An Integrated Module Performs Selective 'On-Line' Epoxidation in the Biosynthesis of the Antibiotic Mupirocin.

The delineation of the complex biosynthesis of the potent antibiotic mupirocin, which consists of a mixture of pseudomonic acids (PAs) isolated from Pseudomonas fluorescens NCIMB 10586, presents significant challenges and the timing and mechanisms of several key transformations remain elusive.   Particularly intriguing are the steps that process the linear backbone from the initial polyketide assembly phase to generate the first cyclic intermediate PA-B. These include epoxidation as well as incorporation of the tetrahydropyran (THP) ring and fatty acid sidechain required for biological activity. Here, we show that the mini-module MmpE performs a rare online (ACP-substrate) epoxidation and is integrated ('in-cis') into the polyketide synthase via a docking domain. A linear polyketide fragment with 6 asymmetric centres was synthesised using a convergent approach and used to demonstrate substrate flux via an atypical KS0 and a previously unannotated ACP (MmpE_ACP). MmpE_ACP-bound synthetic substrates were critical in demonstrating successful epoxidation in vitro by the purified MmpE oxidoreductase domain. Alongside feeding studies, these results confirm the timing as well as chain length dependence of this selective epoxidation. These mechanistic studies pinpoint the location and nature of the polyketide substrate prior to the key formation of the THP ring and esterification that generate PA-B.

Antibiotic origami: selective formation of spirotetronates in abyssomicin biosynthesis.

The abyssomicins are a structurally intriguing family of bioactive natural products that include compounds with potent antibacterial, antitumour and antiviral activities. The biosynthesis of the characteristic abyssomicin spirotetronate core occurs via an enzyme-catalysed intramolecular Diels-Alder reaction, which proceeds via one of two distinct stereochemical pathways to generate products differing in configuration at the C15 spirocentre. Using the purified spirotetronate cyclases AbyU (from abyssomicin C/atrop-abyssomicin C biosynthesis) and AbmU (from abyssomicin 2/neoabyssomicin biosynthesis), in combination with synthetic substrate analogues, here we show that stereoselectivity in the spirotetronate-forming [4 + 2]-cycloaddition is controlled by a combination of factors attributable to both the enzyme and substrate. Furthermore, an achiral substrate was enzymatically cyclised to a single enantiomer of a spirocyclic product. X-ray crystal structures, molecular dynamics simulations, and assessment of substrate binding affinity and reactivity in both AbyU and AbmU establish the molecular determinants of stereochemical control in this important class of biocatalysts.

Delineation of the complete reaction cycle of a natural Diels-Alderase.

The Diels-Alder reaction is one of the most effective methods for the synthesis of substituted cyclohexenes. The development of protein catalysts for this reaction remains a major priority, affording new sustainable routes to high value target molecules. Whilst a small number of natural enzymes have been shown capable of catalysing [4 + 2] cycloadditions, there is a need for significant mechanistic understanding of how these prospective Diels-Alderases promote catalysis to underpin their development as biocatalysts for use in synthesis. Here we present a molecular description of the complete reaction cycle of the bona fide natural Diels-Alderase AbyU, which catalyses formation of the spirotetronate skeleton of the antibiotic abyssomicin C. This description is derived from X-ray crystallographic studies of AbyU in complex with a non-transformable synthetic substrate analogue, together with transient kinetic analyses of the AbyU catalysed reaction and computational reaction simulations. These studies reveal the mechanistic intricacies of this enzyme system and establish a foundation for the informed reengineering of AbyU and related biocatalysts.

Combining total synthesis and genetic engineering to probe dihydropyran formation in ambruticin biosynthesis.

