Toward scalable production of biobased N-substituted furfurylamines by engineered imine reductases

Abstract

N-substituted furfurylamines (FAs) are valuable precursors for producing pharmacologically active compounds and polymers. However, enzymatic synthesis of the type of chemicals is still in its infancy. Here we report an imine reductase from Streptomyces albidoflavus (SaIRED) for the reductive amination of biobased furans. A simple, fast and interference-resistant high-throughput screening (HTS) method was developed, based on the coloration reaction of carbonyl compounds with 2,4-dinitrophenylhydrazine. The reductive amination activity of IREDs can be directly indicated by a colorimetric assay. With the reductive amination of furfural with allylamine as the model reaction, SaIRED with the activity of 4.8 U mg^–1 was subjected to three rounds of protein engineering and screening by this HTS method, affording a high-activity tri-variant I127V/D241A/A242T (named M3, 20.2 U mg^–1). The variant M3 showed broad substrate scope, and enabled efficient reductive amination of biobased furans with a variety of amines including small aliphatic amines and sterically hindered amines, giving the target FAs in yields up to >99%. In addition, other variants were identified for preparative-scale synthesis of commercially interesting amines such as N-2-(methylsulfonyl)ethyl-FA by the screen method, with isolated yields up to 87% and turnover numbers up to 9700 for enzyme. Gram-scale synthesis of N-allyl-FA, a valuable building block and potential polymer monomer, was implemented at 0.25 mol L^–1 substrate loading by a whole-cell catalyst incorporating variant M3, with 4.7 g L^–1 h^–1 space-time yield and 91% isolated yield.