Genetic engineering approaches for the fermentative production of phenylglycines.

L-phenylglycine (L-Phg) is a rare non-proteinogenic amino acid, which only occurs in some natural compounds, such as the streptogramin antibiotics pristinamycin I and virginiamycin S or the bicyclic peptide antibiotic dityromycin. Industrially, more interesting than L-Phg is the enantiomeric D-Phg as it plays an important role in the fine chemical industry, where it is used as a precursor for the production of semisynthetic ß-lactam antibiotics. Based on the natural L-Phg operon from Streptomyces pristinaespiralis and the stereo-inverting aminotransferase gene hpgAT from Pseudomonas putida, an artificial D-Phg operon was constructed. The natural L-Phg operon, as well as the artificial D-Phg operon, was heterologously expressed in different actinomycetal host strains, which led to the successful production of Phg. By rational genetic engineering of the optimal producer strains S. pristinaespiralis and Streptomyces lividans, Phg production could be improved significantly. Here, we report on the development of a synthetic biology-derived D-Phg pathway and the optimization of fermentative Phg production in actinomycetes by genetic engineering approaches. Our data illustrate a promising alternative for the production of Phgs.

Characterization of the phenylglycine aminotransferase PglE from Streptomyces pristinaespiralis.

l-phenylglycine is a rare non-proteinogenic amino acid, which only occurs in a few natural compounds, such as the streptogramin antibiotics pristinamycin I and virginiamycin S or the bicyclic peptide antibiotic dityromycin. Here we report on the biochemical characterization of the aminotransferase PglE that catalyzes the transamination from phenylglyoxylate to l-phenylglycine, which represents the final reaction step during phenylglycine biosynthesis. Enzyme assays with the purified PglE enzyme revealed that l-phenylalanine is used as an amino group donor for the transamination reaction, leading to the formation of phenylpyruvate, which may re-enter phenylglycine biosynthesis as a precursor. Based on these results, we postulate a novel l-phenylglycine biosynthetic pathway.