The ambruticins are a family of potent antifungal polyketide derived natural products isolated from the myxobacterium Sorangium cellulosum. Their unusual structures include a trisubstituted cyclopropyl group and two oxygen heterocycles, a tetrahydropyran (THP) and dihydropyran (DHP). Herein we report a flexible modular approach for the total synthesis of ambruticins which is used to prepare ambruticins F and S as well as in the first total synthesis of 20,21-dihydroambruticin F. The flexible strategy unites 3 fragments via Julia-Kocienski olefinations and provides important standards for investigation of dihydropyran formation in ambruticin biosynthesis. Cultures of wild-type S. cellulosum So ce10 produce mainly ambruticin S and the VS series of metabolites. An efficient electroporation method enabled gene knockout experiments which revealed that the ΔambP-S mutant of S. cellulosum accumulated the bisTHP polyketide 20,21-dihydroambruticin F. In contrast, the ΔambN-S mutant gave ambruticin F with the 20,21-alkene as the major metabolite confirming that AmbP and AmbO (a Rieske enzyme and flavin-dependent monooxygenase respectively) are implicated in 20,21-alkene formation. The results of feeding studies to a Sorangium strain containing only ambP and ambO are in accord with formation of the 20,21-alkene occurring prior to generation of the C3 to C7 dihydroxylated tetrahydropyran in ambruticin biosynthesis.

Structure and Function of the α-Hydroxylation Bimodule of the Mupirocin Polyketide Synthase.

Mupirocin is a clinically important antibiotic produced by a trans-AT Type I polyketide synthase (PKS) in Pseudomonas fluorescens. The major bioactive metabolite, pseudomonic acid A (PA-A), is assembled on a tetrasubstituted tetrahydropyran (THP) core incorporating a 6-hydroxy group proposed to be introduced by α-hydroxylation of the thioester of the acyl carrier protein (ACP) bound polyketide chain. Herein, we describe an in vitro approach combining purified enzyme components, chemical synthesis, isotopic labelling, mass spectrometry and NMR in conjunction with in vivo studies leading to the first characterisation of the α-hydroxylation bimodule of the mupirocin biosynthetic pathway. These studies reveal the precise timing of hydroxylation by MupA, substrate specificity and the ACP dependency of the enzyme components that comprise this α-hydroxylation bimodule. Furthermore, using purified enzyme, it is shown that the MmpA KS0 shows relaxed substrate specificity, suggesting precise spatiotemporal control of in trans MupA recruitment in the context of the PKS. Finally, the detection of multiple intermodular MupA/ACP interactions suggests these bimodules may integrate MupA into their assembly.

Interrogation of an Enzyme Library Reveals the Catalytic Plasticity of Naturally Evolved [4+2] Cyclases.

Stereoselective carbon-carbon bond forming reactions are quintessential transformations in organic synthesis. One example is the Diels-Alder reaction, a [4+2] cycloaddition between a conjugated diene and a dienophile to form cyclohexenes. The development of biocatalysts for this reaction is paramount for unlocking sustainable routes to a plethora of important molecules. To obtain a comprehensive understanding of naturally evolved [4+2] cyclases, and to identify hitherto uncharacterised biocatalysts for this reaction, we constructed a library comprising forty-five enzymes with reported or predicted [4+2] cycloaddition activity. Thirty-one library members were successfully produced in recombinant form. In vitro assays employing a synthetic substrate incorporating a diene and a dienophile revealed broad-ranging cycloaddition activity amongst these polypeptides. The hypothetical protein Cyc15 was found to catalyse an intramolecular cycloaddition to generate a novel spirotetronate. The crystal structure of this enzyme, along with docking studies, establishes the basis for stereoselectivity in Cyc15, as compared to other spirotetronate cyclases.

Biosynthesis of pleuromutilin congeners using an Aspergillus oryzae expression platform.

