The biocatalytic reductive amination of ketone to chiral amine is one of the most challenging reactions. Using a genome-mining approach, we found proteins catalyzing the reductive amination of ketones without a carboxylic function in the α or β position. The synthesis of (4S)-4-aminopentanoic acid (ee ≥99.5%) was achieved with the thermoactive amine dehydrogenase (AmDH) AmDH4 from Petrotoga mobilis in 88% yield. The high stability and substrate tolerance make AmDH4 a very good starting point for further discovery of reductive amination biocatalysts with an enlarged substrate range. This is the first report of wild-type enzymes with related genes having proper NAD(P)H–AmDH activity.

Access to chiral amines with more than one stereocentre remains challenging, although an increasing number of methods are emerging. Here we developed a proof‐of‐concept bi‐enzymatic cascade, consisting of an ene reductase and amine dehydrogenase (AmDH), to afford chiral diastereomerically enriched amines in one pot. The asymmetric reduction of unsaturated ketones and aldehydes by ene reductases from the Old Yellow Enzyme family (OYE) was adapted to reaction conditions for the reductive amination by amine dehydrogenases. By studying the substrate profiles of both reported biocatalysts, thirteen unsaturated carbonyl substrates were assayed against the best duo OYE/AmDH. Low (5 %) to high (97 %) conversion rates were obtained with enantiomeric and diastereomeric excess of up to 99 %. We expect our established bi‐enzymatic cascade to allow access to chiral amines with both high enantiomeric and diastereomeric excess from varying alkene substrates depending on the combination of enzymes.