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The colinearity of canonical modular polyketide synthases, which creates a direct link between multienzyme structure and the chemical structure of the biosynthetic end-product, has become a cornerstone of knowledge-based genome mining. Herein, we report genetic and enzymatic evidence for the remarkable role of an enoylreductase in the polyketide synthase for azalomycin F biosynthesis. This internal enoylreductase domain, previously identified as acting only in the second of two chain extension cycles on an initial iterative module, is shown to also catalyze enoylreduction in trans within the next module. The mechanism for this rare deviation from colinearity appears to involve direct cross-modular interaction of the reductase with the longer acyl chain, rather than back transfer of the substrate into the iterative module, suggesting an additional and surprising plasticity in natural PKS assembly-line catalysis., (© 2020 Wiley-VCH GmbH.)

The type I polyketide SF2487/A80577 (herein referred to as tetromadurin) is a polyether tetronate ionophore antibiotic produced by the terrestrial Gram-positive bacterium Actinomadura verrucosospora. Tetromadurin is closely related to the polyether tetronates tetronasin (M139603) and tetronomycin, all of which are characterised by containing a tetronate, cyclohexane, tetrahydropyran, and at least one tetrahydrofuran ring. We have sequenced the genome of Actinomadura verrucosospora to identify the biosynthetic gene cluster responsible for tetromadurin biosynthesis (the mad gene cluster). Based on bioinformatic analysis of the 32 genes present within the cluster a plausible biosynthetic pathway for tetromadurin biosynthesis is proposed. Functional confirmation of the mad gene cluster is obtained by performing in-frame deletions in each of the genes mad10 and mad31, which encode putative cyclase enzymes responsible for cyclohexane and tetrahydropyran formation, respectively. Furthermore, the A. verrucosospora Δmad10 mutant produces a novel tetromadurin metabolite that according to mass spectrometry analysis is analogous to the recently characterised partially cyclised tetronasin intermediate lacking its cyclohexane and tetrahydropyran rings. Our results therefore elucidate the biosynthetic machinery of tetromadurin biosynthesis and lend support for a conserved mechanism of cyclohexane and tetrahydropyran biosynthesis across polyether tetronates., Competing Interests: The authors have declared that no competing interests exist.

Ossamycin from Streptomyces hygroscopicus var. ossamyceticus is an antifungal and cytotoxic polyketide and a potent inhibitor of the mitochondrial ATPase. Analysis of a near-complete genome sequence of the ossamycin producer has allowed the identification of the 127-kbp ossamycin biosynthetic gene cluster. The presence in the cluster of a specific crotonyl-CoA carboxylase/reductase homologue suggests that the 5-methylhexanoate extension unit used in construction of the macrocyclic core is incorporated intact from the unusual precursor isobutyrylmalonyl-CoA. Surprisingly, the modular polyketide synthase uses only 14 extension modules to accomplish 15 cycles of polyketide chain extension, a rare example of programmed iteration on a modular polyketide synthase. Specific deletion of genes encoding cytochrome P450 enzymes has given insight into the late-stage tailoring of the ossamycin macrocycle required for the attachment of the unusual 2,3,4,6-deoxyaminohexose sugar l-ossamine to C-8 of the ossamycin macrocycle. The ossamycin cluster also encodes a putative spirocyclase enzyme, OssO, which may play a role in establishing the characteristic spiroketal moiety of the natural product., Competing Interests: The authors have declared that no competing interests exist.

Detailed analysis of the modular Type I polyketide synthase (PKS) involved in the biosynthesis of the marginolactone azalomycin F in mangrove Streptomyces sp. 211726 has shown that only nineteen extension modules are required to accomplish twenty cycles of polyketide chain elongation. Analysis of the products of a PKS mutant specifically inactivated in the dehydratase domain of extension-module 1 showed that this module catalyzes two successive elongations with different outcomes. Strikingly, the enoylreductase domain of this module can apparently be "toggled" off and on : it functions in only the second of these two cycles. This novel mechanism expands our understanding of PKS assembly-line catalysis and may explain examples of apparent non-colinearity in other modular PKS systems., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Desertomycin A is an aminopolyol polyketide containing a macrolactone ring. We have proposed that desertomycin A and similar compounds (marginolactones) are formed by polyketide synthases primed not with γ-aminobutanoyl-CoA but with 4-guanidinylbutanoyl-CoA, to avoid facile cyclization of the starter unit. This hypothesis requires that there be a final-stage de-amidination of the corresponding guanidino-substituted natural product, but no enzyme for such a process has been described. We have now identified candidate amidinohydrolase genes within the desertomycin and primycin clusters. Deletion of the putative desertomycin amidinohydrolase gene dstH in Streptomyces macronensis led to the accumulation of desertomycin B, the guanidino form of the antibiotic. Also, purified DstH efficiently catalyzed the in vitro conversion of desertomycin B into the A form. Hence this amidinohydrolase furnishes the missing link in this proposed naturally evolved example of protective-group chemistry., (© 2015 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

