Resting Cell Biotransformation.

All necessary plasmids were inserted into strain MX102 R⁰ by electroporation and plated on 2x YT with 200 mg/L ampicillin, 50 mg/L spectinomycin, and 50 mg/L kanamycin (ASK). Triplicate seed cultures of 4 mL 2x YT-ASK with 5 g/L D-mannitol were prepared from single colonies and grown at 37 °C for 16 h. Cell cultivation cultures were normalized to 0.04 OD₆₀₀ in 50 mL 2x YT-ASK with 10 g/L D-mannitol and 0.5 mM 5-aminolevunic acid. The cells were incubated shaking for 4 h at 30 °C and then induced with 0.5 mM IPTG and 0.1% (w/v) L-arabinose. After induction, cells were incubated for 12 h while shaking at 30 °C before harvest. At harvest the OD₆₀₀ of each culture was determined and the volume of cells necessary to prepare a final suspension of 100 OD₆₀₀ in 3 mL final volume was transferred to 50 mL conical tubes and pelleted for 10 min at room temperature, 2500 g. The pelleted cells were washed by gentle resuspension and centrifugation twice in 40 mL 100 mM KP_i pH 7.5 at room temperature, then washed one more time in 10 mL of the same and transferred to 15 mL conical tubes. The conical tubes were pelleted a final time and resuspended to a total volume of 3 mL.

The resting cell reactions were initiated by the addition of 600 µL washed cell suspension to 2.4 mL concentrated reaction master mix in 15 mL conical tubes. Reaction mixture was composed of 200 mM D-glucose, 0 or 10 mM NMN⁺, 5 g/L m-Bdo, 0.5 mM IPTG, 100 mM KP_i dibasic, 200 mM MOPS, ASK, and 0.1% (w/v) L-arabinose. Resting cell reactions were then incubated horizontally at 30 °C, 250 rpm, for 48 h. Resting cell reactions with Tp Nox expressed were instead incubated at 18 °C for 152 h.