Biosynthesis of Biscognienyne B Involving a Cytochrome P450-Dependent Alkynylation

The alkyne is a biologically significant moiety found in many natural products and a versatile functional group widely used in modern chemistry. Recent studies have revealed the biosynthesis of acetylenic bonds in fatty acids and amino acids. However, the molecular basis for the alkynyl moiety in acetylenic prenyl chains occurring in a number of meroterpenoids remains obscure. Here, we identify the biosynthetic gene cluster and characterize the biosynthetic pathway of an acetylenic meroterpenoid biscognienyne B based on heterologous expression, feeding experiments, and in vitro assay. This work shows that the alkyne moiety is constructed by an unprecedented cytochrome P450 enzyme BisI, which shows promiscuous activity towards C5 and C15 prenyl chains. This finding provides an opportunity for discovery of new compounds, featuring acetylenic prenyl chains, through genome mining, and it also expands the enzyme inventory for de novo biosynthesis of alkynes.     

Discovery of a Dual Function Cytochrome P450 that Catalyzes Enyne Formation in Cyclohexanoid Terpenoid Biosynthesis

The 1,3‐enyne moiety is commonly found in cyclohexanoid natural products produced by endophytic and plant pathogenic fungi. Asperpentyn ( 1 ) is a 1,3‐enyne‐containing cyclohexanoid terpenoid isolated from Aspergillus and Pestalotiopsis. The genetic basis and biochemical mechanism of 1,3‐enyne biosynthesis in 1 , and other natural products containing this motif, has remained enigmatic despite their potential ecological roles. Identified here is the biosynthetic gene cluster and characterization of two crucial enzymes in the biosynthesis of 1 . A P450 monooxygenase that has a dual function, to first catalyze dehydrogenation of the prenyl chain to generate a cis‐diene intermediate and then serve as an acetylenase to yield an alkyne moiety, and thus the 1,3‐enyne, was discovered. A UbiA prenyltransferase was also characterized and it is unusual in that it favors transferring a five‐carbon prenyl chain, rather than a polyprenyl chain, to a p‐hydroxybenzoic acid acceptor.     

Discovery of a Dual Function Cytochrome P450 that Catalyzes Enyne Formation in Cyclohexanoid Terpenoid Biosynthesis

The 1,3-enyne moiety is commonly found in cyclohexanoid natural products produced by endophytic and plant pathogenic fungi. Asperpentyn ( 1 ) is a 1,3-enyne-containing cyclohexanoid terpenoid isolated from Aspergillus and Pestalotiopsis. The genetic basis and biochemical mechanism of 1,3-enyne biosynthesis in 1 , and other natural products containing this motif, has remained enigmatic despite their potential ecological roles. Identified here is the biosynthetic gene cluster and characterization of two crucial enzymes in the biosynthesis of 1 . A P450 monooxygenase that has a dual function, to first catalyze dehydrogenation of the prenyl chain to generate a cis-diene intermediate and then serve as an acetylenase to yield an alkyne moiety, and thus the 1,3-enyne, was discovered. A UbiA prenyltransferase was also characterized and it is unusual in that it favors transferring a five-carbon prenyl chain, rather than a polyprenyl chain, to a p-hydroxybenzoic acid acceptor.     

Influence of acetylene-containing peripheral chains on the mesomorphic properties of triphenylene-based liquid crystals

Three series of novel acetylene-containing triphenylene-based liquid crystals have been prepared. One class of material is symmetrical with the acetylenic unit located at the terminus of a peripheral alkynyloxy chain in a peripheral position at the terminus of an alkoxy chain. These symmetrical hexa-substituted materials possess high melting points which renders mesomorphism monotropic. A second class comprises materials where only one of the six peripheral units contains a terminal alkyne chain. The asymmetry confers relatively low melting points and an enantiotropic hexagonal columnar mesophase is exhibited for all materials, albeit to a lower temperature than the known parent systems. The third series of materials also comprises unsymmetrical structures, here containing one acetylenic unit conjugated with the triphenylene core and five alkoxy chains. This third series of materials show enhanced π–π interactions and enhanced space-filling effects of the acetylenic unit close to the core which enhance mesophase stability markedly over the hexaalkoxy analogues. This result shows that rigidity of peripheral chains, as opposed to molecular symmetry, is the significant factor for the reduction of mesophase stabilities.     

