• DOI:10.1038/73773
• Corpus ID: 20991257

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

@article{May2000InvertingEB,
  title={Inverting enantioselectivity by directed evolution of hydantoinase for improved production of l-methionine},
  author={Oliver S. May and Peter T. Nguyen and Frances H. Arnold},
  journal={Nature Biotechnology},
  year={2000},
  volume={18},
  pages={317-320}
}

• O. May, Peter T. Nguyen, F. Arnold
• Published 1 March 2000
• Biology
• Nature Biotechnology

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.