Insights into the Pamamycin Biosynthesis

Pamamycins are macrodiolides of polyketide origin with antibacterial activities. Their biosynthesis has been proposed to utilize succinate as a building block. However, the mechanism of succinate incorporation into a polyketide was unclear. Here, we report identification of a pamamycin biosynthesis gene cluster by aligning genomes of two pamamycin‐producing strains. This unique cluster contains polyketide synthase (PKS) genes encoding seven discrete ketosynthase (KS) enzymes and one acyl‐carrier protein (ACP)‐encoding gene. A cosmid containing the entire set of genes required for pamamycin biosynthesis was successfully expressed in a heterologous host. Genetic and biochemical studies allowed complete delineation of pamamycin biosynthesis. The pathway proceeds through 3‐oxoadipyl‐CoA, a key intermediate in the primary metabolism of the degradation of aromatic compounds. 3‐Oxoadipyl‐CoA could be used as an extender unit in polyketide assembly to facilitate the incorporation of succinate.     

Enzymatic Polyketide Chain Branching To Give Substituted Lactone,Lactam, and Glutarimide Heterocycles

Polyketides typically result from head‐to‐tail condensation of acyl thioesters to produce highly functionalized linear chains. The biosynthesis of the phytotoxin rhizoxin, however, involves a polyketide synthase (PKS) module that introduces a δ‐lactone chain branch through Michael addition of a malonyl extender to an α,β‐unsaturated intermediate unit. To evaluate the scope of the branching module, polyketide mimics were synthesized and their biotransformation by the reconstituted PKS module from the Rhizopus symbiont Burkholderia rhizoxinica was monitored in vitro. The impact of the type and configuration of the δ‐substituents was probed and it was found that amino‐substituted surrogates yield the corresponding lactams. A carboxamide analogue was transformed into a glutarimide unit, which can be found in many natural products. Our findings illuminate the biosynthesis of glutarimide‐bearing polyketides and also demonstrate the utility of this branching module for synthetic biology.     

Oxalyl‐CoA Decarboxylase Enables Nucleophilic One‐Carbon Extension of Aldehydes to Chiral α‐Hydroxy Acids

The synthesis of complex molecules from simple, renewable carbon units is the goal of a sustainable economy. Here we explored the biocatalytic potential of the thiamine‐diphosphate‐dependent (ThDP) oxalyl‐CoA decarboxylase (OXC)/2‐hydroxyacyl‐CoA lyase (HACL) superfamily that naturally catalyzes the shortening of acyl‐CoA thioester substrates through the release of the C₁‐unit formyl‐CoA. We show that the OXC/HACL superfamily contains promiscuous members that can be reversed to perform nucleophilic C₁‐extensions of various aldehydes to yield the corresponding 2‐hydroxyacyl‐CoA thioesters. We improved the catalytic properties of Methylorubrum extorquens OXC by rational enzyme engineering and combined it with two newly described enzymes—a specific oxalyl‐CoA synthetase and a 2‐hydroxyacyl‐CoA thioesterase. This enzymatic cascade enabled continuous conversion of oxalate and aromatic aldehydes into valuable (S)‐α‐hydroxy acids with enantiomeric excess up to 99 %.     

The stereochemistry of complex polyketide biosynthesis by modular polyketide synthases

Polyketides are a diverse class of medically important natural products whose biosynthesis is catalysed by polyketide synthases (PKSs), in a fashion highly analogous to fatty acid biosynthesis. In modular PKSs, the polyketide chain is assembled by the successive condensation of activated carboxylic acid-derived units, where chain extension occurs with the intermediates remaining covalently bound to the enzyme, with the growing polyketide tethered to an acyl carrier domain (ACP). Carboxylated acyl-CoA precursors serve as activated donors that are selected by the acyltransferase domain (AT) providing extender units that are added to the growing chain by condensation catalysed by the ketosynthase domain (KS). The action of ketoreductase (KR), dehydratase (DH), and enoylreductase (ER) activities can result in unreduced, partially reduced, or fully reduced centres within the polyketide chain depending on which of these enzymes are present and active. The PKS-catalysed assembly process generates stereochemical diversity, because carbon-carbon double bonds may have either cis- or trans- geometry, and because of the chirality of centres bearing hydroxyl groups (where they are retained) and branching methyl groups (the latter arising from use of propionate extender units). This review shall cover the studies that have determined the stereochemistry in many of the reactions involved in polyketide biosynthesis by modular PKSs.     

