A Long‐Range Acting Dehydratase Domain as the Missing Link for C17‐Dehydration in Iso‐Migrastatin Biosynthesis

The dehydratase domains (DHs) of the iso‐migrastatin (iso‐MGS) polyketide synthase (PKS) were investigated by systematic inactivation of the DHs in module‐6, ‐9, ‐10 of MgsF (i.e., DH6, DH9, DH10) and module‐11 of MgsG (i.e., DH11) in vivo, followed by structural characterization of the metabolites accumulated by the mutants, and biochemical characterization of DH10 in vitro, using polyketide substrate mimics with varying chain lengths. These studies allowed us to assign the functions for all four DHs, identifying DH10 as the dedicated dehydratase that catalyzes the dehydration of the C17 hydroxy group during iso‐MGS biosynthesis. In contrast to canonical DHs that catalyze dehydration of the β‐hydroxy groups of the nascent polyketide intermediates, DH10 acts in a long‐range manner that is unprecedented for type I PKSs, a novel dehydration mechanism that could be exploited for polyketide structural diversity by combinatorial biosynthesis and synthetic biology.     

Biosynthesis of l‐4‐Chlorokynurenine,an Antidepressant Prodrug and a Non‐Proteinogenic Amino Acid Found in Lipopeptide Antibiotics

l ‐4‐Chlorokynurenine (l ‐4‐Cl‐Kyn) is a neuropharmaceutical drug candidate that is in development for the treatment of major depressive disorder. Recently, this amino acid was naturally found as a residue in the lipopeptide antibiotic taromycin. Herein, we report the unprecedented conversion of l ‐tryptophan into l ‐4‐Cl‐Kyn catalyzed by four enzymes in the taromycin biosynthetic pathway from the marine bacterium Saccharomonospora sp. CNQ‐490. We used genetic, biochemical, structural, and analytical techniques to establish l ‐4‐Cl‐Kyn biosynthesis, which is initiated by the flavin‐dependent tryptophan chlorinase Tar14 and its flavin reductase partner Tar15. This work revealed the first tryptophan 2,3‐dioxygenase (Tar13) and kynurenine formamidase (Tar16) enzymes that are selective for chlorinated substrates. The substrate scope of Tar13, Tar14, and Tar16 was examined and revealed intriguing promiscuity, thereby opening doors for the targeted engineering of these enzymes as useful biocatalysts.     

Synthesis,Structural Reassignment,and Antibacterial Evaluation of 2,18‐Seco‐Lankacidinol B

Lankacidins are a group of polyketide natural products with activity against several strains of Gram‐positive bacteria. We developed a route to stereochemically diverse variants of 2,18‐seco‐lankacidinol B and found that the stereochemical assignment at C4 requires revision. This has interesting implications for the biosynthesis of natural products of the lankacidin class, all of which possessed uniform stereochemistry prior to this finding. We have evaluated 2,18‐seco‐lankacidinol B and three stereochemical derivatives against a panel of pathogenic Gram‐positive and Gram‐negative bacteria.     

15‐Hydroperoxy‐PGE2: Intermediate in Mammalian and Algal Prostaglandin Biosynthesis

Arachidonic‐acid‐derived prostaglandins (PGs), specifically PGE₂, play a central role in inflammation and numerous immunological reactions. The enzymes of PGE₂ biosynthesis are important pharmacological targets for anti‐inflammatory drugs. Besides mammals, certain edible marine algae possess a comprehensive repertoire of bioactive arachidonic‐acid‐derived oxylipins including PGs that may account for food poisoning. Described here is the analysis of PGE₂ biosynthesis in the red macroalga Gracilaria vermiculophylla that led to the identification of 15‐hydroperoxy‐PGE₂, a novel precursor of PGE₂ and 15‐keto‐PGE₂. Interestingly, this novel precursor is also produced in human macrophages where it represents a key metabolite in an alternative biosynthetic PGE₂ pathway in addition to the well‐established arachidonic acid‐PGG₂‐PGH₂‐PGE₂ route. This alternative pathway of mammalian PGE₂ biosynthesis may open novel opportunities to intervene with inflammation‐related diseases.     

Guanitrypmycin Biosynthetic Pathways Imply Cytochrome P450 Mediated Regio‐ and Stereospecific Guaninyl‐Transfer Reactions

Mining microbial genomes including those of Streptomyces reveals the presence of a large number of biosynthetic gene clusters. Unraveling this genetic potential has proved to be a useful approach for novel compound discovery. Here, we report the heterologous expression of two similar P450‐associated cyclodipeptide synthase‐containing gene clusters in Streptomyces coelicolor and identification of eight rare and novel natural products, the C3‐guaninyl indole alkaloids guanitrypmycins. Expression of different gene combinations proved that the cyclodipeptide synthases assemble cyclo‐l ‐Trp‐l ‐Phe and cyclo‐l ‐Trp‐l ‐Tyr, which are consecutively and regiospecifically modified by cyclodipeptide oxidases, cytochrome P450 enzymes, and N‐methyltransferases. In vivo and in vitro results proved that the P450 enzymes function as key biocatalysts and catalyze the regio‐ and stereospecific 3α‐guaninylation at the indole ring of the tryptophanyl moiety. Isotope‐exchange experiments provided evidence for the non‐enzymatic epimerization of the biosynthetic pathway products via keto–enol tautomerism. This post‐pathway modification during cultivation further increases the structural diversity of guanitrypmycins.     

In vitro Reconstitution of the Biosynthetic Pathway to the Nitroimidazole Antibiotic Azomycin

Nitroimidazoles are one of the most effective ways to treat anaerobic bacterial infections. Synthetic nitroimidazoles are inspired by the structure of azomycin, isolated from Streptomyces eurocidicus in 1953. Despite its foundational role, no biosynthetic gene cluster for azomycin has been found. Guided by bioinformatics, we identified a cryptic biosynthetic gene cluster in Streptomyces cattleya and then carried out in vitro reconstitution to deduce the enzymatic steps in the pathway linking l ‐arginine to azomycin. The gene cluster we discovered is widely distributed among soil‐dwelling actinobacteria and proteobacteria, suggesting that azomycin and related nitroimidazoles may play important ecological roles. Our work sets the stage for development of biocatalytic approaches to generate azomycin and related nitroimidazoles.     

Biosynthesis of the Carbonylmethylene Structure Found in the Ketomemicin Class of Pseudotripeptides

We recently discovered novel pseudotripeptides, the ketomemicins, which possess a C‐terminal pseudodipeptide connected with a carbonylmethylene instead of an amide bond, through heterologous expression of gene clusters identified in actinobacteria. The carbonylmethylene structure is a stable isostere of the amide bond and its biological significance has been shown in several natural and synthetic products. Despite the biological importance of these compounds, little is known about how the carbonylmethylene structure is biosynthesized. In this work, we fully characterized the biosynthetic machinery of the pseudodipeptide. An aldolase, dehydratase, PLP‐dependent glycine‐C‐acetyltransferase, and dehydrogenase were involved in the formation of the pseudodipeptide, with malonyl‐CoA and phenylpyruvate as starter substrates.