One-Pot Conversion of L-Threonine into L-Homoalanine:Biocatalytic Production of an Unnatural Amino Acid from a Natural One

摘要

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 co-transaminase (co-TA). TD catalyzes the dehydration/deamination of L-threonine, leading to the generation of 2-oxobu-tyrate which is asymmetrically converted to L-homoalanine via transamination with benzylamine executed by co-TA. To make up the coupled reaction system, we cloned and overexpressed a TD from Escherichia coli and an (S)-specific co-TA from Para-coccus denitrificans. In the coupled reactions, l-threonine serves as a precursor of 2-oxobutyrate for the co-TA reaction, eliminating the need for employing the expensive oxo acid as a starting reactant. In contrast to a-transaminase reactions in which use of amino acids as an exclusive amino donor limits complete conversion, amines are exploited-in the co-TA reaction and thus maximum conversion could reach 100%. The co-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 co-TA-only reaction did not produce any detectable amount of L-homoalanine from 10 mM l-threonine and 20 mM benzylamine, whereas the co-TA reaction coupled with TD led to 91% conversion of L-threonine to L-homoalanine.