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.     

Tetrathiafulvalene–Oligo(para‐phenyleneethynylene) Conjugates: Formation of Multiple Mixed‐Valence Complexes upon Electrochemical Oxidation

Short monodisperse oligo‐ (para‐phenyleneethynylene) (pOPE) units bearing laterally attached tetrathio‐substituted tetrathiofulvalene (TTF) units have been synthesised from functionalised aromatic building blocks by using the Sonogashira cross‐coupling methodology. The unusual redox properties of these TTF–pOPE conjugates were observed by employing electrochemical methods, such as cyclic voltammetry and exhaustive electrolysis. We found that formally one half of the TTF units in the pOPE monomer 1 , dimer 2 , and trimer 3 (with 2, 4, and 6 TTF units, respectively) are electrochemically silent during the first‐step oxidation at 0.49 V. We propose the formation of persistent mixed‐valence complexes from the TTF and TTF^+. units present in an equal ratio. Such mixed‐valence dyads (single or multiple in the partially oxidised 1 – 3 ) exhibit an unusual stability towards oxidation until the potential of the second oxidation at 0.84 V is achieved. This finding suggests that below this potential the oxidation of the respective mix‐valence complexes is extremely slow.     

MCAT is not required for in vitro polyketide synthesis in a minimal actinorhodin polyketide synthase from Streptomyces coelicolor

Background: It has been proposed that Streptomyces malonyl CoA:holo acyl carrier protein transacylases (MCATs) provide a link between fatty acid and polyketide biosynthesis. Two recent studies have provided evidence that the presence of MCAT is essential for polyketide synthesis to proceed in reconstituted minimal polyketide synthases (PKSs). In contrast to this, we previously showed that the holo acyl carrier proteins (ACPs) from type II PKSs are capable of catalytic self -malonylation in the presence of malonyl CoA, which suggests that MCAT might not be necessary for polyketide biosynthesis.Results: We reconstituted a homologous actinorhodin (act) type II minimal PKS in vitro, When act holo-ACP is present in limiting concentrations, MCAT is required by the synthase complex in order for polyketide biosynthesis to proceed. When holo-ACP is present in excess, however, efficient polyketide synthesis proceeds without MCAT, The rate of polyketide production increases with holo-ACP concentration, but at low ACP concentration or equimolar ACP:KS:CLF (KS, ketosynthase; CLF, chain length determining factor) concentrations this rate is significantly lower than expected, indicating that free holo-ACP is sequestered by the KS/CLF complex.Conclusions: The rate of polyketide biosynthesis is dictated by the ratio of holo-ACP to KS and CLF, as well as by the total protein concentration, There is no absolute requirement for MCAT in polyketide biosynthesis in vitro, although the role of MCAT during polyketide synthesis in vivo remains an open question. MCAT might be responsible for the rate enhancement of malonyl transfer at very low free holo-ACP concentrations or it could be required to catalyse the transfer of malonyl groups from malonyl CoA to sequestered holo-ACP.     