Elaiophylin is an unusual C2 -symmetric antibiotic macrodiolide produced on a bacterial modular polyketide synthase assembly line. To probe the mechanism and selectivity of diolide formation, we sought to reconstitute ring formation in vitro by using a non-natural substrate. Incubation of recombinant elaiophylin thioesterase/cyclase with a synthetic pentaketide analogue of the presumed monomeric polyketide precursor of elaiophylin, specifically its N-acetylcysteamine thioester, produced a novel 16-membered C2 -symmetric macrodiolide. A linear dimeric thioester is an intermediate in ring formation, which indicates iterative use of the thioesterase active site in ligation and subsequent cyclization. Furthermore, the elaiophylin thioesterase acts on a mixture of pentaketide and tetraketide thioesters to give both the symmetric decaketide diolide and the novel asymmetric hybrid nonaketide diolide. Such thioesterases have potential as tools for the in vitro construction of novel diolides., (© 2015 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.)

Mycolactones are polyketide-derived lipid virulence factors made by the slow-growing human pathogen, Mycobacterium ulcerans. Three unusually large and homologous plasmid-borne genes (mlsA1: 51 kb, mlsB: 42 kb and mlsA2: 7 kb) encode the mycolactone type I polyketide synthases (PKS). The extreme size and low sequence diversity of these genes has posed significant barriers for exploration of the genetic and biochemical basis of mycolactone synthesis. Here, we have developed a truncated, more tractable 3-module version of the 18-module mycolactone PKS and we show that this engineered PKS functions as expected in the natural host M. ulcerans to produce an additional polyketide; a triketide lactone (TKL). Cell fractionation experiments indicated that this 3-module PKS and the putative accessory enzymes encoded by mup045 and mup038 associated with the mycobacterial cell wall, a finding supported by confocal microscopy. We then assessed the capacity of the faster growing, Mycobacterium marinum to harbor and express the 3-module Mls PKS and accessory enzymes encoded by mup045 and mup038. RT-PCR, immunoblotting, and cell fractionation experiments confirmed that the truncated Mls PKS multienzymes were expressed and also partitioned with the cell wall material in M. marinum. However, this heterologous host failed to produce TKL. The systematic deconstruction of the mycolactone PKS presented here suggests that the Mls multienzymes are necessary but not sufficient for mycolactone synthesis and that synthesis is likely to occur (at least in part) within the mycobacterial cell wall. This research is also the first proof-of-principle demonstration of the potential of this enzyme complex to produce tailored small molecules through genetically engineered rearrangements of the Mls modules.

Mycolactone is a diffusible lipid secreted by the human pathogen Mycobacterium ulcerans, which induces the formation of open skin lesions referred to as Buruli ulcers. Here, we show that mycolactone operates by hijacking the Wiskott-Aldrich syndrome protein (WASP) family of actin-nucleating factors. By disrupting WASP autoinhibition, mycolactone leads to uncontrolled activation of ARP2/3-mediated assembly of actin in the cytoplasm. In epithelial cells, mycolactone-induced stimulation of ARP2/3 concentrated in the perinuclear region, resulting in defective cell adhesion and directional migration. In vivo injection of mycolactone into mouse ears consistently altered the junctional organization and stratification of keratinocytes, leading to epidermal thinning, followed by rupture. This degradation process was efficiently suppressed by coadministration of the N-WASP inhibitor wiskostatin. These results elucidate the molecular basis of mycolactone activity and provide a mechanism for Buruli ulcer pathogenesis. Our findings should allow for the rationale design of competitive inhibitors of mycolactone binding to N-WASP, with anti-Buruli ulcer therapeutic potential.

Modular biocatalysis is responsible for the generation of countless bioactive products and its mining remains a major focus for drug discovery purposes. One of the enduring hurdles is the isolation of biosynthetic intermediates in a readily-analysed form. We prepared a series of nonhydrolysable pantetheine and N-acetyl cysteamine mimics of the natural (methyl)malonyl extender units recruited for polyketide formation. Using these analogues as competitive substrates, we were able to trap and off-load diketide and triketide species directly from an in vitro reconstituted type I polyketide synthase, the 6-deoxyerythronolide B synthase 3 (DEBS3). The putative intermediates, which were extracted in organic solvent and characterised by LC-HR-ESI-MS, are the first of their kind and prove that small-molecule chain terminators can be used as convenient probes of the biosynthetic process.

