Open AccessRecombinant Polycistronic Structure of Hydantoinase Process Genes in Escherichia coli for the Production of Optically Pure d -Amino Acids
Author(s) -
Ana Isabel Martínez-Gómez,
Sergio MartínezRodríguez,
Josefa María ClementeJiménez,
Joaquín Pozo-Dengra,
Felipe RodríguezVico,
Francisco Javier Las HerasVázquez
Publication year - 2007
Publication title -
applied and environmental microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.552
H-Index - 324
eISSN - 1070-6291
pISSN - 0099-2240
DOI - 10.1128/aem.02365-06
Subject(s) - hydantoin , norleucine , norvaline , escherichia coli , amino acid , biochemistry , biology , recombinant dna , valine , methionine , enzyme , stereochemistry , phenylalanine , chemistry , gene
Two recombinant reaction systems for the production of optically pured -amino acids from differentd,l -5-monosubstituted hydantoins were constructed. Each system contained three enzymes, two of which wered -hydantoinase andd -carbamoylase fromAgrobacterium tumefaciens BQL9. The third enzyme was hydantoin racemase 1 for the first system and hydantoin racemase 2 for the second system, both fromA. tumefaciens C58. Each system was formed by using a recombinantEscherichia coli strain with one plasmid harboring three genes coexpressed with one promoter in a polycistronic structure. Thed -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 pured -methionine,d -leucine,d -norleucine,d -norvaline,d -aminobutyric acid,d -valine,d -phenylalanine,d -tyrosine, andd -tryptophan from the corresponding hydantoin racemic mixture. For the production of almost alld -amino acids studied in this work, system 1 hydrolyzed the 5-monosubstituted hydantoins faster than system 2.