Acyl coa

Malonyl coenzyme A (malonyl-CoA), acyl-CoAs, fatty acids, NADH, NADPH, and antibiotics were purchased from Sigma-Aldrich (St. Louis, MO, USA); Takara Biotechnology Co., Ltd. (Dalian, China) provided molecular biology reagents; the Ni²⁺-agarose columns were purchased from Invitrogen (Shanghai, China); American Radiolabeled Chemicals, Inc. (St. Louis, MO, USA) provided sodium [1-¹⁴C] acetate (specific activity, 50 mCi/mM); and Bio-Rad (Hercules, CA, USA) provided the Quick Start Bradford dye reagent. All other reagents were of the highest available quality.

Acyl-CoAs were purchased from Sigma, and E. coli CAT was purchased from Promega (part # E1051). The reaction contained 1.25 unit of CAT (one unit is defined as the amount of enzyme required to transfer 1 nmol of acetate to chloramphenicol in one minute at 37 °C), 250 μM acyl-CoA, 280 μM chloramphenicol, in 100 μl of 100 mM Tris buffer, pH 7.8. A reaction without CAT was served as control. The reaction was incubated at 37 °C for 2 h, and ethyl acetate (100 μL) was added to stop the reaction. The mixture was centrifuged (10,000 × g) for 10 min, and the ethyl acetate phase was collected and dried under a N₂ flow. Methanol (100 μL) was added to dissolve the residues, and a fraction (20 μL) of the methanol solution was injected into HPLC for analysis (Agilent 1200 with a ZORBAX SB-C18 column, 4.6 mm × 150 mm, 5 μ). Acetonitrile (solvent B) and water (solvent A) were used as the mobile phases with a flow rate of 1.0 mL/min. The program was as follow: 10% B for 5 min, 10–90% B in A over 20 min, 90–100% B in A over 1 min, 100% B for 4 min, 100–10% B in A over 4 min, 10% B for 4 min. The detection wavelength was 220 nm. The peak integration area of the acylated chloramphenicol products on HPLC was used to calculate the conversion rate of each of the reactions with a different acyl-CoA substrate.

Malonyl-CoA, acetyl-CoA, acyl-CoAs, fatty acids, NADH, NADPH, and antibiotics were purchased from Sigma-Aldrich. Takara Biotechnology Co. provided molecular biology reagents. Novagen provided the pET vectors. Ni-agarose columns were obtained from Invitrogen. GE healthcare provided the HiTrap Q strong anion-exchange column and Bio-Rad provided the Quick Start Bradford dye reagent. All other reagents were of the highest available quality. Takara Biotechnology Co. synthesized the oligonucleotide primers.