A Monooxygenase from Boreostereum vibrans Catalyzes Oxidative Decarboxylation in a Divergent Vibralactone Biosynthesis Pathway

The oxidative decarboxylation of prenyl 4‐hydroxybenzoate to prenylhydroquinone has been frequently proposed for the biosynthesis of prenylated (hydro)quinone derivates (sometimes meroterpenoids), yet no corresponding genes or enzymes have so far been reported. A FAD‐binding monooxygenase (VibMO1) was identified that converts prenyl 4‐hydroxybenzoate into prenylhydroquinone and is likely involved in the biosynthesis of vibralactones and other meroterpenoids in the basidiomycete Boreostereum vibrans. Feeding of 3‐allyl‐4‐hydroxybenzylalcohol, an analogue of the vibralactone pathway intermediate 3‐prenyl‐4‐hydroxybenzylalcohol, generated 20 analogues with different scaffolds. This demonstrated divergent pathways to skeletally distinct compounds initiating from a single precursor, thus providing the first insight into a novel biosynthetic pathway for 3‐substituted γ‐butyrolactones from a shikimate origin.     

Mechanistic Understanding of the Divergent Cyclizations of o‐Alkynylbenzaldehyde Acetals and Thioacetals Catalyzed by Metal Halides

The mechanisms of regiodivergent cyclizations of o‐alkynylbenzaldehyde acetals and thioacetals catalyzed by Pd and Pt halides are studied. DFT calculations found that both reactions are initiated by electrophilic activation of the acetylenic moiety instead of the previously proposed metal‐triggered C?X (X=O, S) cleavage. Both the regioselective cyclization of the π‐alkyne complex and the chemoselective [1,2]‐migration in the carbenoid intermediate were determined as key steps to achieving the observed divergence. For acetal derivatives containing an internal alkyne, the 6‐endo‐dig cyclization is more favorable and leads to the carbenoid intermediate easily through further steps of C?X fragmentation and carbocation cyclization. Then, from the carbenoid intermediate, the [1,2]‐migration of sulfur is easier than that of H, Me, and Ph; whereas, a reversed aptitude was predicted for the oxygen analogue, which is consistent with the greater ability of sulfur atoms to stabilize β‐carbocations. However, for precursors containing a terminal alkyne, the 5‐exo‐dig pathway is preferred and only the 1,2‐disubstituted indene product is seen, irrespective of the nature of the acetal; thus, a different product from that reported in the literature is predicted for benzaldehyde acetal with a terminal alkyne at the ortho position. This prediction led us to reconsider some of the reported results and hidden realities were uncovered with solid new experimental evidence.     

Perquinolines A–C: Unprecedented Bacterial Tetrahydroisoquinolines Involving an Intriguing Biosynthesis

Metabolic profiling of Streptomyces sp. IB2014/016‐6 led to the identification of three new tetrahydroisoquinoline natural products, perquinolines A–C ( 1 – 3 ). Labelled precursor feeding studies and the cloning of the pqr biosynthetic gene cluster revealed that 1 – 3 are assembled by the action of several unusual enzymes. The biosynthesis starts with the condensation of succinyl‐CoA and l ‐phenylalanine catalyzed by the amino‐7‐oxononanoate synthase‐like enzyme PqrA, representing rare chemistry in natural product assembly. The second condensation and cyclization events are conducted by PqrG, an enzyme resembling an acyl‐CoA ligase. Last, ATP‐grasp RimK‐type ligase PqrI completes the biosynthesis by transferring a γ‐aminobutyric acid or β‐alanine moiety. The discovered pathway represents a new route for assembling the tetrahydroisoquinoline cores of natural products.     

Enantioselective intramolecular [2 + 2 + 2] cycloaddition of enediynes for the synthesis of chiral cyclohexa-1,3-dienes

The enantioselective intramolecular [2 + 2 + 2] cycloaddition of various enediynes, where two acetylenic moieties are connected by a trans-olefinic moiety, gave chiral tricyclic cyclohexa-1,3-dienes using Rh-H8-BINAP catalyst. In the case of carbon-atom-tethered enediynes, enantioselectivity was generally good-to-high regardless of the substituents on their alkyne termini. In contrast, with heteroatom-tethered enediynes, appropriate substituents were required to induce the oxidative coupling of alkyne and alkene moieties before that of two alkyne moieties, which would be important for highly enantioselective intramolecular cycloaddition.     

