Linking orphan clusters to molecules

Linking orphan clusters to molecules. (a) Flowchart of the “genomisotopic approach” for the isotope-guided isolation of orfamide A biosynthesized from the ofa gene cluster. C, condensation domain; A, adenylation domain; TE, thioesterase domain; small circles symbolize the thiolation (T) domain. (b) Phylogeny of trans-AT KS domains, KS domains that accept similar substrates fall into the same clade type. (c) Cloning of the psy gene cluster and the elucidation of its biosynthetic pathway from a highly complicated metagenome by the chemical structure-based gene targeting approach. The KS with one asterisk is rare and no clade exists so far. The KS with two asterisks falls into the cis-AT group. Domains shown in white lack conserved catalytic residues and are non-functional. For the abbreviation for each domain, see reference []. (d) Flowchart of PrISM for the identification of robustly expressed NRPSs. The high molecular weight proteins from microbial cultures are subjected to in-gel digestion and proteomics analysis by LC/MSn. Peptides that contain Ppant arms can be traced by the Ppant ejection assay. Peptide sequences obtained from de novo peptide sequencing are used to design degenerate primers to amplify regions of the expressed gene cluster. The A domains of the identified NRPS cluster are used to predict the substrates of amino acid incorporated. The metabolites with the predicted amino acid sequences are ultimately confirmed by LC/MSn in the culture supernatant.

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Recent advances in awakening silent biosynthetic gene clusters and linking orphan clusters to natural products in microorganisms. () Yi-Ming Chiang, et al. Curr Opin Chem Biol. ;15(1):137-143. Figure: F2. Drosophila melanogaster

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