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Applied and Environmental Microbiology, March 2007, p. 1525-1531, Vol. 73, No. 5
0099-2240/07/$08.00+0     doi:10.1128/AEM.02365-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Recombinant Polycistronic Structure of Hydantoinase Process Genes in Escherichia coli for the Production of Optically Pure D-Amino Acids{triangledown} ,{dagger}

Ana Isabel Martínez-Gómez, Sergio Martínez-Rodríguez, Josefa María Clemente-Jiménez, Joaquín Pozo-Dengra, Felipe Rodríguez-Vico, and Francisco Javier Las Heras-Vázquez*

Departamento de Química-Física, Bioquímica y Química Inorgánica, Edificio C.I.T.E.I., Universidad de Almería, La Cañada de San Urbano, Almería E-04120, Spain

Received 6 October 2006/ Accepted 1 January 2007

Two recombinant reaction systems for the production of optically pure D-amino acids from different D,L-5-monosubstituted hydantoins were constructed. Each system contained three enzymes, two of which were D-hydantoinase and D-carbamoylase from Agrobacterium tumefaciens BQL9. The third enzyme was hydantoin racemase 1 for the first system and hydantoin racemase 2 for the second system, both from A. tumefaciens C58. Each system was formed by using a recombinant Escherichia coli strain with one plasmid harboring three genes coexpressed with one promoter in a polycistronic structure. The D-carbamoylase gene was cloned closest to the promoter in order to obtain the highest level of synthesis of the enzyme, thus avoiding intermediate accumulation, which decreases the reaction rate. Both systems were able to produce 100% conversion and 100% optically pure D-methionine, D-leucine, D-norleucine, D-norvaline, D-aminobutyric acid, D-valine, D-phenylalanine, D-tyrosine, and D-tryptophan from the corresponding hydantoin racemic mixture. For the production of almost all D-amino acids studied in this work, system 1 hydrolyzed the 5-monosubstituted hydantoins faster than system 2.

* Corresponding author. Mailing address: Departamento de Química-Física, Bioquímica y Química Inorgánica, Edificio C.I.T.E.I., Universidad de Almería, La Cañada de San Urbano, Almería E-04120, Spain. Phone: 34 950 015055. Fax: 34 950 015008. E-mail: fjheras{at}ual.es.

{triangledown} Published ahead of print on 12 January 2007.

{dagger} Supplemental material for this article may be found at http://aem.asm.org/.

Applied and Environmental Microbiology, March 2007, p. 1525-1531, Vol. 73, No. 5
0099-2240/07/$08.00+0     doi:10.1128/AEM.02365-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Martinez-Gomez, A. I., Martinez-Rodriguez, S., Pozo-Dengra, J., Tessaro, D., Servi, S., Clemente-Jimenez, J. M., Rodriguez-Vico, F., Las Heras-Vazquez, F. J. (2009). Potential Application of N-Carbamoyl-{beta}-Alanine Amidohydrolase from Agrobacterium tumefaciens C58 for {beta}-Amino Acid Production. Appl. Environ. Microbiol. 75: 514-520 [Abstract] [Full Text]  


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