Peptide-derived cyclophanes inhabit a unique niche in the chemical space of macrocyclic peptides with several examples of pharmaceutical importance. Although both synthetic and biocatalytic methods are available for constructing these macrocycles, versatile (bio)­catalysts able to incorporate a variety of amino acids that compose the macrocycle would be useful for the creation of diverse peptide cyclophanes. In this report, we synergized the use of bioinformatic tools to map the biosynthetic landscape of radical SAM enzymes (3-CyFEs) that catalyze three-residue cyclophane formation in the biosynthesis of a new family of RiPP natural products, the triceptides. This analysis revealed 3940 (3113 unique) putative precursor sequences predicted to be modified by 3-CyFEs. Several uncharacterized maturase systems were identified that encode unique precursor types. Functional studies were carried out in vivo in Escherichia coli to identify modified precursors containing His and Tyr residues. NMR analysis of the products revealed that Tyr and His can also be incorporated into cyclophane macrocycles by 3-CyFEs. Collectively, all aromatic amino acids can be incorporated by 3-CyFEs, and the cyclophane formation strictly occurs via a C­(sp2)–C­(sp3) cross-link between the (hetero)­aromatic ring to Cβ. In addition to 3-CyFEs, we functionally validated an Fe­(II)/α-ketoglutarate-dependent hydroxylase, resulting in β-hydroxylated residues within the cyclophane rings. This study reveals the potential breadth of triceptide precursors and a systematic approach for studying these enzymes to broaden the diversity of peptide macrocycles.

Ribosomally synthesized and posttranslationally modified peptide natural products (RiPPs) exhibit diverse structures and bioactivities and are classified into distinct biosynthetic families. A recently reported family is the proteusins, with the prototype members polytheonamides being generated by almost 50 maturation steps, including introduction of d-residues at multiple positions by an unusual radical SAM epimerase. A region in the protein-like N-terminal leader of proteusin precursors is identified that is crucial for epimerization. It resembles a precursor motif previously shown to mediate interaction in thioether bridge-formation in class I lanthipeptide biosynthesis. Beyond this region, similarities were identified between proteusin and further RiPP families, including class I lanthipeptides. The data suggest that common leader features guide distinct maturation types and that nitrile hydratase-like enzymes are ancestors of several RiPP classes.

Heterologous expression of a unique biosynthetic gene cluster (BGC) comprising a highly reducing polyketide synthase and stand-alone thioesterase genes in Aspergillus oryzae enabled us to isolate a novel 34-membered polyene macrolide, phaeospelide A (1). This is the first isolation of a fungal polyene macrolide and the first demonstration of fungal aliphatic macrolide biosynthetic machinery. In addition, sequence similarity network analysis demonstrated the existence of a large number of BGCs for novel fungal macrolides.

A highly reducing polyketide synthase (HR-PKS) collaborates with a type III PKS to produce alkylresorcinols via unusual biosynthetic machinery.