Oxygenated Metabolites of n‐3 Polyunsaturated Fatty Acids as Potential Oxidative Stress Biomarkers: Total Synthesis of 8‐F3t‐IsoP, 10‐F4t‐NeuroP and [D4]‐10‐F4t‐NeuroP

A wide variety of metabolic products of polyunsaturated fatty acids is of paramount importance for improving our medical knowledge in the field of oxidized lipids. Two novel metabolites of n‐3 polyunsaturated fatty acids, 8‐F_3t‐IsoP and 10‐F_4t‐NeuroP as well as a deuterated derivative thereof were synthesized based on an acetylenic intermediate. An original approach achieved lateral chain insertion of 8‐F_3t‐IsoP by a ring‐closing alkyne metathesis/semi‐reduction strategy together with a temporary tether.     

Biosynthesis of the isoprenoid moieties of furanonaphthoquinone I and endophenazine A in Streptomyces cinnamonensis DSM 1042

Streptomyces cinnamonensis DSM 1042 produces the polyketide-isoprenoid compound furanonaphthoquinone I (FNQ I) and isoprenylated phenazines, predominantly endophenazine A. However, the recently identified biosynthetic gene cluster for these compounds only contains a single gene for a mevalonate pathway enzyme, that is, a putative mevalonate kinase gene. This is in strong contrast to all Streptomyces strains examined so far, where all six genes encoding the mevalonate pathway enzymes are clustered in a single operon of 6.8 kb and, thus, raised the question about the biosynthetic origin of the isoprenoid moieties of FNQ I and endophenazine A. In this study, we investigated the incorporation of [13C2]acetate and [2-13C]glycerol into FNQ I and endophenazine A. The results unequivocally prove that the isoprenoid building blocks of both compounds are predominantly formed via the mevalonate pathway (approximately 80%) but that the MEP pathway (approximately 20%) contributes to the biosynthesis of these molecules, too. In actinomycetes, this is the first experimentally proven example of the utilization of both biosynthetic routes for the formation of one single secondary metabolite. The incorporation pattern of [2-13C]glycerol was consistent with a "reverse" prenyl transfer, that is, with the formation of a C-C bond from C-3 of GPP to the polyketide nucleus of FNQ I.     

The antibiotic CJ-15,801 is an antimetabolite that hijacks and then inhibits CoA biosynthesis

The natural product CJ-15,801 is an inhibitor of Staphylococcus aureus, but not other bacteria. Its close structural resemblance to pantothenic acid, the vitamin precursor of coenzyme A (CoA), and its Michael acceptor moiety suggest that it irreversibly inhibits an enzyme involved in CoA biosynthesis or utilization. However, its mode of action and the basis for its specificity have not been elucidated to date. We demonstrate that CJ-15,801 is transformed by the uniquely selective S. aureus pantothenate kinase, the first CoA biosynthetic enzyme, into a substrate for the next enzyme, phosphopantothenoylcysteine synthetase, which is inhibited through formation of a tight-binding structural mimic of its native reaction intermediate. These findings reveal CJ-15,801 as a vitamin biosynthetic pathway antimetabolite with a mechanism similar to that of the sulfonamide antibiotics and highlight CoA biosynthesis as a viable antimicrobial drug target.     

Two Distinct Substrate Binding Modes for the Normal and Reverse Prenylation of Hapalindoles by the Prenyltransferase AmbP3

The cyanobacterial prenyltransferase AmbP3 catalyzes the reverse prenylation of the tetracyclic indole alkaloid hapalindole U at its C‐2 position. Interestingly, AmbP3 also accepts hapalindole A, a halogenated C‐10 epimer of hapalindole U, and catalyzes normal prenylation at its C‐2 position. The comparison of the two ternary crystal structures, AmbP3‐DMSPP/hapalindole U and AmbP3‐DMSPP/hapalindole A, at 1.65–2.00 Å resolution revealed two distinct orientations for the substrate binding that define reverse or normal prenylation. The tolerance of the enzyme for these altered orientations is attributed to the hydrophobicity of the substrate binding pocket and the plasticity of the amino acids surrounding the allyl group of the prenyl donor. This is the first study to provide the intimate structural basis for the normal and reverse prenylations catalyzed by a single enzyme, and it offers novel insight into the engineered biosynthesis of prenylated natural products.     

