Combinatorialization of Fungal Polyketide Synthase–Peptide Synthetase Hybrid Proteins

The programming of the fungal polyketide synthase (PKS) is quite complex, with a simple domain architecture leading to elaborate products. An additional level of complexity has been found within PKS-based pathways where the PKS is fused to a single module nonribosomal peptide synthetase (NRPS) to synthesize polyketides conjugated to amino acids. Here, we sought to understand the communication between these modules that enable correct formation of polyketide-peptide hybrid products. To do so, we fused together the genes that are responsible for forming five highly chemically diverse fungal natural products in a total of 57 different combinations, comprising 34 distinct module swaps. Gene fusions were formed with the idea of testing the connection and compatibility of the PKS and NRPS modules mediated by the acyl carrier protein (ACP), condensation (C) and ketoreductase (KR) domains. The resulting recombinant gene fusions were analyzed in a high-yielding expression platform to avail six new compounds, including the first successful fusion between a PKS and NRPS that make highly divergent products, and four previously reported molecules. Our results show that C domains are highly selective for a subset of substrates. We discovered that within the highly reducing (hr) PKS class, noncognate ACPs of closely related members complement PKS function. We intercepted a pre-Diels–Alder intermediate in lovastatin synthesis for the first time, shedding light on this canonical fungal biochemical reaction. The results of these experiments provide a set of ground rules for the successful engineering of hr-PKS and PKS-NRPS products in fungi

Two Related Pyrrolidinedione Synthetase Loci in <i>Fusarium heterosporum</i> ATCC 74349 Produce Divergent Metabolites

Equisetin synthetase (EqiS), from the filamentous fungus <i>Fusarium heterosporum</i> ATCC 74349, was initially assigned on the basis of genetic knockout and expression analysis. Increasing inconsistencies in experimental results led us to question this assignment. Here, we sequenced the <i>F. heterosporum</i> genome, revealing two hybrid polyketide-peptide proteins that were candidates for the equisetin synthetase. The surrounding genes in both clusters had the needed auxiliary genes that might be responsible for producing equisetin. Genetic mutation, biochemical analysis, and recombinant expression in the fungus enabled us to show that the initially assigned EqiS does not produce equisetin but instead produces a related 2,4-pyrrolidinedione, fusaridione A, that was previously unknown. Fusaridione A is methylated in the 3-position of the pyrrolidinedione, which has not otherwise been found in natural products, leading to spontaneous reverse-Dieckmann reactions. A newly described gene cluster, <i>eqx</i>, is responsible for producing equisetin

Aestuaramides, a Natural Library of Cyanobactin Cyclic Peptides Resulting from Isoprene-Derived Claisen Rearrangements

We report 12 cyanobactin cyclic peptides, the aestuaramides, from the cultivated cyanobacterium <i>Lyngbya aestuarii</i>. We show that aestuaramides are synthesized enzymatically as reverse <i>O</i>-prenylated tyrosine ethers that subsequently undergo a Claisen rearrangement to produce forward <i>C</i>-prenylated tyrosine. These results reveal that a nonenzymatic Claisen rearrangement dictates isoprene regiochemistry in a natural system. They also reveal one of the mechanisms that organisms use to generate structurally diverse compound libraries starting from simple ribosomal peptide pathways (RiPPs)

Enzymatic N- and C‑Protection in Cyanobactin RiPP Natural Products

Recent innovations in peptide natural product biosynthesis reveal a surprising wealth of previously uncharacterized biochemical reactions that have potential applications in synthetic biology. Among these, the cyanobactins are noteworthy because these peptides are protected at their N- and C-termini by macrocyclization. Here, we use a novel bifunctional enzyme AgeMTPT to protect linear peptides by attaching prenyl and methyl groups at their free N- and C-termini. Using this peptide protectase in combination with other modular biosynthetic enzymes, we describe the total synthesis of the natural product aeruginosamide B and the biosynthesis of linear cyanobactin natural products. Our studies help to define the enzymatic mechanism of macrocyclization, providing evidence against the water exclusion hypothesis of transpeptidation and favoring the kinetic lability hypothesis

Small Molecules in the Cone Snail Arsenal

Cone snails are renowned for producing peptide-based venom, containing conopeptides and conotoxins, to capture their prey. A novel small-molecule guanine derivative with unprecedented features, genuanine, was isolated from the venom of two cone snail species. Genuanine causes paralysis in mice, indicating that small molecules and not just polypeptides may contribute to the activity of cone snail venom

Modulating the Serotonin Receptor Spectrum of Pulicatin Natural Products

Serotonin (5-HT) receptors are important in health and disease, but the existence of 14 subtypes necessitates selective ligands. Previously, the pulicatins were identified as ligands that specifically bound to the subtype 5-HT_2B in the 500 nM to 10 μM range and that exhibited <i>in vitro</i> effects on cultured mouse neurons. Here, we examined the structure–activity relationship of 30 synthetic and natural pulicatin derivatives using binding, receptor functionality, and <i>in vivo</i> assays. The results reveal the 2-arylthiazoline scaffold as a tunable serotonin receptor-targeting pharmacophore. Tests in mice show potential antiseizure and antinociceptive activities at high doses without motor impairment

Post-Translational Tyrosine Geranylation in Cyanobactin Biosynthesis

Prenylation is a widespread modification that improves the biological activities of secondary metabolites. This reaction also represents a key modification step in biosyntheses of cyanobactins, a family of ribosomally synthesized and post-translationally modified peptides (RiPPs) produced by cyanobacteria. In cyanobactins, amino acids are commonly isoprenylated by ABBA prenyltransferases that use C₅ donors. Notably, mass spectral analysis of piricyclamides from a fresh-water cyanobacterium suggested that they may instead have a C₁₀ geranyl group. Here we characterize a novel geranyltransferase involved in piricyclamide biosynthesis. Using the purified enzyme, we show that the enzyme PirF catalyzes Tyr <i>O</i>-geranylation, which is an unprecedented post-translational modification. In addition, the combination of enzymology and analytical chemistry revealed the structure of the final natural product, piricyclamide 7005E1, and the regioselectivity of PirF, which has potential as a synthetic biological tool providing drug-like properties to diverse small molecules

Oxazinin A, a Pseudodimeric Natural Product of Mixed Biosynthetic Origin from a Filamentous Fungus

A racemic, prenylated polyketide dimer, oxazinin A (<b>1</b>), was isolated from a novel filamentous fungus in the class Eurotiomycetes, and its structure was elucidated spectroscopically. The pentacyclic structure of oxazinin A (<b>1</b>) is a unique combination of benzoxazine, isoquinoline, and a pyran ring. Oxazinin A (<b>1</b>) exhibited antimycobacterial activity and modestly antagonized transient receptor potential (TRP) channels

Totopotensamides, Polyketide–Cyclic Peptide Hybrids from a Mollusk-Associated Bacterium <i>Streptomyces</i> sp.

Two new compounds, the peptide–polyketide glycoside totopotensamide A (<b>1</b>) and its aglycone totopotensamide B (<b>2</b>), were isolated from a <i>Streptomyces</i> sp. cultivated from the gastropod mollusk <i>Lienardia totopotens</i> collected in the Philippines. The compounds contain a previously undescribed polyketide component, a novel 2,3-diaminobutyric acid-containing macrolactam, and a new amino acid, 4-chloro-5,7-dihydroxy-6-methylphenylglycine. The application of Marfey’s method to phenylglycine derivatives was explored using quantum mechanical calculations and NMR