Experimental equipment Analytical TLC were performed on silica gel 60 F254 (Merck) and RP-18 F254 (Merck). Column chromatography was carried out on silica gel 60 (70-230 and 40-50 mesh). The NMR spectra were recorded on a Bruker AVANCE III 500 spectrometer (1 H NMR, 500 MHz; 13 C NMR, 125 MHz) or a JEOL ECA-600 (1 H NMR, 600 MHz; 13 C NMR, 150 MHz). Chemical shifts for 1 H and 13 C NMR are given in parts per million (d) relative to tetramethylsilane (dH 0.00) and residual solvent signals (dC 77.0) for CDCl3, (dH 3.30, dC 49.0) for CD3OD and (dH 7.16, dC 128.4) for C6D6 as internal standards. Mass spectra were measured on Exactive Orbitrap Mass Spectrometer (Thermo Fischer Scientific). IR was measured on a JASCO FVS-6000 spectrometer. UV spectra were recorded on a JASCO-V-730 spectrophotometer. HPLC analysis was performed on a JASCO AS-1555-10 Intelligent Sampler, a JASCO PU-4180 RHPLC Pump, a JASCO MD-4017 Photo Diode Array Detector (JASCO), which equipped with a COSMOSIL Packed Column 5C18-MS-II (φ4.6 mm×150 mm) (Nacalai tesque). Fungal material Aspergillus kawachii IFO 4308 and Colletotrichum incanum MAFF 238704 were obtained from Biological Resource Center, NITE (NBRC) and Ministry of Agriculture, Forestry Fisheries (MAFF) respectively. Each fungus was cultured in a PDB liquid medium for 3 days and the cultured mycelium was ground to a fine powder in liquid N2. To the mycelial powder in 1.5 mL tube, 100 µL TE buffer and lysis solution (SDS 1g, 2.5 mL Tris-HCl buffer (1 M), EDTA•2Na•2H2O 186 mg, NaCl 292 mg, 50 mL mQ) were added, gently inverted and incubated 5 min on ice. After centrifugation at 15,000 rpm, 4ºC, for 20 min, 100 µL of the supernatants were transferred to a new 1.5 mL tube, followed by phenol extraction using Phenol: Chloroform: Isoamyl Alcohol 25:24:1 (pH 5.2, Nacalai tesque) and ethanol precipitation. After centrifugation again, TE buffer were added to the pellet of DNA. The DNA solutions of A. kawachii and C. incanum were used as a template for cloning of akmlA-D and cimlA-D respectively. Heterologous host strains Aspergillus oryzae NSAR1 (niaD − , sC − , DargB, adeA −) S1 was used as the host for fungal expression. Escherichia coli Dh5a (TAKARA) was used for the cloning of akmlA-D and cimlA-D. Culture medium Culture medium for A. kawachii IFO 4308 and C. incanum MAFF 238704 PDB (agar) medium: Potato-Dextrose Broth (DIFCO) 7.2 g (and agarose (Nacalai tesque) 4.5 g) in 300 mL distilled water. CPS medium (0.015% adenine): Czapek-Dox Broth (Difco) 1.05 (2.63) g, peptone* 0.3 (0.75) g, Soluble Starch (Nacalai tesque) 0.6 (1.5) g, Maltose Monohydrate (Nacalai tesque) 0.3 (0.75) g and adenine 9 (22.5) mg in 60 (150) mL distilled water. *peptone: mixture of 0.2 (0.5) g of soy peptone, casein peptone and meat peptone (Nacalai tesque). MYG medium: Bacto Peptone (DIFCO) 0.06 g, Bacto Malt Extract (DIFCO) 1.2 g, D-(+)glucose (Nacalai tesque) in 60 mL distilled water. Culture medium for A. oryzae NSAR1 PDB agar medium (adenine 0.05%, arginine 0.1%): Potato-Dextrose Broth (DIFCO) 7.2 g and agarose (Nacala...

Genome mining and heterologous biosynthesis led to the discovery of a 12 membered macrolide from Macrophomina phaseolina.

Diterpenoid pyrones are a type of mainly fungal meroterpenoid metabolite consisting of a diterpene connected to a pyrone, some of which show potent bioactivity. Through genome mining and heterologous expression, nine new diterpenoid pyrones, shearones A−I (1−9), were discovered from the fungus Eupenicillium shearii IFM 42152, and their biosynthetic enzyme activities were revealed. Some of these heterologously biosynthesized diterpenoid pyrones exhibited moderate antiaggregative ability against amyloid β42 in vitro.

Ribosomally synthesized and posttranslationally modified peptide natural products (RiPPs) exhibit diverse structures and bioactivities and are classified into distinct biosynthetic families. A recently reported family is the proteusins, with the prototype members polytheonamides being generated by almost 50 maturation steps, including introduction of d‐residues at multiple positions by an unusual radical SAM epimerase. A region in the protein‐like N‐terminal leader of proteusin precursors is identified that is crucial for epimerization. It resembles a precursor motif previously shown to mediate interaction in thioether bridge‐formation in class I lanthipeptide biosynthesis. Beyond this region, similarities were identified between proteusin and further RiPP families, including class I lanthipeptides. The data suggest that common leader features guide distinct maturation types and that nitrile hydratase‐like enzymes are ancestors of several RiPP classes.