Fig. 6: Production of alkaloids (S)-45,46,48 and 49 using CnTYR variants.
a Production of (S)-45 using WT-CnTYR, variants N201S, M1 (N201S/G205R/V206I) and M25 (N201S/G205R). b Production of (S)-46 using WT-CnTYR and M1 (N201S/G205R/V206I) together with MTs RnCOMT/MxSafC/Cj6OMT, EcMAT (SAM supply), and EcMTAN (SAH degradation). c Production of (S)-47 using WT- CnTYR, N201S, M8 (N201S/H202N) and M26 (N201S/G205K). d Production of (S)-48 using WT-CnTYR, N201S, M1 (N201S/G205R/V206I) and M8 (N201S/H202N). e Enzyme cascades to alkaloids (S)−45, 46, 48, and 49. Reaction conditions: Details are provided in the Supplementary Information and are specific to each cascade. E.g., for (S)-46, a 20 mL reaction mixture of 11 (10 mM, 1 equiv.), CnTYR variants and EfTyrDC lysates (10% (v/v)), 8 (4 equiv.) PLP (5 mM) and CuSO4 (5 μM) at 25 °C for 8 h. Compound 47 (1.5 equiv.) and TfNCS lysates (10% (v/v)) were then added for another 16 h reaction. For the methylation step, EcMAT lysates (10% (v/v)), EcMTAN lysates (2.5% (v/v)) and RnCOMT/MxSafC/Cj6OMT lysates (10% (v/v)) were added for another 8 h reaction. aYields were determined by HPLC analysis at 280 nm against products standards. Experiments were performed in triplicate, and the error bars indicate the standard error of the triplicate reactions; bIsolated yields are given in parenthesis for preparative-scale reactions and products were purified by preparative HPLC or an extraction method; cees were determined by chiral HPLC.