The glycosylation of natural product scaffolds with highly modified deoxysugars is often essential for their biological activity, being responsible for specific contacts to molecular targets and significantly affecting their pharmacokinetic properties. In order to provide tools for the targeted alteration of natural product glycosylation patterns, significant strides have been made to understand the biosynthesis of activated deoxysugars and their transfer. We report here efforts towards the production of plasmid-borne biosynthetic gene cassettes capable of producing TDP-activated forms of D-mycaminose, D-angolosamine and D-desosamine. We additionally describe the transfer of these deoxysugars to macrolide aglycones using the glycosyl transferases EryCIII, TylMII and AngMII, which display usefully broad substrate tolerance.

The biosynthetic pathway to the unusual tetronate ring of certain polyketide natural products, including the antibiotics abyssomicin and tetronomycin (TMN) and the antitumour compound chlorothricin (CHL), is presently unknown. The gene clusters governing chlorothricin and tetronomycin biosynthesis both contain a gene encoding an atypical member of the FkbH family of enzymes, which has previously been shown to synthesise glyceryl-S-acyl carrier protein (ACP) as the first step in production of unusual extender units for modular polyketide biosynthesis. We show here that purified recombinant FkbH-like protein, Tmn16, from the TMN gene cluster catalyses the efficient transfer of a glyceryl moiety from D-1,3-bisphosphoglycerate (1,3-BPG) to either of the dedicated ACPs, Tmn7a and ChlD2, to form glyceryl-S-ACP, which directly implicates this compound as an intermediate in tetronate biosynthesis as well. Neither Tmn16 nor Tmn7a produced glyceryl-S-ACP when incubated, respectively, with analogous ACP and FkbH-like proteins from a known extender-unit pathway; this indicates a highly selective channelling of glycolytic metabolites into tetronate biosynthesis.

Meridamycin is a non-immunosuppressant, FKBP-binding macrocyclic polyketide, which has major potential as a neuroprotectant in a range of neurodegenerative disorders including dementia, Parkinson's disease and ischaemic stroke. A biosynthetic cluster predicted to encode biosynthesis of meridamycin was cloned from the prolific secondary-metabolite-producing strain Streptomyces sp. DSM 4137, not previously known to produce this compound, and specific gene deletion was used to confirm the role of this cluster in the biosynthesis of meridamycin. The meridamycin modular polyketide synthase consists of 14 extension modules distributed between three giant multienzyme proteins. The terminal module is flanked by a highly unusual cytochrome P450-like domain. The characterization of the meridamycin biosynthetic locus in this readily manipulated streptomycete species opens the way to the engineering of new, altered meridamycins of potential therapeutic importance.

The spinosyns are a family of potent and highly selective insect control agents that display a favorable environmental profile. As some regions of the spinosyn molecule are recalcitrant to chemical modification, a targeted genetic approach was carried out to generate new analogues. The polyketide synthase (PKS) loading modules from the avermectin PKS of Streptomyces avermitilis and the erythromcyin PKS of Saccharopolyspora erythraea were each used to replace the spinosyn PKS loading module. Both of the resulting strains containing hybrid PKS pathways produced the anticipated spinosyn analogues. Supplementation of the culture media with a range of exogenous carboxylic acids led to the successful incorporation of these novel elements to yield further novel spinosyn molecules, some of which demonstrated potent and new insecticidal activities. Furthermore, it has been demonstrated that semisynthesis of such novel metabolites can then be used to generate active analogues, demonstrating the effectiveness of utilizing these complementary methods to search the chemical space around this template.

Polyether ionophores, such as monensin A, are known to be biosynthesised, like many other antibiotic polyketides, on giant modular polyketide synthases (PKSs), but the intermediates and enzymes involved in the subsequent steps of oxidative cyclisation remain undefined. In particular there has been no agreement on the mechanism and timing of the final polyketide chain release. We now report evidence that MonCII from the monensin biosynthetic gene cluster in Streptomyces cinnamonensis, which was previously thought to be an epoxide hydrolase, is a novel thioesterase that belongs to the alpha/beta-hydrolase structural family and might catalyse this step. Purified recombinant MonCII was found to hydrolyse several thioester substrates, including an N-acetylcysteamine thioester derivative of monensin A. Further, incubation with a hallmark inhibitor of such enzymes, phenylmethanesulfonyl fluoride, led to inhibition of the thioesterase activity and to the accumulation of an acylated form of MonCII. These findings require a reassessment of the role of other enzymes implicated in the late stages of polyether ionophore biosynthesis.

The bacterial multienzyme polyketide synthases (PKSs) produce a diverse array of products that have been developed into medicines, including antibiotics and anticancer agents. The modular genetic architecture of these PKSs suggests that it might be possible to engineer the enzymes to produce novel drug candidates, a strategy known as 'combinatorial biosynthesis'. So far, directed engineering of modular PKSs has resulted in the production of more than 200 new polyketides, but key challenges remain before the potential of combinatorial biosynthesis can be fully realized.