Pleuromutilin is an antibiotic diterpenoid made by Clitopilus passeckerianus and related fungi, and it is the progenitor of a growing class of semi-synthetic antibiotics used in veterinary and human medicine. To harness the biotechnological potential of this natural product class, a full understanding of its biosynthetic pathway is essential. Previously, a linear pathway for pleuromutilin biosynthesis was established. Here we report two shunt pathways involving Pl-sdr and Pl-atf that were identified through the rational heterologous expression of combinations of pleuromutilin biosynthetic genes in Aspergillus oryzae. Three novel pleuromutilin congeners were isolated, and their antimicrobial activity was investigated, alongside that of an additional derivative produced through a semi-synthetic approach. It was observed that the absence of various functional groups - 3 ketone, 11 hydroxyl group or 21 ketone - from the pleuromutilin framework affected the antibacterial activity of pleuromutilin congeners. This study expands our knowledge on the biosynthesis of pleuromutilin and provides avenues for the development of novel pleuromutilin analogues by combining synthetic biology and synthetic chemistry.

Correction: Back et al. A New Micromonospora Strain with Antibiotic Activity Isolated from the Microbiome of a Mid-Atlantic Deep-Sea Sponge. Mar. Drugs 2021, 19, 105.

After publication of this article [...].

Maleidride biosynthesis - construction of dimeric anhydrides - more than just heads or tails.

Covering: up to early 2022Maleidrides are a family of polyketide-based dimeric natural products isolated from fungi. Many maleidrides possess significant bioactivities, making them attractive pharmaceutical or agrochemical lead compounds. Their unusual biosynthetic pathways have fascinated scientists for decades, with recent advances in our bioinformatic and enzymatic understanding providing further insights into their construction. However, many intriguing questions remain, including exactly how the enzymatic dimerisation, which creates the diverse core structure of the maleidrides, is controlled. This review will explore the literature from the initial isolation of maleidride compounds in the 1930s, through the first full structural elucidation in the 1960s, to the most recent in vivo, in vitro, and in silico analyses.

The Role of Cytochrome†P450 AbyV in the Final Stages of Abyssomicin†C Biosynthesis.

Abyssomicin C and its atropisomer are potent inhibitors of bacterial folate metabolism. They possess complex polycyclic structures, and their biosynthesis has been shown to involve several unusual enzymatic transformations. Using a combination of synthesis and in vitro assays we reveal that AbyV, a cytochrome P450 enzyme from the aby gene cluster, catalyses a key late-stage epoxidation required for the installation of the characteristic ether-bridged core of abyssomicin C. The X-ray crystal structure of AbyV has been determined, which in combination with molecular dynamics simulations provides a structural framework for our functional data. This work demonstrates the power of combining selective carbon-13 labelling with NMR spectroscopy as a sensitive tool to interrogate enzyme-catalysed reactions in vitro with no need for purification.

The Role of Cytochrome†P450 AbyV in the Final Stages of Abyssomicin†C Biosynthesis.

Abyssomicin C and its atropisomer are potent inhibitors of bacterial folate metabolism. They possess complex polycyclic structures, and their biosynthesis has been shown to involve several unusual enzymatic transformations. Using a combination of synthesis and in vitro assays we reveal that AbyV, a cytochrome P450 enzyme from the aby gene cluster, catalyses a key late-stage epoxidation required for the installation of the characteristic ether-bridged core of abyssomicin C. The X-ray crystal structure of AbyV has been determined, which in combination with molecular dynamics simulations provides a structural framework for our functional data. This work demonstrates the power of combining selective carbon-13 labelling with NMR spectroscopy as a sensitive tool to interrogate enzyme-catalysed reactions in vitro with no need for purification.

Structure and Function of the α-Hydroxylation Bimodule of the Mupirocin Polyketide Synthase.

