FD-CF reactions for the expression of ATF1 enzyme were incubated at 37°C for 20 hours in the cell-free reaction. The completed FD-CF reaction containing the enzymes was then added into a separate freshly prepared catalysis reaction. The total catalysis reaction volume was 300 μl and included 50 mM HEPES (pH 7.5), 100 mM KCl, 5 mM EDTA, the relevant substrates (25 mM isoamyl alcohol for ATF1), and freshly prepared cofactor (5 mM acetyl-CoA for ATF1), and 10% of the volume was the FD-CF reaction containing the enzyme. These reactions were allowed to proceed 20 hours in capped vials at room temperature. For GC-MS analysis, the stir bar sorptive extraction method (52) was used. Polydimethylsiloxane stir bars (GERSTEL 011222-001-00) were held in the headspace of the reaction vial by a magnet during the catalysis reaction to absorb volatile components. After the completion of the reaction, the stir bar was added to a headspace vial containing 100 μl of dodecane/ethanol (10:1) and analyzed on a GC-MS headspace sampler (Agilent 7697A) to confirm the identity of the converted product. The GC-MS total ion count signal was converted to parts per million by generating a standard curve using the same process described above, but the FD-CF reactions did not contain DNA template or substrates but were spiked instead with known parts per million concentrations of the product isoamyl acetate.

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