The macrolide antibiotic concanamycin A has been identified as an exceptionally potent inhibitor of the vacuolar (V-type) ATPase. Such compounds have been mooted as the basis of a potential drug treatment for osteoporosis, since the V-ATPase is involved in the osteoclast-mediated bone resorption that underlies this common condition. To enable combinatorial engineering of altered concanamycins, the biosynthetic gene cluster governing the biosynthesis of concanamycin A has been cloned from Streptomyces neyagawaensis and shown to span a region of over 100 kbp of contiguous DNA. An efficient transformation system has been developed for S. neyagawaensis and used to demonstrate the role of the cloned locus in the formation of concanamycin A. Sequence analysis of the 28 ORFs in the region has revealed key features of the biosynthetic pathway, in particular the biosynthetic origin of portions of the backbone, which arise from the unusual polyketide building blocks ethylmalonyl-CoA and methoxymalonyl-ACP, and the origin of the pendant deoxysugar moiety 4'-O-carbamoyl-2'-deoxyrhamnose, as well as the presence of a modular polyketide synthase (PKS) encoded by six giant ORFs. Examination of the methoxymalonyl-specific acyltransferase (AT) domains has led to recognition of an amino acid sequence motif which can be used to distinguish methylmalonyl-CoA- from methoxymalonyl-ACP-specific AT domains in natural PKSs.

A type I PKS gene probe obtained from RAPB of the rapamycin producer Streptomyces hygroscopicus, strongly hybridised to 92 out of 1120 cosmids from a genomic library of the elaiophylin-producing strain Streptomyces sp. DSM4137. Partial cosmid sequencing suggested the presence of 10 separate sequences encoding type I PKS genes. One entire DNA sequence was obtained and found exactly to match the gene organisation expected for the biosynthesis of the unusual macrodiolide polyketide elaiophylin. The putative elaiophylin gene cluster contains five large open-reading frames encoding typical modular polyketide synthases, which together catalyse the synthesis of the octaketide monomer of elaiophylin. Other genes were identified that would be required for provision of the ethylmalonate extender unit, for the synthesis and attachment of 2-deoxy-L-fucose and in regulation, or in export of the product. Immediately adjacent to the putative elaiophylin biosynthetic gene cluster is a 30-kbp region containing the gene for adenosylcobalamin-dependent methylmalonyl CoA mutase and also genes involved in the biosynthesis of the cobalamin cofactor. Analysis of the latter gene set confirms the view that cbiD of the anaerobic pathway and cobF in the aerobic pathway catalyse the same methylation of precorrin-5. The proximity of these genes to the putative elaiophylin gene cluster can best be rationalised if in this organism succinyl-CoA is a significant source of the methylmalonate units for complex polyketide biosynthesis.

The glycosyltransferases OleG1 and OleG2 and the cytochrome P450 oxidase OleP from the oleandomycin biosynthetic gene cluster of Streptomyces antibioticus have been expressed, either separately or from artificial gene cassettes, in strains of Saccharopolyspora erythraea blocked in erythromycin biosynthesis, to investigate their potential for the production of diverse novel macrolides from erythronolide precursors. OleP was found to oxidize 6-deoxyerythronolide B, but not erythronolide B. However, OleP did oxidize derivatives of erythronolide B in which a neutral sugar is attached at C-3. The oxidized products 3-O-mycarosyl-8a-hydroxyerythronolide B, 3-O-mycarosyl-8,8a-epoxyerythronolide B, 6-deoxy-8-hydroxyerythronolide B and the olefin 6-deoxy-8,8a-dehydroerythronolide B were all isolated and their structures determined. When oleP and the mycarosyltransferase eryBV were co-expressed in a gene cassette, 3-O-mycarosyl-6-deoxy-8,8a-dihydroxyerythronolide B was directly obtained. When oleG2 was co-expressed in a gene cassette together with oleP, 6-deoxyerythronolide B was converted into a mixture of 3-O-rhamnosyl-6-deoxy-8,8a-dehydroerythronolide B and 3-O-rhamnosyl-6-deoxy-8,8a-dihydroxyerythronolide B, confirming previous reports that OleG2 can transfer rhamnose, and confirming that oxidation by OleP and attachment of the neutral sugar to the aglycone can occur in either order. Similarly, four different 3-O-mycarosylerythronolides were found to be substrates for the desosaminyltransferase OleG1. These results provide additional insight into the nature of the intermediates in OleP-mediated oxidation, and suggest that oleandomycin biosynthesis might follow parallel pathways in which epoxidation either precedes or follows attachment of the neutral sugar.