Engineering the enantioselectivity of a novel imine reductase from Streptomyces viridochromogenes for the dynamic kinetic reductive amination of cyclic β-ketoester

N -substituted β-amino esters as an important class of chiral amines, are widely used as building blocks in pharmaceuticals. A novel imine reductase Sv IRED was identified through gene mining from Streptomyces viridochromogenes with high enantioselectivity of > 99 % ee (1 R , 2 S ), 95 % de ( cis:trans ), towards 2-oxocyclohexane-1-carboxylate and cyclopropylamine. Sv IRED exhibited the highest activity at pH 7.0 and 40 °C. A variety of amine donors could be used by Sv IRED and produce chiral N -substituted β-amino esters with different enantioselectivity. Employing methylamine ( i ) and pyrrolidine ( n ) as amine donors, Sv IRED exhibited reversed enantioselectivity of 23 % ee (1 S , 2 R ) and 79 % ee (1 S , 2 R ) respectively. Through alanine scanning, M183 and H244 were identified with remarkable roles in governing enantioselectivity towards i and n . Compared with WT Sv IRED, the enantioselectivity of M183A was inverted to ee value of >99 % (1 R , 2 S ) and de value of >99 % ( cis:trans ) towards i , and the enantioselectivity of H244A was inverted to ee value of 88 % (1 R , 2 S ) and de value of >99 % ( cis:trans ) towards n . Interaction analysis indicated the orientation of ester group was vital for manipulation of enantioselectivity. This study provides novel Imine reductase mutants for enantioselective synthesis of N -substituted β -amino esters with different amine donors.