Identification of the Cirratiomycin Biosynthesis Gene Cluster in Streptomyces Cirratus: Elucidation of the Biosynthetic Pathways for 2,3-Diaminobutyric Acid and Hydroxymethylserine

Cirratiomycin, a heptapeptide with antibacterial activity, was isolated and characterized in 1981; however, its biosynthetic pathway has not been elucidated. It contains several interesting nonproteinogenic amino acids, such as (2S,3S)-2,3-diaminobutyric acid ((2S,3S)-DABA) and alpha-(hydroxymethyl)serine, as building blocks. Here, we report the identification of a cirratiomycin biosynthetic gene cluster in Streptomyces cirratus. Bioinformatic analysis revealed that several Streptomyces viridifaciens and Kitasatospora aureofaciens strains also have this cluster. One S. viridifaciens strain was confirmed to produce cirratiomycin. The biosynthetic gene cluster was shown to be responsible for cirratiomycin biosynthesis in S. cirratus in a gene inactivation experiment using CRISPR-cBEST. Interestingly, this cluster encodes a nonribosomal peptide synthetase (NRPS) composed of 12 proteins, including those with an unusual domain organization: a stand-alone adenylation domain, two stand-alone condensation domains, two type II thioesterases, and two NRPS modules that have no adenylation domain. Using heterologous expression and in vitro analysis of recombinant enzymes, we revealed the biosynthetic pathway of (2S,3S)-DABA: (2S,3S)-DABA is synthesized from l-threonine by four enzymes, CirR, CirS, CirQ, and CirB. In addition, CirH, a glycine/serine hydroxymethyltransferase homolog, was shown to synthesize alpha-(hydroxymethyl)serine from d-serine in vitro. These findings broaden our knowledge of nonproteinogenic amino acid biosynthesis.