Biohydrogenation of C(20) polyunsaturated fatty acids by anaerobic bacteria.

1 The polyunsaturated fatty acids (PUFAs) include many bioactive lipids. The microbial 2 metabolism of C 18 PUFAs is known to produce their bioactive isomers, such as conjugated fatty 3 acids and hydroxy fatty acids, but there is little information on that of C 20 PUFAs. In this study, 4 we aimed to obtain anaerobic bacteria for the ability to produce novel PUFA from C 20 PUFAs. 5 Through the screening of about 100 strains of anaerobic bacteria, Clostridium bifermentans JCM 6 1386 was selected as a strain with the ability to saturate PUFAs during anaerobic cultivation. 7 This strain converted arachidonic acid ( cis -5, cis -8, cis -11, cis -14-eicosatetraenoic acid) and 8 eicosapentaenoic acid ( cis -5, cis -8, cis -11, cis -14, cis -17-eicosapentaenoic acid) into 9 cis -5, cis -8, trans -13-eicosatrienoic acid and cis -5, cis -8, trans -13, cis -17-eicosatetraenoic acid, 10 giving yields of 57% and 67% against the added PUFAs, respectively. This is the first report of 11 the isolation of a bacterium transforming C 20 PUFAs into corresponding 12 non-methylene-interrupted fatty acids. We further investigated the substrate specificity of the 13 biohydrogenation by this strain and revealed that it can convert two cis double bonds at the ω6 14 and ω9 positions in various C 18 and C 20 PUFAs into a trans double bond at the ω7 position. This 15 study should serve to open up the development of novel potentially bioactive PUFAs.


5
Microorganism and cultivation 6 The identified anaerobic bacteria used for this study (Supplementary Table S1) were 7 preserved in our laboratory (AKU Culture Collection, Division of Applied Life Science, Faculty 8 of Agriculture, Kyoto University, Kyoto, Japan) and those obtained from other culture collections 9 (JCM, Japan Collection of Microorganisms, Saitama, Japan; and ATCC, American Type Culture 10 Collection, VA, USA). The unidentified anaerobic bacteria used for this study were isolated from 11 pond, wastewater, fish viscera, and so on. The medium was GAM broth (pH 7.0) (Nissui 12 Pharmaceutical co., Ltd., Tokyo, Japan) supplemented with 0.03% (w/v) LA, AA or 0.02% (w/v) 13 EPA. Each strain was inoculated into 15 mL of the medium in screw-capped tubes (16.5 × 215 14 mm) and then incubated in an anaerobic chamber (98% nitrogen and 2% hydrogen) at 37°C for 15 2-3 days. After the cultivation, the culture medium was separated into supernatant and cells by 16 centrifugation (8,000 g, 10 min), and the supernatant was used for lipid analysis.
(ULBON HR-SS-10, 50 m × 0.25 mm I.D., Shinwa Kako, Kyoto, Japan). The column 1 temperature was initially 180°C and was raised to 220°C at a rate of 2°C/min and maintained at 2 that temperature for 20 min. The injector and detector were operated at 250°C. Helium was used 3 as a carrier gas at 0.97 ml/min. 4 Isolation, derivatization, and identification of products 5 For the isolation of the newly generated fatty acid in a culture of C. bifermentans JCM 6 1386 with 0.03% (w/v) AA (UK1), its methyl esters were purified by high-performance liquid 7 chromatography (HPLC, monitored at 205 and 233 nm) using a Shimadzu LC-VP system fitted 8 with a Cosmosil column 5C18-ARII (20 × 250 mm, Nacalai tesque, Kyoto, Japan). The mobile 9 phase was acetonitrile-water (80:20, v/v) at a flow rate of 5.0 mL/min and the column 10 temperature of 30°C. The fraction containing UK1 was further purified by HPLC on Inertsil ODS 11 SQ5-1385 (4.6 × 250 mm, GL Science Inc., CA, USA) joined with Capcelpak C18 UG20 (4.6 × 12 250 mm, Shiseido, Tokyo, Japan). Acetonitrile-water (80:20, v/v) was used as the mobile phase at 13 a flow rate of 1.2 mL/min. For the isolation of the newly generated fatty acid in a culture of C. 14 bifermentans JCM 1386 with 0.02% (w/v) EPA (UK2), its methyl esters were purified using a 15 same procedure as described for UK1 except that the mobile phase used for the latter step was 16 acetonitrile-water (80:20, v/v) at a flow rate of 1.0 mL/min.
1 Preparation of pyrrolidide fatty acids 2 Pyrrolidide derivatives were prepared by direct treatment of the isolated methyl esters 3 with pyrrolidine-acetic acid (10:1, v/v) in screw-cap tubes for 1 h at 115°C followed by extraction 4 according to the method of Andersson and Holman (25). The organic extract was washed with 5 water and dried over anhydrous Na2SO4, and then the solvent was removed by a vacuum in a 6 rotary evaporator.

