Discovery and Engineering Imine Reductase for Gram-Scale Synthesis of ( <i>S</i> )-Nicotine

An engineered imine reductase variant capable of synthesizing (<i>S</i>)-nicotine was developed through structure-guided directed evolution. Enzyme library screening identified IR-55 as an ideal parent, exhibiting high yield and good enantioselectivity. Targeted combinatorial mutagenesis of a crucial loop (residues 227-236) produced mutant M6 with 97.7% ee. By dynamically regulating the pH within the range of 7.5-8.0, a substrate loading of 250 g/L was achieved, resulting in a conversion rate of 94% after 4 h with space-time yield (STY) of 58.7 g/L/h. Scale-up with 2.5 g of substrate in 20 mL for 6 h yielded 1.23 g of product (97.2% ee, 75.9% isolated yield STY 10.3 g/L/h), demonstrating superior space-time yield and operational efficiency compared to existing biocatalytic routes. This work not only offers an efficient pathway for the industrial synthesis of (<i>S</i>)-nicotine through rational enzyme engineering but also provides a comprehensive strategy to overcome substrate inhibition and enzyme inactivation under high substrate concentration.