The AusAB non-ribosomal peptide synthase in Staphylococcus aureus preferentially incorporates exogenous phenylalanine and tyrosine into the aureusimine natural products

Abstract

Non-ribosomal peptide synthases (NRPS) are modular multidomain enzymes, responsible for the biosynthesis of various secondary metabolites, in a mRNA-template independent manner. They are predominantly present in bacteria and fungi, where they synthesize a variety of products, including antibiotics, siderophores, toxins and signalling molecules. The human pathogen Staphylococcus aureus possesses one single NRPS, AusA, highly conserved in all sequenced S. aureus strains. AusA incorporates the aromatic amino acids (AAA) phenylalanine or tyrosine, as well as the branched-chain amino acids (BCAA) valine and leucine into three cyclic dipeptides collectively called aureusimines: phevalin, tyrvalin and leuvalin. By using targeted metabolomics, we found that AusA preferentially incorporates phenylalanine and tyrosine from an exogenous source into aureusimines, whereas the source of valine can be either endo- or exogenous. Upon cultivation in a chemically defined medium (CDM) lacking phenylalanine, the amino acid was not incorporated into phevalin, despite de novo phenylalanine biosynthesis. Tyrosine production remained unaffected. Conversely, upon cultivation in medium lacking tyrosine, tyrvalin production was not detected, despite tyrosine de novo biosynthesis. Phevalin production, however, remained unaltered. By contrast, omission of valine in the culture medium not only resulted in de novo valine biosynthesis but also was accompanied by phevalin production. To our knowledge, this is the first report of a selective incorporation of AAAs by a bacterial NRPS, which provides useful basis for linking bacterial cell metabolic status to the biosynthesis of secondary metabolites. IMPORTANCEPeptide and protein synthesis are fundamental processes in nature, which are largely mediated by the ribosomal machinery. An alternative pathway for peptide synthesis is non-ribosomal mRNA-template independent synthesis, performed by so-called non-ribosomal peptide synthases (NRPS). NRPSs are multi-enzyme complexes, which serve the simultaneous role of template and biosynthetic machinery. They are mostly found in bacteria and fungi and are responsible for the biosynthesis of many pharmacologically significant products, including antibiotics, anticancer compounds or immunosuppressants. The human pathogen S. aureus possesses one such NRPS, AusA, which synthesizes three cyclic dipeptides termed "aureusimines" using the aromatic amino acids phenylalanine and tyrosine, and the branched-chain amino acid valine. Although the biological role of aureusimines remains unknown, AusA appears to play a role in the interaction of S. aureus with the host. In addition, owing to its minimal canonical NRPS structure and autonomous function (i.e. most NRPS pathways require the assembly of several NRPS proteins), AusA represents an excellent model system for studying such molecular assembly lines. Our observation is, to our knowledge, the first report of a NRPS incorporating phenylalanine and tyrosine only from exogenous sources (e.g. environment, culture medium), but not from de novo "self-made" pool. This opens up new avenues in understanding and modulating the function of NRPSs (e.g. for biotechnological purposes).