Mupirocin is a clinically important antibiotic produced by a trans-AT Type I polyketide synthase (PKS) in Pseudomonas fluorescens. The major bioactive metabolite, pseudomonic acid A (PA-A), is assembled on a tetrasubstituted tetrahydropyran (THP) core incorporating a 6-hydroxy group proposed to be introduced by α-hydroxylation of the thioester of the acyl carrier protein (ACP) bound polyketide chain. Herein, we describe an in vitro approach combining purified enzyme components, chemical synthesis, isotopic labelling, mass spectrometry and NMR in conjunction with in vivo studies leading to the first characterisation of the α-hydroxylation bimodule of the mupirocin biosynthetic pathway. These studies reveal the precise timing of hydroxylation by MupA, substrate specificity and the ACP dependency of the enzyme components that comprise this α-hydroxylation bimodule. Furthermore, using purified enzyme, it is shown that the MmpA KS0 shows relaxed substrate specificity, suggesting precise spatiotemporal control of in trans MupA recruitment in the context of the PKS. Finally, the detection of multiple intermodular MupA/ACP interactions suggests these bimodules may integrate MupA into their assembly.

Programmed Iteration Controls the Assembly of the Nonanoic Acid Side Chain of the Antibiotic Mupirocin.

Mupirocin is a clinically important antibiotic produced by Pseudomonas fluorescens NCIMB 10586 that is assembled by a complex trans-AT polyketide synthase. The polyketide fragment, monic acid, is esterified by a 9-hydroxynonanoic acid (9HN) side chain which is essential for biological activity. The ester side chain assembly is initialised from a 3-hydroxypropionate (3HP) starter unit attached to the acyl carrier protein (ACP) MacpD, but the fate of this species is unknown. Herein we report the application of NMR spectroscopy, mass spectrometry, chemical probes and in vitro assays to establish the remaining steps of 9HN biosynthesis. These investigations reveal a complex interplay between a novel iterative or "stuttering" KS-AT didomain (MmpF), the multidomain module MmpB and multiple ACPs. This work has important implications for understanding the late-stage biosynthetic steps of mupirocin and will be important for future engineering of related trans-AT biosynthetic pathways (e.g. thiomarinol).

Cladobotric Acids: Metabolites from Cultures of Cladobotryum sp., Semisynthetic Analogues and Antibacterial Activity.

Three new polyketide-derived natural products, cladobotric acids G-I (1-3), and six known metabolites (4, 5, 8-11) were isolated from fermentation of the fungus Cladobotryum sp. grown on rice. Their structures were elucidated by extensive spectroscopic methods. Two metabolites, cladobotric acid A (4) and pyrenulic acid A (10), were converted to a series of new products (12-20) by semisynthesis. The antibacterial activities of all these compounds were investigated against the Gram-positive pathogen Staphylococcus aureus including methicillin-susceptible (MSSA), methicillin-resistant and vancomycin-intermediate (MRSA/VISA), and heterogeneous vancomycin-intermediate (hVISA) strains. Results of these antibacterial assays revealed structural features of the unsaturated decalins important for biological activity.

In silico analyses of maleidride biosynthetic gene clusters.

Maleidrides are a family of structurally related fungal natural products, many of which possess diverse, potent bioactivities. Previous identification of several maleidride biosynthetic gene clusters, and subsequent experimental work, has determined the 'core' set of genes required to construct the characteristic medium-sized alicyclic ring with maleic anhydride moieties. Through genome mining, this work has used these core genes to discover ten entirely novel putative maleidride biosynthetic gene clusters, amongst both publicly available genomes, and encoded within the genome of the previously un-sequenced epiheveadride producer Wicklowia aquatica CBS 125634. We have undertaken phylogenetic analyses and comparative bioinformatics on all known and putative maleidride biosynthetic gene clusters to gain further insights regarding these unique biosynthetic pathways.

Synthetic and biosynthetic methods for selective cyclisations of 4,5-epoxy alcohols to tetrahydropyrans.