Genome-based deletion analysis reveals the prenyl xanthone biosynthesis pathway in Aspergillus nidulans

Xanthones are a class of molecules that bind to a number of drug targets and possess a myriad of biological properties. An understanding of xanthone biosynthesis at the genetic level should facilitate engineering of second-generation molecules and increasing production of first-generation compounds. The filamentous fungus Aspergillus nidulans has been found to produce two prenylated xanthones, shamixanthone and emericellin, and we report the discovery of two more, variecoxanthone A and epishamixanthone. Using targeted deletions that we created, we determined that a cluster of 10 genes including a polyketide synthase gene, mdpG, is required for prenyl xanthone biosynthesis. mdpG was shown to be required for the synthesis of the anthraquinone emodin, monodictyphenone, and related compounds, and our data indicate that emodin and monodictyphenone are precursors of prenyl xanthones. Isolation of intermediate compounds from the deletion strains provided valuable clues as to the biosynthetic pathway, but no genes accounting for the prenylations were located within the cluster. To find the genes responsible for prenylation, we identified and deleted seven putative prenyltransferases in the A. nidulans genome. We found that two prenyltransferase genes, distant from the cluster, were necessary for prenyl xanthone synthesis. These genes belong to the fungal indole prenyltransferase family that had previously been shown to be responsible for the prenylation of amino acid derivatives. In addition, another prenyl xanthone biosynthesis gene is proximal to one of the prenyltransferase genes. Our data, in aggregate, allow us to propose a complete biosynthetic pathway for the A. nidulans xanthones.     

Promysalin, a salicylate-containing Pseudomonas putida antibiotic, promotes surface colonization and selectively targets other Pseudomonas

Under control of the Gac regulatory system, Pseudomonas putida RW10S1 produces promysalin to promote its own swarming and biofilm formation, and to selectively inhibit many other pseudomonads, including the opportunistic pathogen Pseudomonas aeruginosa. This amphipathic antibiotic is composed of salicylic acid and 2,8-dihydroxymyristamide bridged by a unique 2-pyrroline-5-carboxyl moiety. In addition to enzymes for salicylic acid synthesis and activation, the biosynthetic gene cluster encodes divergent type II fatty acid biosynthesis components, unusual fatty acid-tailoring enzymes (two Rieske-type oxygenases and an amidotransferase), an enzyme resembling a proline-loading module of nonribosomal peptide synthetases, and the first prokaryotic member of the BAHD family of plant acyltransferases. Identification of biosynthetic intermediates enabled to propose a pathway for synthesis of this bacterial colonization factor.     

One‐Pot Enzymatic Synthesis of Merochlorin A and B

The polycycles merochlorin A and B are complex halogenated meroterpenoid natural products with significant antibacterial activities and are produced by the marine bacterium Streptomyces sp. strain CNH‐189. Heterologously produced enzymes and chemical synthesis are employed herein to fully reconstitute the merochlorin biosynthesis in vitro. The interplay of a dedicated type III polyketide synthase, a prenyl diphosphate synthase, and an aromatic prenyltransferase allow formation of a highly unusual aromatic polyketide‐terpene hybrid intermediate which features an unprecedented branched sesquiterpene moiety from isosesquilavandulyl diphosphate. As supported by in vivo experiments, this precursor is furthermore chlorinated and cyclized to merochlorin A and isomeric merochlorin B by a single vanadium‐dependent haloperoxidase, thus completing the remarkably efficient pathway.     

Two distinct mechanisms for TIM barrel prenyltransferases in bacteria

The reactions of two bacterial TIM barrel prenyltransferases (PTs), MoeO5 and PcrB, were explored. MoeO5, the enzyme responsible for the first step in moenomycin biosynthesis, catalyzes the transfer of farnesyl to 3-phosphoglyceric acid (3PG) to give a product containing a cis-allylic double bond. We show that this reaction involves isomerization to a nerolidyl pyrophosphate intermediate followed by bond rotation prior to attack by the nucleophile. This mechanism is unprecedented for a prenyltransferase that catalyzes an intermolecular coupling. We also show that PcrB transfers geranyl and geranylgeranyl groups to glycerol-1-phosphate (G1P), making it the first known bacterial enzyme to use G1P as a substrate. Unlike MoeO5, PcrB catalyzes prenyl transfer without isomerization to give products that retain the trans-allylic bond of the prenyl donors. The TIM barrel family of PTs is unique in including enzymes that catalyze prenyl transfer by distinctly different reaction mechanisms.     

