Inverting enantioselectivity by directed evolution of hydantoinase for improved production of L-methionine

Using directed evolution, we have improved the hydantoinase process for pro duction of L-methionine (L-met) in Escherichia coli. This was accomplished by inverting the enantioselectivity and increasing the total activity of a key enzyme in a whole-cell catalyst. The selectivity of all known hydantoin ases for D-5-(2-methylthioethyl)hydantoin (D-MTEH) over the L-enantiomer le ads to the accumulation of intermediates and reduced productivity for the L -amino acid. We used random mutagenesis, saturation mutagenesis, and screen ing to convert the D-selective hydantoinase from Arthrobacter sp. DSM 9771 into an L-selective enzyme and increased its total activity fivefold. Whole E. coli cells expressing the evolved L-hydantoinase, an L-N-carbamoylase, and a hydantoin racemase produced 91 mM L-met from 100 mM D,L-MTEH in less than 2 h. The improved hydantoinase increased productivity fivefold for >90 % conversion of the substrate. The accumulation of the unwanted intermediat e D-carbamoyl-methionine was reduced fourfold compared to cells with the wi ld-type pathway. Highly D-selective hydantoinase mutants were also discover ed. Enantioselective enzymes rapidly optimized by directed evolution and in troduced into multienzyme pathways may lead to improved whole-cell catalyst s for efficient production of chiral compounds.