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Natural products are a diverse class of biologically produced compounds that participate in fundamental biological processes such as cell signaling, nutrient acquisition, and interference competition. Unique triple-bond functionalities like isonitriles and alkynes often drive bioactivity and may serve as indicators of novel chemical logic and enzymatic machinery. Yet, the biosynthetic underpinnings of these groups remain only partially understood, constraining the opportunity to rationally engineer biomolecules with these functionalities for applications in pharmaceuticals, bioorthogonal chemistry, and other value-added chemical processes. Here, we focus our review on characterized biosynthetic pathways for isonitrile and alkyne functionalities, their bioorthogonal transformations, and prospects for engineering their biosynthetic machinery for biotechnological applications.

Small molecule natural products are a chemically diverse class of biomolecules that fulfill myriad biological functions, including autoregulation, communication with microbial neighbors and the host, interference competition, nutrient acquisition, and resistance to oxidative stress. Human commensal bacteria are increasingly recognized as a potential source of new natural products, which may provide insight into the molecular ecology of many different human body sites as well as novel scaffolds for therapeutic development. Here, we review the scientific literature on natural products derived from residents of the human nasal/oral cavity, discuss their discovery, biosynthesis, and ecological roles, and identify key questions in the study of these compounds.

Triacsins are a family of natural products having in common an N-hydroxytriazene moiety not found in any other known secondary metabolites. Though many studies have examined the biological activity of triacsins in lipid metabolism, their biosynthesis has remained unknown. Here we report the identification of the triacsin biosynthetic gene cluster in Streptomyces aureofaciens ATCC 31442. Bioinformatic analysis of the gene cluster led to the discovery of the tacrolimus producer Streptomyces tsukubaensis NRRL 18488 as a new triacsin producer. In addition to targeted gene disruption to identify necessary genes for triacsin production, stable isotope feeding was performed in vivo to advance the understanding of N-hydroxytriazene biosynthesis.

The electron-rich isonitrile is an important functionality in bioactive natural products, but its biosynthesis has been restricted to the IsnA family of isonitrile synthases. We herein provide the first structural and biochemical evidence of an alternative mechanism for isonitrile formation. ScoE, a putative non-heme iron(II)-dependent enzyme from Streptomyces coeruleorubidus, was shown to catalyze the conversion of (R)-3-((carboxymethyl)amino)butanoic acid to (R)-3-isocyanobutanoic acid through an oxidative decarboxylation mechanism. This work further provides a revised scheme for the biosynthesis of a unique class of isonitrile lipopeptides, of which several members are critical for the virulence of pathogenic mycobacteria.

Meroterpenoids of the ochraceopones family featuring a linear tetracyclic scaffold exhibit exceptional antiviral and anti‐inflammatory activities. The biosynthetic pathway and chemical logic to generate this linear tetracycle, however, remain unknown. In this study, we identified and characterized all biosynthetic enzymes to afford ochraceopones and elucidated the complete biosynthetic pathway. We demonstrated that the linear tetracyclic scaffold of ochraceopones was derived from an angular tetracyclic precursor. A multifunctional cytochrome P450 OchH was validated to catalyze the free‐radical‐initiated carbon‐carbon bond cleavage of the angular tetracycle. Then, a new carbon‐carbon bond was verified to be constructed using a new aldolase OchL, which catalyzes an intramolecular aldol reaction to form the linear tetracycle. This carbon‐carbon bond fragmentation and aldol reaction cascade features an unprecedented strategy for converting a common angular tetracycle to a distinctive linear tetracyclic scaffold in meroterpenoid biosynthesis. [ABSTRACT FROM AUTHOR]

Epoxyketone proteasome inhibitors have attracted much interest due to their potential as anticancer drugs. Although the biosynthetic gene clusters for several peptidyl epoxyketone natural products have recently been identified, the enzymatic logic involved in the formation of the terminal epoxyketone pharmacophore has been relatively unexplored. Here, we report the identification of the minimal set of enzymes from the eponemycin gene cluster necessary for the biosynthesis of novel metabolites containing a terminal epoxyketone pharmacophore in Escherichia coli, a versatile and fast-growing heterologous host. This set of enzymes includes a non-ribosomal peptide synthetase (NRPS), a polyketide synthase (PKS), and an acyl-CoA dehydrogenase (ACAD) homologue. In addition to the in vivo functional reconstitution of these enzymes in E. coli, in vitro studies of the eponemycin NRPS and (13) C-labeled precursor feeding experiments were performed to advance the mechanistic understanding of terminal epoxyketone formation.

