A robust and stereocomplementary panel of ene-reductase variants for gram-scale asymmetric hydrogenation

We report an engineered panel of ene-reductases (ERs) from Thermus scotoductus SA-01 (TsER) that combines control over facial selectivity in the reduction of electron deficient C??C double bonds with thermostability (up to 70 ?C), organic solvent tolerance (up to 40 % v/v) and a broad substrate scope (23 compounds, three new to literature). Substrate acceptance and facial selectivity of 3-methylcyclohexenone was rationalized by crystallisation of TsER C25D/I67T and in silico docking. The TsER variant panel shows excellent enantiomeric excess (ee) and yields during bi-phasic preparative scale synthesis, with isolated yield of up to 93 % for 2R,5S-dihydrocarvone (3.6 g). Turnover frequencies (TOF) of approximately 40 000 h-1 were achieved, which are comparable to rates in hetero- and homogeneous metal catalysed hydrogenations. Preliminary batch reactions also demonstrated the reusability of the reaction system by consecutively removing the organic phase (n-pentane) for product removal and replacing with fresh substrate. Four consecutive batches yielded ca. 27 g L-1 R-levodione from a 45 mL aqueous reaction, containing less than 17 mg (10 ?M) enzyme and the reaction only stopping because of acidification. The TsER variant panel provides a robust, highly active and stereocomplementary base for further exploitation as a tool in preparative organic synthesis.

Automated summary

The engineered panel of ene-reductases (ERs) from Thermus scotoductus SA-01 shows control over facial selectivity, thermostability, organic solvent tolerance, and a broad substrate scope. The TsER variant panel exhibits excellent enantiomeric excess (ee) and yields during bi-phasic preparative scale synthesis.