Enantioselective Biosynthesis of (+)- and (−)-Auranthines

In nature, organisms produce various bioactive natural products (NPs). Most NPs target naturally occurring macromolecules, such as proteins and nucleotides. Thus, NPs are produced in a stereocontrolled manner except for the cases where nonenzymatic reactions are involved in the biosynthesis. This is why stereoisomers, especially enantiomers, are rarely found among metabolites from natural sources. During the biosynthetic study of auranthine, a fungal NP containing a nitrile group, we discovered that the (-)-isomer of auranthine (<b>1</b>) is produced by <i>Aspergillus lentulus</i> strains isolated in Japan, while a previously unreported (+)-enantiomer <b>2</b> is produced by those isolated elsewhere. The biosynthetic genes for both isomers were determined by transcriptomic, gene deletion, and heterologous expression experiments, revealing that two different nonribosomal peptide synthetases (NRPSs) NitA and NitC were involved in the biosynthesis of <b>1</b> and <b>2</b>, respectively. Both NitA and NitC are bimodular NRPSs, as is the case for the asperlicin-synthesizing enzyme AspA. All incorporate two molecules of anthranilic acid and one molecule of amino acid to form the peptide core. However, only NitC contains an epimerization domain, suggesting that is how the enantiomeric pair is biosynthesized by NitA and NitC. Furthermore, biosynthesis of the nitrile-bearing l-γ-cyanohomoalanine that is incorporated into <b>1</b> and <b>2</b> was found to be catalyzed by an argininosuccinate synthetase-like NitB using l-glutamine as a substrate. This study reports not only the unique mechanism of nitrile-containing amino acid biosynthesis but also the intriguing production of an enantiomeric pair of secondary metabolites by different strains of the same fungal species (250/250).