Bioorthogonal chemistry has recently emerged to be one of the most powerful tools in drug discovery and chemical biology. The exploration of it has successfully advanced the field of natural product research. In this Perspective, we survey current strategies for the installation of chemical handles into the molecular scaffolds of several major classes of natural products, including polyketides (PKs), non-ribosomal peptides (NRPs), and their hybrids. By tagging these natural products with chemical handles and coupling them with subsequent bioorthogonal reactions, researchers have visualized and studied the mode of action of natural products, as well as synthesized derivatives with better pharmaceutical properties. We conclude this Perspective by considering two questions: is there a general way to synthesize tagged PKs/NRPs? Does natural product labeling have a broader impact in the field of natural product research beyond current known applications?

Aliphatic medium-chain 1-alkenes (MCAEs, ∼10 carbons) are "drop-in" compatible next-generation fuels and precursors to commodity chemicals. Mass production of MCAEs from renewable resources holds promise for mitigating dependence on fossil hydrocarbons. An MCAE, such as 1-undecene, is naturally produced by Pseudomonas as a semivolatile metabolite through an unknown biosynthetic pathway. We describe here the discovery of a single gene conserved in Pseudomonas responsible for 1-undecene biosynthesis. The encoded enzyme is able to convert medium-chain fatty acids (C10-C14) into their corresponding terminal olefins using an oxygen-activating, nonheme iron-dependent mechanism. Both biochemical and X-ray crystal structural analyses suggest an unusual mechanism of β-hydrogen abstraction during fatty acid substrate activation. Our discovery unveils previously unidentified chemistry in the nonheme Fe(II) enzyme family, provides an opportunity to explore the biology of 1-undecene in Pseudomonas, and paves the way for tailored bioconversion of renewable raw materials to MCAE-based biofuels and chemical commodities.

Siderophores belonging to the ferrichrome family are essential for the viability of fungal species and play a key role for virulence of numerous pathogenic fungi. Despite their biological significance, our understanding of how these iron-chelating cyclic hexapeptides are assembled by non-ribosomal peptide synthetase (NRPS) enzymes remains poorly understood, primarily due to the nonlinearity exhibited by the domain architecture. Herein, we report the biochemical characterization of the SidC NRPS, responsible for construction of the intracellular siderophore ferricrocin. In vitro reconstitution of purified SidC reveals its ability to produce ferricrocin and its structural variant, ferrichrome. Application of intact protein mass spectrometry uncovers several non-canonical events during peptidyl siderophore biosynthesis, including inter-modular loading of amino acid substrates and an adenylation domain capable of poly-amide bond formation. This work expands the scope of NRPS programming, allows biosynthetic assignment of ferrichrome NRPSs, and sets the stage for reprogramming towards novel hydroxamate scaffolds. Fungal siderophores are biosynthesised by nonribosomal peptide synthetases (NRPSs) with highly unusual domain architectures. Here, the authors characterise cryptic programming events occurring within SidC NRPS, responsible for ferricrocin biosynthesis in Aspergillus nidulans. [ABSTRACT FROM AUTHOR]

The alkyne functionality has attracted much interest due to its diverse chemical and biological applications. We recently elucidated an acyl carrier protein (ACP)‐dependent alkyne biosynthetic pathway, however, little is known about ACP interactions with the alkyne biosynthetic enzymes, an acyl‐ACP ligase (JamA) and a membrane‐bound bifunctional desaturase/acetylenase (JamB). Here, we showed that JamB has a more stringent interaction with ACP than JamA. In addition, site‐directed mutagenesis of a non‐cognate ACP significantly improved its compatibility with JamB, suggesting a possible electrostatic interaction at the ACP‐JamB interface. Finally, error‐prone PCR and screening of a second non‐cognate ACP identified hot spots on the ACP that are important for interacting with JamB and yielded mutants which were better recognized by JamB. Our data thus not only provide insights into the ACP interactions in alkyne biosynthesis, but it also potentially aids in future combinatorial biosynthesis of alkyne‐tagged metabolites for chemical and biological applications. © 2018 American Institute of Chemical Engineers AIChE J, 64: 4255–4262, 2018 [ABSTRACT FROM AUTHOR]