Precursor‐Directed Biosynthesis: Stereospecificity for Branched‐Chain Diketides of the β‐Ketoacyl‐ACP Synthase Domain 2 of 6‐Deoxyerythronolide B Synthase

Modular polyketide synthases such as 6‐deoxyerythronolide B synthase (DEBS) catalyze the biosynthesis of structurally complex natural products. Streptomyces coelicolor CH999/pJRJ2 harbors a plasmid encoding DEBS(KS1⁰), a mutant form of 6‐deoxyerythronolide B synthase that is blocked in the formation of 6‐deoxyerythronolide B ( 1 , 6‐dEB) due to a mutation in the active site of the ketosynthase (KS1) domain that normally catalyzes the first polyketide chain‐elongation step of 6‐dEB biosynthesis. Administration of (2S,3R,4S)‐ and (2S,3R,4R)‐3‐hydroxy‐2,4‐dimethylhexanoic acid N‐acetylcysteamine (SNAC) thioesters (= S‐[2‐(acetylamino)ethyl] (2S,3R,4S)‐ and (2S,3R,4R)‐3‐hydroxy‐2,4‐dimethylhexanethioates) 3 and 4 in separate experiments to cultures of Streptomyces coelicolor CH999/pJRJ2 led to production of the corresponding (14S)‐ and (14R)‐14‐methyl analogues of 6‐dEB, 10 and 11 , respectively. Unexpectedly, when a 3 : 2 mixture of 4 and 3 was fed under the same conditions, exclusively branched‐chain macrolactone 11 was isolated. In similar experiments, feeding of 3 and 4 to S. coelicolor CH999/pCK16, an engineered strain harboring DEBS1+TE(KS1⁰), resulted in formation of the branched‐chain triketide lactones 13 and 14 , while feeding of the 3 : 2 mixture of 4 and 3 gave exclusively 14 . The biochemical basis for this stereochemical discrimination was established by using purified DEBS module 2+TE to determine the steady‐state kinetic parameters for 3 and 4 , with the k_cat/K_M for 4 shown to be sevenfold greater than that of 3 .     

Enzymatic Formation of a Skipped Methyl‐Substituted Octaprenyl Side Chain of Longestin (KS‐505a): Involvement of Homo‐IPP as a Common Extender Unit

Longestin (KS‐505a), a specific inhibitor of phosphodiesterase, is a meroterpenoid that consists of a unique octacyclic terpene skeleton with branched methyl groups at unusual positions (C1 and C12). Biochemical analysis of Lon23, a methyltransferase involved in the biosynthesis of longestin, demonstrated that it methylates homoisopentenyl diphosphate (homo‐IPP) to afford (3Z)‐3‐methyl IPP. This compound, along with IPP, is selectively accepted as extender units by Lon22, a geranylgeranyl diphosphate (GGPP) synthase homologue, to yield dimethylated GGPP (dmGGPP). The absolute configuration of dmGGPP was determined to be (4R,12R) by degradation and chiral GC analysis. These findings allowed us to propose an enzymatic sequence for key steps of the biosynthetic pathway of the unusual homoterpenoid longestin.     

Enzymatic extender unit generation for in vitro polyketide synthase reactions: structural and functional showcasing of Streptomyces coelicolor MatB

In vitro experiments with modular polyketide synthases (PKSs) are often limited by the availability of polyketide extender units. To determine the polyketide extender units that can be biocatalytically accessed via promiscuous malonyl-CoA ligases, structural and functional studies were conducted on Streptomyces coelicolor MatB. We demonstrate that this adenylate-forming enzyme is capable of producing most CoA-linked polyketide extender units as well as pantetheine- and N-acetylcysteamine-linked analogs useful for in?vitro PKS studies. Two ternary product complex structures, one containing malonyl-CoA and AMP and the other containing (2R)-methylmalonyl-CoA and AMP, were solved to 1.45?? and 1.43?? resolution, respectively. MatB crystallized in the thioester-forming conformation, making extensive interactions with the bound extender unit products. This first structural characterization of an adenylate-forming enzyme that activates diacids reveals the molecular details for how malonate and its derivatives are accepted. The orientation of the α-methyl group of bound (2R)-methylmalonyl-CoA, indicates that it is necessary to epimerize α-substituted extender units formed by MatB before they can be accepted by PKS acyltransferase domains. We demonstrate the in?vitro incorporation of methylmalonyl groups ligated by MatB to CoA, pantetheine, or N-acetylcysteamine into a triketide pyrone by the terminal module of the 6-deoxyerythronolide B synthase. Additionally, a means for quantitatively monitoring certain in?vitro PKS reactions using MatB is presented.     