Tetrahydropyrans (THPs) are common structural motifs found in natural products and synthetic therapeutic molecules. In Nature these 6-membered oxygen heterocycles are often assembled via intramolecular reactions involving either oxy-Michael additions or ring opening of epoxy-alcohols. Indeed, the polyether natural products have been particularly widely studied due to their fascinating structures and important biological properties; these are commonly formed via endo-selective epoxide-opening cascades. In this review we outline synthetic approaches for endo-selective intramolecular epoxide ring opening (IERO) of 4,5-epoxy-alcohols and their applications in natural product synthesis. In addition, the biosynthesis of THP-containing natural products which utilise IERO reactions are reviewed.

Programmed Iteration Controls the Assembly of the Nonanoic Acid Side Chain of the Antibiotic Mupirocin.

Mupirocin is a clinically important antibiotic produced by Pseudomonas fluorescens NCIMB 10586 that is assembled by a complex trans-AT polyketide synthase. The polyketide fragment, monic acid, is esterified by a 9-hydroxynonanoic acid (9HN) side chain which is essential for biological activity. The ester side chain assembly is initialised from a 3-hydroxypropionate (3HP) starter unit attached to the acyl carrier protein (ACP) MacpD, but the fate of this species is unknown. Herein we report the application of NMR spectroscopy, mass spectrometry, chemical probes and in vitro assays to establish the remaining steps of 9HN biosynthesis. These investigations reveal a complex interplay between a novel iterative or "stuttering" KS-AT didomain (MmpF), the multidomain module MmpB and multiple ACPs. This work has important implications for understanding the late-stage biosynthetic steps of mupirocin and will be important for future engineering of related trans-AT biosynthetic pathways (e.g. thiomarinol).

Synthesis and pharmacological characterisation of arctigenin analogues as antagonists of AMPA and kainate receptors.

(-)-Arctigenin and a series of new analogues have been synthesised and then tested for their potential as AMPA and kainate receptor antagonists of human homomeric GluA1 and GluK2 receptors expressed in HEK293 cells using a Ca2+ influx assay. In general, these compounds showed antagonist activity at both receptors with greater activity evident at AMPARs. Schild analysis indicates that a spirocyclic analogue 6c acts as a non-competitive antagonist. Molecular docking studies in which 6c was docked into the X-ray crystal structure of the GluA2 tetramer suggest that (-)-arctigenin and its analogues bind in the transmembrane domain in a similar manner to the known AMPA receptor non-competitive antagonists GYKI53655 and the antiepileptic drug perampanel. The arctigenin derivatives described herein may serve as novel leads for the development of drugs for the treatment of epilepsy.

Ambruticins: tetrahydropyran ring formation and total synthesis.

The ambruticins are a family of polyketide natural products which exhibit potent antifungal activity. Gene knockout experiments are in accord with the proposal that the tetrahydropyran ring of the ambruticins is formed via the AmbJ catalysed epoxidation of the unsaturated 3,5-dihydroxy acid, ambruticin J, followed by regioselective cyclisation to ambruticin F. Herein, a convergent approach to the total synthesis of ambruticin J is described as well as model studies involving epoxidation and cyclisations of unsaturated hydroxy esters to give tetrahydropyrans and tetrahydrofurans. The total synthesis involves preparation of three key fragments which were united via a Suzuki-Miyaura cross-coupling and Julia-Kocienski olefination to generate the required carbon framework. Global deprotection to a triol and selective oxidation of the primary alcohol gave, after hydrolysis of the lactone, ambruticin J.

Core Steps to the Azaphilone Family of Fungal Natural Products.

Azaphilones are a family of polyketide-based fungal natural products that exhibit interesting and useful bioactivities. This minireview explores the literature on various characterised azaphilone biosynthetic pathways, which allows for a proposed consensus scheme for the production of the core azaphilone structure, as well as identifying early diversification steps during azaphilone biosynthesis. A consensus understanding of the core enzymatic steps towards a particular family of fungal natural products can aid in genome-mining experiments. Genome mining for novel fungal natural products is a powerful technique for both exploring chemical space and providing new insights into fungal natural product pathways.