Vaccenic acid

Methyl esters of DHA (C22:6 ω3), DPAω3 (C22:5 ω3), DPAω6 (C22:5 ω6), EPA (C20:5 ω3), ETA (C20:4 ω3), vaccenic acid (C18:1 ω7), palmitic acid (C16:0), GLA (C18:3 ω6), SDA (C18:4 ω3), and other chemicals were purchased from Sigma-Aldrich Japan K.K. (Tokyo, Japan) or Cayman Chemical Company (Ann Arbor, MI, USA). Primers were purchased from FASMAC Co. Ltd. (Kanagawa, Japan). Heptadecanoic acid (C17:0) was obtained from Tokyo Chemical Industry Co. Ltd. (Tokyo, Japan). Enzymes and kits for DNA manipulation were purchased from Takara Bio Inc. (Shiga, Japan) or New England Biolabs Japan Inc. (Tokyo, Japan). PCR reactions were carried out using a GeneAmp PCR System 9700 thermal cycler (Thermo Fisher Scientific Inc., Waltham, MA, USA) with Tks Gflex DNA polymerase (Takara Bio). General genetic manipulations of E. coli were performed according to standard protocols.

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.