Syntheses,Structure, Electrochemistry,and Optical Properties of 1,3‐Diethyl‐2,3‐dihydro‐1‐H‐1,3,2‐pyrido‐[4,5‐b]‐diazaboroles

Reaction of 2,5‐bis(dibromoboryl)thiophene ( 4 ) or 1,4‐bis(dibromoboryl)benzene ( 6 ) with two equivalents of N,N′‐dilithiated 2,3‐diaminopyridine ( 3 ) led to the generation of the pyridodiazaboroles 5 and 7 in which the two diazaborole rings are linked by 2,5‐thiophen‐diyl or 1,4‐phenylene units via the boron atom. The novel compounds were characterized by elemental analyses and spectroscopy (¹H‐, ¹¹B‐, ¹³C‐NMR, MS, and UV‐VIS). The molecular structure of 5 was elucidated by X‐ray diffraction. Cyclovoltammograms of 5 and 7 show two irreversible oxidation waves at 0.76 and 0.73 V, respectively vs Fc/Fc⁺. The novel compounds display intense blue luminescence with Stokes shifts of 76 and 74 nm and relative quantum yields of 39 and 43 % vs Coumarin 120 (Φ = 50 %).     

Synthesis and properties of conjugated polymers containing 3,9‐ and 2,9‐linked carbazole units in the main chain

Novel conjugated polymers containing 3,9‐ or 2,9‐linked carbazole units in the main chain were synthesized by the polycondensation of ethynyl‐ and iodo‐substituted 9‐arylenecarbazolylene monomers, and their optical and electrical properties were studied. Polymers with weight‐average molecular weights of 3400–12,000 were obtained in 76–99% yields by the Sonogashira coupling polycondensation in piperidine or tetrahydrofuran (THF)/piperidine at 30 °C for 48 h. All the 3,9‐linked polymers absorbed light around 300 nm. The para‐phenylene‐linked polymer also absorbed light around 350 nm, while meta‐phenylene‐linked one did not. The 3,9‐linked polymers absorbed light at a wavelength longer than the 2,9‐linked one. The polymers emitted blue fluorescence with high quantum yields (0.21–0.78) upon excitation at the absorption maxima. The polymers were oxidized around 0.6 V, and reduced around 0.5 V. Poly( 1 ) showed the dark conductivity of 3.7 × 10^?11 S/cm (10³ V/cm). © 2009 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 47: 3506–3517, 2009     

N‐Bearing Furanone Derivatives from an Endophytic Fungus in Huperzia serrata

Two new polyketide derivatives, huaspenones C and D ( 1 and 2 , resp.), were isolated from the cultures of an endophytic fungus Peyronellaea sp. HS‐12, derived from the stems of Huperzia serrata. They share N‐bearing furan‐3(2H)‐one backbone, and 2 has an unprecedented furo[3,2‐c]pyridine skeleton. Their structures including the absolute configuration were elucidated by extensive spectroscopic analysis combined with quantum‐chemical calculations. (2E,4E)‐6‐hydroxy‐2‐methylocta‐2,4‐dienoic acid ( 3 ), a key intermediate of the biosynthesis of 1 and 2 , was also obtained from the endophyte.     

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.     

