γ linolenic acid

Ethyl acetate (EtOAc), methanol (MeOH), acetonitrile (ACN), chloroform (CHCl₃), sulfuric acid 96% (H₂SO₄) and propan-2-ol were purchased from Carlo Erba (Val de Reuil, France). Ammonium formate and the following standards were purchased from Sigma Aldrich (Saint-Quentin Fallavier, France): dodecanoic acid, myristic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, γ-linolenic acid, arachidic acid, arachidonic acid, nervonic acid, eicosapentaenoic acid and eicosatrienoic acid. Dimethyl carbonate (DMC) and lauric acid 99% were purchased from Acros Organics (Geel, Belgium). Formic acid was purchased from Fisher Scientific SAS (Illkirch, France). Nonanoic acid 98% and decanoic acid 99% were purchased from Alfa Aesar (Haverhill, MA, USA). Water was purified using a Milli-Q system (Millipore Corporation, Bedford, MA, USA). Phosphoric acid 85–90% (H₃PO₄) and Formic acid were purchased from Fisher Scientific SAS (Illkirch, France). Vanillin was purchased from Extrasynthese (Genay, France).

Ethyl acetate (EtOAc), methanol (MeOH), toluene, hexane, formic acid, diethylether, glacial acetic acid, petroleum ether, sulfuric acid 96% (H₂SO₄) and dimethylsulfoxide (DMSO) were purchased from Carlo Erba (Val de Reuil, France). Dimethyl carbonate (DMC), sodium alginate, (±)-α-tocophérol 96% (vitamin E), oleic acid, linoleic acid, palmitic acid, myristic acid, stearic acid, palmitoleic acid, γ-linolenic acid, 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT), menadione and glucose were purchased from Sigma Aldrich (Saint-Quentin Fallavier, France). Phosphoric acid 85% was purchased from Merck pro analysis (Darmstad, Germany). Labrafac^®® WL 1349 was purchased from Gattefossé (Saint-Priest, France). Montane 80^®® and Montanox 80^®® were purchased from Seppic (Castres, France). Copper nitrate Cu(NO₃)₂ was purchased from Fisher Scientific SAS (Illkirch, France). Vanillin, acetic acid trihydrate 99+% were purchased from Acros Organics (Geel, Belgium). Water was purified using a Milli-Q system (Millipore Corporation, Bedford, MA, USA).

As host cells, we used two strains of E. coli, namely JM109 (Yanisch-Perron et al., 1985 (link)) for the construction of plasmids and Rosetta^TM 2 (Merck Millipore, Darmstadt, Germany) for the expression of the target genes. The cells were grown in 2 mL of Luria–Bertani (LB) medium (Bertani, 1951 (link)) at 37°C with shaking at 180 rpm. All transformants were maintained in LB medium solidified with 1.5% (w/v) Bacto^® agar (BD Biosciences Japan, Tokyo, Japan) in the presence of 100 μg/mL sodium ampicillin, 50 μg/mL chloramphenicol, or 50 μg/mL spectinomycin dihydrochloride pentahydrate, depending on the selection markers on the plasmids. To exogenously supply fatty acids to the culture of E. coli cells, 1 mM of each sodium salt of palmitoleic acid (16:1Δ9, Wako Pure Chemicals, Osaka, Japan), 18:1Δ9 (Tokyo Chemical Industry, Tokyo, Japan), linoleic acid (18:2Δ9,12, Funakoshi, Tokyo, Japan), γ-linolenic acid (18:3Δ6,9.12, Sigma-Aldrich Japan, Tokyo, Japan), α-linolenic acid (18:3Δ9,12,15, Funakoshi), or vaccenic acid (18:1Δ11, Sigma-Aldrich Japan) was added to the liquid LB medium. Corynebacterium urealyticum ATCC 43042 was grown on R agar¹ with 0.5% (v/v) TWEEN 80 (Wako Pure Chemicals) and incubated at 37°C for 18 h.

Fatty acids were named using the formula Cx:yΔnc, as described previously (Simopoulos, 2006 (link)). FAME of myristic acid (C14:0), palmitic acid (C16:0), palmitoleic acid (C16:1Δ9c), heptadecanoic acid (C17:0), stearic acid (C18:0), oleic acid (C18:1Δ9c), LA (C18:2Δ9c,12c), ALA (C18:3Δ9c,12c,15c), γ-linolenic acid (C18:3 D6c,9c,12c) and Supelco^® 37-component FAMEs Mix (C4–C24 unsaturated) were purchased from Sigma–Aldrich (St. Louis, MO, United States). HPLC-grade methanol, chloroform, n-hexane, and n-pentane were used in this study, which were purchased from Alltech Scientific (Chaoyang District, Beijing, China). n-hexane was used as a solvent to prepare standard stock solutions and dilutions. Heptadecanoate (C17:0) was used as an internal standard because it is generally not present in biological samples. All compounds and stock solutions were stored according to manufacturer’s protocol.