Corynebacterium glutamicum as a host for synthesis and export of D-Amino Acids.
A number of d-amino acids occur in nature, and there is growing interest in their function and metabolism, as well as in their production and use. Here we use the well-established l-amino-acid-producing bacterium Corynebacterium glutamicum to study whether d-amino acid synthesis is possible and whether mechanisms for the export of these amino acids exist. In contrast to Escherichia coli, C. glutamicum tolerates d-amino acids added extracellularly. Expression of argR (encoding the broad-substrate-specific racemase of Pseudomonas taetrolens) with its signal sequence deleted results in cytosolic localization of ArgR in C. glutamicum. The isolated enzyme has the highest activity with lysine (100%) but also exhibits activity with serine (2%). Upon overexpression of argR in an l-arginine, l-ornithine, or l-lysine producer, equimolar mixtures of the d- and l-enantiomers accumulated extracellularly. Unexpectedly, argR overexpression in an l-serine producer resulted in extracellular accumulation of a surplus of d-serine (81 mM d-serine and 37 mM l-serine) at intracellular concentrations of 125 mM d-serine plus 125 mM l-serine. This points to a nonlimiting ArgR activity for intracellular serine racemization and to the existence of a specific export carrier for d-serine. Export of d-lysine relies fully on the presence of lysE, encoding the exporter for l-lysine, which is apparently promiscuous with respect to the chirality of lysine. These data show that d-amino acids can also be produced with C. glutamicum and that in special cases, due to specific carriers, even a preferential extracellular accumulation of this enantiomer is possible.
Institut für Bio- und Geowissenschaften, IBG-1: Biotechnologie, Forschungszentrum Jülich, D-52425 Jülich, Germany. l.eggeling@fz-juelich.de
Norma
N
Stäbler
Tadao
T
Oikawa
Michael
M
Bott
Lothar
L
Eggeling
D000591:Amino Acid Isomerases / Q000235:genetics / Q000378:metabolism*; D000596:Amino Acids / Q000096:biosynthesis* / Q000737:chemistry*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D048230:Corynebacterium glutamicum / Q000201:enzymology / Q000254:growth & development / Q000378:metabolism*; D003470:Culture Media; D003600:Cytosol; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology*; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology; D008239:Lysine / Q000096:biosynthesis / Q000737:chemistry; D021381:Protein Transport; D011549:Pseudomonas / Q000201:enzymology; D012694:Serine / Q000096:biosynthesis / Q000737:chemistry
D000596:Amino Acids; D001426:Bacterial Proteins; D003470:Culture Media; D012694:Serine; D000591:Amino Acid Isomerases; D008239:Lysine
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