Polyketide synthases (PKSs) are one of the most profound biosynthetic factories for producing polyketides with diverse structures and biological activities. These enzymes have been historically studied and engineered to make un-natural polyketides for drug discovery, and have also recently been explored for synthesizing biofuels and industrial chemicals due to their versatility and customizability. Here, we review recent advances in the mechanistic understanding and engineering of modular PKSs for producing polyketide-derived chemicals, and provide perspectives on this relatively new application of PKSs. [ABSTRACT FROM AUTHOR]

Surging energy consumption and environmental concerns have stimulated interest in the production of chemicals and fuels through sustainable and renewable approaches. Fatty acid-based hydrocarbons, such as alkanes and alkenes, are of particular interest to directly replace fossil fuels. Towards this effort, understanding of hydrocarbon-producing enzymes is the first indispensable step to bio-production of hydrocarbons. Here, we review recent advances in the discovery and mechanistic study of enzymes capable of converting fatty acid precursors into hydrocarbons, and provide perspectives on the future of this rapidly growing field. [ABSTRACT FROM AUTHOR]

Covering: up to 2016 Antimycin-type depsipeptides are a family of natural products with great structural diversity and outstanding biological activities. These compounds have typically been isolated from actinomycetes and are generated from hybrid nonribosomal peptide synthetase (NRPS)-polyketide synthase (PKS) assembly lines. This review covers the literature on the four classes of antimycin-type depsipeptides, which differ by macrolactone ring size, and it discusses the discovery, biosynthesis, chemical synthesis, and biological activities of this family of compounds. [ABSTRACT FROM AUTHOR]

Directed evolution was used to improve the activity of JamB, a membrane-bound bifunctional desaturase/acetylenase. To quickly assess the protein engineering outcomes, we developed a new platform for quantifying extracellular alkyne-tagged metabolites through a fluorogenic click reaction. Random mutagenesis yielded the best JamB variant with ∼20-fold increased activity in E. coli. [ABSTRACT FROM AUTHOR]

Ikarugamycin is a member of the polycyclic tetramate macrolactams (PTMs) family of natural products with diverse biological activities. The biochemical mechanisms for the formation of polycyclic ring systems in PTMs remain elusive. The enzymatic mechanism of constructing an inner five-membered ring in ikarugamycin is reported. A three-gene-cassette ikaABC from the marine-derived Streptomyces sp. ZJ306 is sufficient for conferring ikarugamycin production in a heterologous host. IkaC catalyzes a reductive cyclization reaction to form the inner five-membered ring by a Michael addition-like reaction. This study provides the first biochemical evidence for polycycle formation in PTMs and suggests a reductive cyclization strategy which may be potentially applicable in general to the corresponding ring formation in other PTMs. [ABSTRACT FROM AUTHOR]

The piericidin A1 (1) gene cluster was identifiedfrom the deep-sea derived Streptomycessp. SCSIO03032. Our in vivo and in vitro experiments verified PieE as a 4′-hydroxylaseand PieB2 as a 4′-O-methyltransferase, allowingthe elucidation of the post-PKS modification steps involved in 1biosynthesis. In addition, the shunt metabolite piericidinE1 (7) was identified as a novel analogue featuring aC-2/C-3 epoxy ring. [ABSTRACT FROM AUTHOR]

Physostigmine is a parasympathomimetic drug used to treat a variety of neurological disorders, including Alzheimer's disease and glaucoma. Because of its potent biological activity and unique pyrroloindole skeleton, physostigmine has been the target of many organic syntheses. However, the biosynthesis of physostigmine has been relatively understudied. In this study, we identified a biosynthetic gene cluster for physostigmine by genome mining. The 8.5 kb gene cluster encodes eight proteins (PsmA-H), seven of which are required for the synthesis of physostigmine from 5-hydroxytryptophan, as shown by in vitro total reconstitution. Further genetic and enzymatic studies enabled us to delineate the biosynthetic pathway for physostigmine. The pathway features an unusual reaction cascade consisting of highly coordinated methylation and acetylation/deacetylation reactions. [ABSTRACT FROM AUTHOR]

