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

May, Oliver and Nguyen, Peter T. and Arnold, Frances H. (2000) Inverting enantioselectivity by directed evolution of hydantoinase for improved production of L-methionine. Nature Biotechnology, 18 (3). pp. 317-320. ISSN 1087-0156. doi:10.1038/73773.

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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.

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Arnold, Frances H.0000-0002-4027-364X
Additional Information:© 2000 Nature America Inc. Received 5 October 1999; accepted 4 December 1999. We thank Dr. Joe Altenbuchner and Dr. A. Wiese for kindly providing vector pJOE2702 and the racemase gene from Arthrobacter aurescens DSM3747, respectively, and Dr. W. Günther for his support in HPLC analytics. We also thank Dr. A. Bommarius for reading the manuscript and useful discussions and J. Ladd for his excellent technical assistance. This work was financed by Degussa-Huels AG. We also thank the Caltech SURF program for support of undergraduate research (P.N. and J. Ladd).
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Caltech Summer Undergraduate Research Fellowship (SURF)UNSPECIFIED
Subject Keywords:directed evolution; enantioselectivity; hydantoinase; biocatalysis; chiral chemicals
Issue or Number:3
Record Number:CaltechAUTHORS:20150325-154133315
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Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:56091
Deposited By: Tony Diaz
Deposited On:26 Mar 2015 02:36
Last Modified:10 Nov 2021 20:54

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