahexaenoic acid for neurological develop- by chain extension, and that it was synthesized through ment in a case with Zellweger syndrome. Brain Dev. another pathway. 29（9）, 586-589（2007）. In Mortierella alpina 1S-4, ω9 desaturase, which con- 4） Coupland, K.; Raoul, Y. Nervonic acid derivatives, their verts C24:0 and C26:0 fatty acids to C24（:1 n-9）and C26（:1 n-9） preparation and use. U. S. Patent 6,664,406（2003）. fatty acids, has been identified28）. It is likely that ω9 desatu- 5） Coupland, K. Anti-inflammatory and immunomodula- rase is involved in the synthesis of C23（:1 n-9）fatty acid pro- tory amino acid derivatives, their preparation and use. duced by strain RD000969. In humans, nervonic acid is U. S. Patent 6,956,059（2005）. biosynthesized by the chain extension of oleic acid29）, but 6） Oda, E.; Hatada, K.; Kimura, J.; Aizawa, Y.; Thani- strain RD000969 is likely to biosynthesize nervonic acid by kachalam, P. V.; Watanabe, K. Relationships between introducing a double bond into C24:0 fatty acid. serum unsaturated fatty acids and coronary risk fac- Filamentous fungi of Mortierella species including Mor- tors: negative relations between nervonic acid and tierella alpina have been used for the industrial produc- obesity-related risk factors. Int. Heart J. 46（6）, 975- tion of PUFA because of their ability to produce PUFA in 985（2005）. large amounts, such as arachidonic acid, eicosapentaenoic 7） Mizushina, Y.; Yoshida, S.; Matsukage, A.; Sakaguchi, K. acid, γ-linolenic acid, and dihomo-γ-linolenic acid30）. In con- The inhibitory action of fatty acids on DNA poly- trast, strain RD000969 produced large amounts of long- merase β. Biochim. Biophys. Acta, Gen. Subj. 1336 chain monounsaturated fatty acids, such as nervonic acid, （3）, 509-521（1997）. which has not been observed in other species of Mortierel- 8） Taylor, D. C.; Guo, Y.; Katavic, V.; Mietkiewska, E.; la. Since strain RD000969 produces more nervonic acid Francis, T.; Bettger, W. New Seed Oils for Improved than previously reported nervonic acid-producing filamen- Human and Animal Health: Genetic Manipulation of tous fungi, it is appropriate for production of nervonic acid. the Brassicaceae for Oils Enriched in Nervonic Acid. Modification of Seed Composition to Promote Health and Nutrition, 219-232（2009）. 9） Wassef, M. K.; Ammon, V.; Wyllie, T. D. Polar lipids of 5 CONCLUSION Macrophomina phaseolina. Lipids 10（3）, 185-190 We isolated a high-nervonic acid-producing filamentous （1975）. fungus, M. capitata RD000969. Strain RD000969 produced 10） Jantzen, E.; Berdal, B. P.; Omland, T. Cellular fatty nervonic acid at a maximum concentration of 186.3 mg・L－1. acid composition of Francisella tularensis. J. Clin. Mi- It also produced rare, very-long-chain monounsaturated crobiol. 10（6）, 928-930（1979）. fatty acids, which has not been observed in other fungi of 11） Kemp, P.; Lander, D. J.; Orpin, C. G. The lipids of the the Mortierella species. The nutritional and pharmaceuti- rumen fungus Piromonas communis. J. Gen. Micro- cal use of nervonic acid produced through fermentation by biol. 130（1）, 27-37（1984）. strain RD000969 is expected. 12） Comlekcioglu, U.; Ozkose, E.; Akyol, I.; Ekinci, M. S. Fatty acid analysis of anaerobic ruminal fungi Neocal- limastix, Caecomyces and Orpinomyces. Int. J. Ag- ric. Biol. 12（4）, 635-637（2010）. ACKNOWLEDGEMENT 13） Rˇ ezanka, T.; Cudlín, J.; Podojil, M. Very-long-chain fat- We would like to thank Dr. Toshihiro Nagao, Osaka Mu- ty acids from lower organism. Folia Microbiol. 32（2）, nicipal Technical Research Institute, for technical assis- 149-176（1987）. tance. 14） Chaudhuri, S.; Ghosh, S.; Bhattacharyya, D. K.; Ban- dyopadhyay, S. Effect of mustard meal on the production of arachidonic acid by Mortierella elongata SC- 208. J. Am. Oil Chem. Soc. 75（8）, 1053-1055（1998）. References 15） Degawa, Y.; Gams, W. A new species of Motierella, and 1） O’Brien, J. S.; Sampson E. L. Fatty acid and fatty alde- an associated sporangiiferous mycoparasite in a new hyde composition of the major brain lipids in normal genus, Nothadelphia. Stud. Mycol. 50, 567-572 human gray matter, white matter, and myelin. J. Lipid （2004）.

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679 J. Oleo Sci. 63, (7) 671-679 (2014)