Diazobenzofluorene-containing atypical angucyclines exhibit promising biological activities. Here we report the inactivation of an amidotransferase-encoding gene flsN3 in Micromonospora rosaria SCSIO N160, a producer of fluostatins. Bioinformatics analysis indicated that FlsN3 was involved in the diazo formation. Chemical investigation of the flsN3-inactivation mutant resulted in the isolation of a variety of angucycline aromatic polyketides, including four racemic aminobenzo[b]fluorenes stealthins D–G (9–12) harboring a stealthin C-like core skeleton with an acetone or butanone-like side chain. Their structures were elucidated on the basis of nuclear magnetic resonance (NMR) spectroscopic data and X-ray diffraction analysis. A plausible mechanism for the formation of stealthins D–G (9–12) was proposed. These results suggested a functional role of FlsN3 in the formation/modification of N–N bond-containing fluostatins. [ABSTRACT FROM AUTHOR]

Isonitrile lipopeptides discovered from Actinobacteria have attracted wide attention due to their fascinating biosynthetic pathways and relevance to the virulence of many human pathogens including Mycobacterium tuberculosis. Specifically, the identification of the new class of isonitrile-forming enzymes that belong to non-heme iron (II) and α-ketoglutarate dependent dioxygenases has intrigued several research groups to investigate their catalytic mechanism. Here we summarize the recent studies on the biosynthesis of isonitrile lipopeptides from Streptomyces and Mycobacterium. The latest research on the core and tailoring enzymes involved in the pathway as well as the isonitrile metabolic enzymes are discussed in this review. [ABSTRACT FROM AUTHOR]

Summary: Many natural products contain epoxyquinone pharmacophore with unknown biosynthetic mechanisms. Recent genetic analysis of the asukamycin biosynthetic gene cluster proposed enzyme candidates related to epoxyquinone formation for manumycin-type metabolites. Our biochemical studies reveal that 3-amino-4-hydroxyl benzoic acid (3,4-AHBA) precursor is activated and loaded on aryl carrier protein (AsuC12) by ATP-dependent adenylase (AsuA2). AsuE1 and AsuE3, both single-component flavin-dependent monooxygenases, catalyze the exquisite regio- and enantiospecific postpolyketide synthase (PKS) assembly oxygenations. AsuE1 installs a hydroxyl group on the 3,4-AHB ring to form a 4-hydroxyquinone moiety, which is epoxidized by AsuE3 to yield the epoxyquinone functionality. Despite being a single-component monooxygenase, AsuE1 activity is elicited by AsuE2, a pathway-specific flavin reductase. We further demonstrate that the epoxyquinone moiety is critical for anti-MRSA activity by analyzing the bioactivity of various manumycin-type metabolites produced through mutasynthesis. [Copyright &y& Elsevier]

The in vivoand in vitrocharacterization of AntB, a dedicated acyltransferase encoded in the antimycin biosynthetic gene cluster, which catalyzes the C-8 acyloxy formation is reported. It is demonstrated that AntB has broad substrate specificity toward both the acyl substrate and the acyl carrier and produces more antimycin analogues with varying C-8 acyloxy moieties. [ABSTRACT FROM AUTHOR]

The P450 enzyme XiaM was identified as a candidate to form the C-24 carboxyl group in xiamycin A (1). Alteration of medium composition led to the discovery of four new compounds from the ΔxiaMand the ΔxiaK(encoding an aromatic ring hydroxylase) mutants. Biotransformation experiments revealed that XiaM was capable of converting a methyl group to a carboxyl group through diol and aldehyde intermediates. [ABSTRACT FROM AUTHOR]

Summary: Phenazine-type metabolites arise from either phenazine-1-carboxylic acid (PCA) or phenazine-1,6-dicarboxylic acid (PDC). Although the biosynthesis of PCA has been studied extensively, PDC assembly remains unclear. Esmeraldins and saphenamycin, the PDC originated products, are antimicrobial and antitumor metabolites isolated from Streptomyces antibioticus Tü 2706. Herein, the esmeraldin biosynthetic gene cluster was identified on a dispensable giant plasmid. Twenty-four putative esm genes were characterized by bioinformatics, mutagenesis, genetic complementation, and functional protein expressions. Unlike enzymes involved in PCA biosynthesis, EsmA1 and EsmA2 together decisively promoted the PDC yield. The resulting PDC underwent a series of conversions to give 6-acetylphenazine-1-carboxylic acid, saphenic acid, and saphenamycin through a unique one-carbon extension by EsmB1–B5, a keto reduction by EsmC, and an esterification by EsmD1–D3, the atypical polyketide sythases, respectively. Two transcriptional regulators, EsmT1 and EsmT2, are required for esmeraldin production. [ABSTRACT FROM AUTHOR]