Corynebacterium glutamicum as a Host for Synthesis and Export of d -Amino Acids

Abstract

ABSTRACT 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.