It was found that all enzymes investigated were able to convert 12 and 13 into 1b, with IRED15 and IRED23 showing the best results. The reduction of substrate 14 to 2nitrobenzyl alcohol (15) was observed, which is a known side reaction of the IREDs and AspRedAm 34 . To upscale the reaction to preparative scale, IRED15 was selected for further experiments. This arose from the fact that the expression of IRED15 at 16.22 µmol/L was much better than the expression of IRED23 at around 0.16 µmol/L. By increasing the enzyme load to 40 µM IRED15, more than 86 % conversion to 1b was achieved after 48 hours. When 10 µM NfsA was added to the reaction after 48 hours, more than 90 % of 6b was formed within 3 hours. When the cascade reaction was performed at 50 mg scale of 12, 27 mg of 6b was produced (Figures &), which corresponds to 62 % isolated yield. The final product of the cascade 6b is shown separately (violet), while all compounds that could possibly still be converted into the product (12, 14, and 1b) are grouped together (blue) and by-products such as 15 and other partially unidentified peaks, potentially from the nitro reduction of 12, 14 or 15, are summarized in a grey bar. Reaction conditions: 5 mM 12, 20 mM 13, 40 µM IRED15, 0.5 mM NADP + , 50 mM D-glucose, 1.6 µM BmGDH, 50 mM NaPi + 300 mM NaCl + 10 % glycerol pH 8.0; 25 °C, 800 rpm. After 24 hours, 10 µM NfsA was added and the reaction was incubated for a further 3 hours before gas chromatographic analysis.