Inverting enantioselectivity by directed evolution of hydantoinase for improved production of L-methionine.
Using directed evolution, we have improved the hydantoinase process for production 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 hydantoinases for D-5-(2-methylthioethyl)hydantoin (D-MTEH) over the L-enantiomer leads to the accumulation of intermediates and reduced productivity for the L-amino acid. We used random mutagenesis, saturation mutagenesis, and screening 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 intermediate D-carbamoyl-methionine was reduced fourfold compared to cells with the wild-type pathway. Highly D-selective hydantoinase mutants were also discovered. Enantioselective enzymes rapidly optimized by directed evolution and introduced into multienzyme pathways may lead to improved whole-cell catalysts for efficient production of chiral compounds.
May
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California Institute of Technology, Division of Chemistry and Chemical Engineering 210-41, Pasadena, CA 91125, USA.
Nguyen
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PT
Arnold
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FH
D000581:Amidohydrolases / Q000737:chemistry / Q000378:metabolism*; D019020:Directed Molecular Evolution / Q000379:methods*; D004926:Escherichia coli / Q000201:enzymology / Q000378:metabolism; D008715:Methionine / Q000096:biosynthesis*; D008956:Models, Chemical; D016296:Mutagenesis; D019151:Peptide Library; D016133:Polymerase Chain Reaction; D013237:Stereoisomerism; D013997:Time Factors
D019151:Peptide Library; D008715:Methionine; D000581:Amidohydrolases; C020047:dihydropyrimidinase