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

Nature Biotechnology volume 18, pages 317–320 (2000)Cite this article

Abstract

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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Figure 1: Reactions and enzymes involved in the production of L-—amino acids from racemic hydantoins by the three-enzyme hydantoinase process.
Figure 2: Representative example of screening results: activities toward D- and L-MTEH of 4,000 clones from the first random mutant library.
Figure 3
Figure 4: Evolutionary progression and enantioselectivities of the various mutants.
Figure 5

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Acknowledgements

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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  1. Division of Chemistry and Chemical Engineering, California Institute of Technology, 210-41, Pasadena, 91125, CA

    Oliver May, Peter T. Nguyen & Frances H. Arnold

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  1. Oliver May
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Correspondence to Frances H. Arnold.

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May, O., Nguyen, P. & Arnold, F. Inverting enantioselectivity by directed evolution of hydantoinase for improved production of l-methionine. Nat Biotechnol 18, 317–320 (2000). https://doi.org/10.1038/73773

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