19
Screening of anaerobic bacteria that have the ability to convert C20 PUFAs 20 The ability of anaerobic bacteria to convert the C20 PUFAs of EPA and AA during 21 cultivation was investigated together with LA as a reference of C18 PUFA. We tested about 100 22 strains, including the identified bacteria, which belonged to genera such as Megasphaera, Bifidobacterium, Lactobacillus, Propionibacterium, Clostridium, Bacteroides, Eubacterium, and 1 so on (Supplementary Table S1), and the unidentified bacteria. The peaks of the PUFAs were 2 identified by comparison with the retention time of the reference standards on GC analysis.

3
Of these bacteria, 2 strains of Clostridium bifermentans (JCM 1386 and JCM 7832) 4 showed the activity to convert AA and EPA, while 5 strains (including the two C20 PUFAs 5 converting strains mentioned above) belonging to the genera of Clostridium and 6 Propionibacterium were found to have the ability to convert LA to vaccenic acid (trans-11-18:1, 7 VA) ( Table 1). 8 Figure 1 shows the GC chromatogram of methylated fatty acids produced by C.  (Table 1). 13 However, C. sporogenes JCM 7849, C. sporogenes JCM 7850, and P. acnes JCM 6473 couldn't 14 convert AA and EPA.

22
In this study, we found that C. bifermentans JCM 1386 could convert AA and EPA into cis-5, cis-8,trans-13-20:3 and cis-5,cis-8,trans-13,cis-17-20:4, respectively, which are NMIFAs 1 with a trans double bond at the ω7 position (see Figs. 5 and 8). This is the first report of the 2 isolation of the bacterium transforming C20 PUFAs into corresponding NMIFAs. Considering 3 that similar reactions were observed with LA (see Fig. 1C), this strain can convert two cis double 4 bonds at the ω6 and ω9 positions in PUFAs into a trans double bond at the ω7 position to 5 generate the trans fatty acids regardless of the existence of double bonds at other positions. In 6 addition, similar reactions were also observed of other C18 and C20 free PUFAs (α-linolenic acid, 7 γ-linolenic acid, and dihomo-γ-linolenic acid) (see Fig. 6). They might be converted into the 8 corresponding NMIFAs with a trans double bond at the ω7 position. However, C. bifermentans 9 JCM 1386 could not convert DHA, indicating that C22 PUFAs might not be a substrate for this 10 strain. Thus, we succeeded in the production of various C18 and C20 NMIFAs with a trans double 11 bond at the ω7 position through the biohydrogenation by C. bifermentans JCM 1386.

12
NMIFAs are a class of PUFAs that has received attention because of their unique structure 13 and physiological activity, and they have often been found in plant oils. Pinolenic acid 14 (cis-5,cis-9,cis-12-18:3) and columbinic acid (trans-5,cis-9,cis-12-18:3) are C18 NMIFAs that 15 were found in Pinus koraiensis and Aquilegia hybrida, respectively (8, 9). They are isomers of 16 γ-linolenic acid and show various effects, such as the reduction of platelet aggregation by 17 prostacyclin production, attenuation of the elevation of blood pressure, LDL-lowering and 18 essential fatty acid activity (9, 11, 12). Podocarpic acid (cis-5,cis-11,cis-14-20:3) is a C20 NMIFA 19 that was found in Platycladus orientalis oil (10). It has been reported to show a reduction in the 20 AA concentration in the phosphatidylinositol fraction of rat liver (13), which functions in signal 21 transduction, such as in the phospholipase C-signaling pathway (13, 29). Considering that PUFAs 22 often show an isomer-specific function, novel NMIFAs are expected to show novel interesting physiological effects. Interestingly, several natural plant oils have a high content of PUFAs with 1 a double bond at the ω7 position (30), and the biohydrogenation of PUFAs often produce PUFAs 2 with a double bond at the ω7 position, such as VA (6, 7). These observations enable us to 3 consider that a double bond at the ω7 position may become a key factor for a biological function.

4
In this context, various C18 and C20 NMIFAs obtained in this study could be worthwhile. It is also 5 noted that these NMIFAs were obtained in the high yield (approximately 60%). Therefore, this

A B
A Self-archived copy in Kyoto University Research Information Repository https://repository.kulib.kyoto-u.ac.jp