Synthesis,Bioactivities and Structure Activity Relationship of N‐4‐Methyl‐1,2,3‐thiadiazole‐5‐carbonyl‐N′‐phenyl Ureas

Twenty nine novel N‐4‐methyl‐1,2,3‐thiadiazole‐5‐carbonyl‐N′‐phenyl ureas were designed and synthesized, and their structures were confirmed by proton nuclear magnetic resonance (¹H NMR), infra red spectroscopy (IR) and high‐resolution mass spectroscopy (HRMS). Compounds V‐9 , V‐11 , V‐12 , V‐15 , V‐19 , V‐21 , V‐22 and V‐24 exhibit excellent activity against Culex pipiens pallens. Compounds V‐12 and V‐22 present good insecticidal activity against Plutella xylostella L. Their median lethal concentrations (LC₅₀) are 164.15 and 89.69 mg·L^?1, respectively. Compound V‐11 also has potential wide spectrum of fungicide activity. Its median effective concentrations (EC₅₀) detected from 3.82 µg·mL^?1 against Physalospora piricola to 31.60 µg·mL^?1 against Cercospora arachidicola. Compounds V‐15 and V‐24 show outstanding induction activities as same as positive controls TDL and ningnanmycin, furthermore V‐24 has the highest induction activity of 41.85%±4.43%. To elucidate the structure activity relationship in these compounds, a 3D‐QSAR model has been built. The established model showed a reliable predicting ability with q² values of 0.643 and r² values of 0.982.     

Structural Basis of a Broadly Selective Acyltransferase from the Polyketide Synthase of Splenocin

Polyketides are a large family of pharmaceutically important natural products, and the structural modification of their scaffolds is significant for drug development. Herein, we report high‐resolution X‐ray crystal structures of the broadly selective acyltransferase (AT) from the splenocin polyketide synthase (SpnD‐AT) in the apo form and in complex with benzylmalonyl and pentynylmalonyl extender unit mimics. These structures revealed the molecular basis for the stereoselectivity and substrate specificity of SpnD‐AT, and enabled the engineering of the industrially important Ery‐AT6 to broaden its substrate scope to include three new types of extender units.     

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.     

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.     

Divergent Mechanistic Routes for the Formation of gem‐Dimethyl Groups in the Biosynthesis of Complex Polyketides

The gem‐dimethyl groups in polyketide‐derived natural products add steric bulk and, accordingly, lend increased stability to medicinal compounds, however, our ability to rationally incorporate this functional group in modified natural products is limited. In order to characterize the mechanism of gem‐dimethyl group formation, with a goal toward engineering of novel compounds containing this moiety, the gem‐dimethyl group producing polyketide synthase (PKS) modules of yersiniabactin and epothilone were characterized using mass spectrometry. The work demonstrated, contrary to the canonical understanding of reaction order in PKSs, that methylation can precede condensation in gem‐dimethyl group producing PKS modules. Experiments showed that both PKSs are able to use dimethylmalonyl acyl carrier protein (ACP) as an extender unit. Interestingly, for epothilone module 8, use of dimethylmalonyl‐ACP appeared to be the sole route to form a gem‐dimethylated product, while the yersiniabactin PKS could methylate before or after ketosynthase condensation.     

Genome Mining of Oxidation Modules in trans‐Acyltransferase Polyketide Synthases Reveals a Culturable Source for Lobatamides

Bacterial trans‐acyltransferase polyketide synthases (trans‐AT PKSs) are multimodular megaenzymes that biosynthesize many bioactive natural products. They contain a remarkable range of domains and module types that introduce different substituents into growing polyketide chains. As one such modification, we recently reported Baeyer–Villiger‐type oxygen insertion into nascent polyketide backbones, thereby generating malonyl thioester intermediates. In this work, genome mining focusing on architecturally diverse oxidation modules in trans‐AT PKSs led us to the culturable plant symbiont Gynuella sunshinyii, which harbors two distinct modules in one orphan PKS. The PKS product was revealed to be lobatamide A, a potent cytotoxin previously only known from a marine tunicate. Biochemical studies show that one module generates glycolyl thioester intermediates, while the other is proposed to be involved in oxime formation. The data suggest varied roles of oxygenation modules in the biosynthesis of polyketide scaffolds and support the importance of trans‐AT PKSs in the specialized metabolism of symbiotic bacteria.