Control of β‐Branching in Kalimantacin Biosynthesis: Application of 13C NMR to Polyketide Programming

The presence of β‐branches in the structure of polyketides that possess potent biological activity underpins the widespread importance of this structural feature. Kalimantacin is a polyketide antibiotic with selective activity against staphylococci, and its biosynthesis involves the unprecedented incorporation of three different and sequential β‐branching modifications. We use purified single and multi‐domain enzyme components of the kalimantacin biosynthetic machinery to address in vitro how the pattern of β‐branching in kalimantacin is controlled. Robust discrimination of enzyme products required the development of a generalisable assay that takes advantage of ¹³C NMR of a single ¹³C label incorporated into key biosynthetic mimics combined with favourable dynamic properties of an acyl carrier protein. We report a previously unassigned modular enoyl‐CoA hydratase (mECH) domain and the assembly of enzyme constructs and cascades that are able to generate each specific β‐branch.     

Synthesis of Phenanthrene Derivatives by Intramolecular Cyclization Utilizing the [1,2]‐Phospha‐Brook Rearrangement Catalyzed by a Brønsted Base

The synthesis of functionalized phenanthrene derivatives was achieved by intramolecular cyclization utilizing the [1,2]‐phospha‐Brook rearrangement under Brønsted base catalysis. Treatment of biaryl compounds having an α‐ketoester moiety and an alkyne moiety at the 2 and 2′ positions, respectively, with diisopropyl phosphite in the presence of a catalytic amount of phosphazene base P2‐tBu provides 9,10‐disubstituted phenanthrene derivatives in high yields. This reaction involves the generation of an ester enolate through an umpolung process, that is, addition of diisopropyl phosphite to a keto moiety followed by the [1,2]‐phospha‐Brook rearrangement, the intramolecular addition to an alkyne, and the [3,3] rearrangement of the allylic phosphate moiety in a consecutive fashion.     

Enzymatic C−H Oxidation–Amidation Cascade in the Production of Natural and Unnatural Thiotetronate Antibiotics with Potentiated Bioactivity

The selective activation of unreactive hydrocarbons by biosynthetic enzymes has inspired new synthetic methods in C−H bond activation. Herein, we report the unprecedented two‐step biosynthetic conversion of thiotetromycin to thiotetroamide C involving the tandem oxidation and amidation of an unreactive ethyl group. We detail the genetic and biochemical basis for the terminal amidation in thiotetroamide C biosynthesis, which involves a uniquely adapted cytochrome P450–amidotransferase enzyme pair and highlights the first oxidation–amidation enzymatic cascade reaction leading to the selective formation of a primary amide group from a chemically inert alkyl group. Motivated by the ten‐fold increase in antibiotic potency of thiotetroamide C ascribed to the acetamide group and the unusual enzymology involved, we enzymatically interrogated diverse thiolactomycin analogues and prepared an unnatural thiotetroamide C analogue with potentiated bioactivity compared to the parent molecule.     

Biosynthesis of a novel cyclic C35-terpene via the cyclisation of a Z-type C35-polyprenyl diphosphate obtained from a nonpathogenic Mycobacterium species

Lipid components from 12 nonpathogenic Mycobacterium species were analysed. A novel cyclic C(35)-terpene, named heptaprenylcycline , was obtained from 3 species, while octahydroheptaprenol , which has 3 Z-double bonds, was obtained from 6 species. The amounts of and in the cultured cells increased after the 4- to 6-d stationary phase. The yield of was considerably greater at a higher temperature of 37 degrees C than at an optimal temperature of 28 degrees C, while that of remained unchanged at all temperatures. A feeding experiment with d-[1-(13)C]glucose revealed that was produced via isopentenyl diphosphate, which is a metabolite of glycolysis and the methylerythritol phosphate pathway. The conversion of octahydroheptaprenyl diphosphate to was successful by using the cell-free extracts of M. chlorophenolicum, demonstrating that is the biosynthetic intermediate of . This is the first example of the biosynthesis of a natural terpene via the cyclisation of a linear C(35)-isoprenoid. The substrate for C(35)-terpene cyclase has Z-type prenyl moieties; however, terpene cyclases usually employ E-type isoprenoids. The gene encoding the terpene cyclase that cyclises prenyl diphosphate containing Z-double bonds as the natural substrate has not yet been detected. Despite a careful search using the FASTA3 program, we could not detect any gene that is homologous to the known diphosphate-triggered type of mono-, sesqui- and diterpene cyclases in the genome of M. vanbaalenii, the DNA sequence of which has recently been elucidated. This suggests that a novel type of terpene cyclase might exist in the nonpathogenic Mycobacterium species.