One-pot conversion of L -threonine into L -homoalanine: Biocatalytic production of an unnatural amino acid from a natural one

A novel biocatalytic process for production of L-homoalanine from L-threonine has been developed using coupled enzyme reactions consisting of a threonine deaminase (TD) and an ω-transaminase (ω-TA). TD catalyzes the dehydration/deamination of L-threonine, leading to the generation of 2-oxobutyrate which is asymmetrically converted to L-homoalanine via transamination with benzylamine executed by ω-TA. To make up the coupled reaction system, we cloned and overexpressed a TD from Escherichia coli and an (S)-specific ω-TA from Paracoccus denitrificans. In the coupled reactions, L-threonine serves as a precursor of 2-oxobutyrate for the ω-TA reaction, eliminating the need for employing the expensive oxo acid as a starting reactant. In contrast to α-transaminase reactions in which use of amino acids as an exclusive amino donor limits complete conversion, amines are exploited in the ω-TA reaction and thus maximum conversion could reach 100%. The ω-TA-only reaction with 10 mM 2-oxobutyrate and 20 mM benzylamine resulted in 94% yield of optically pure L-homoalanine (ee>99%). However, the ω-TA-only reaction did not produce any detectable amount of L-homoalanine from 10 mM L-threonine and 20 mM benzylamine, whereas the ω-TA reaction coupled with TD led to 91% conversion of L-threonine to L-homoalanine.