A Dehydratase Domain in Ambruticin Biosynthesis Displays Additional Activity as a Pyran‐Forming Cyclase

Hydropyran rings are a common structural motif in reduced polyketides. Information on their biosynthetic formation and particularly the biochemical characterization of the responsible enzymes has only been reported in few cases. The dehydratase domain AmbDH3 from the ambruticin polyketide synthase was investigated. Through in vitro assay of the recombinant domain with synthetically‐derived substrate surrogates, it was shown that it has a second catalytic activity as a cyclase that performs oxa‐conjugate addition. Probing AmbDH3 with synthetic substrate analogues revealed stereoselectivity and substrate tolerance in both substeps. This is the first characterization of a pyran‐forming cyclase from a cis‐AT PKS system and the first report of a polyketide synthase domain with this kind of dual activity. Finally, it was revealed that this domain shows potential for application in chemoenzymatic synthesis.     

The Interplay between a Multifunctional Dehydratase Domain and a C‐Methyltransferase Effects Olefin Shift in Ambruticin Biosynthesis

The olefin shift is an important modification during polyketide biosynthesis. Particularly for type I cis‐AT PKS, little information has been gained on the enzymatic mechanisms involved. We present our in vitro investigations on the olefin shift occurring during ambruticin biosynthesis. The unique, multifunctional domain AmbDH4 catalyzes consecutive dehydration, epimerization, and enoyl isomerization. The resulting 3‐enethioate is removed from the equilibrium by α‐methylation catalyzed by the highly specific C‐methyltransferase AmbM. This thermodynamically unfavorable overall process is enabled by the high, concerted substrate specificity of the involved enzymes. AmbDH4 shows close relationship to DH domains and initial mechanistic studies suggest that the olefin shift occurs via a similar proton‐shuttling mechanism as previously described for EI domains from trans‐AT‐PKS.     

A Long‐Range Acting Dehydratase Domain as the Missing Link for C17‐Dehydration in Iso‐Migrastatin Biosynthesis

The dehydratase domains (DHs) of the iso‐migrastatin (iso‐MGS) polyketide synthase (PKS) were investigated by systematic inactivation of the DHs in module‐6, ‐9, ‐10 of MgsF (i.e., DH6, DH9, DH10) and module‐11 of MgsG (i.e., DH11) in vivo, followed by structural characterization of the metabolites accumulated by the mutants, and biochemical characterization of DH10 in vitro, using polyketide substrate mimics with varying chain lengths. These studies allowed us to assign the functions for all four DHs, identifying DH10 as the dedicated dehydratase that catalyzes the dehydration of the C17 hydroxy group during iso‐MGS biosynthesis. In contrast to canonical DHs that catalyze dehydration of the β‐hydroxy groups of the nascent polyketide intermediates, DH10 acts in a long‐range manner that is unprecedented for type I PKSs, a novel dehydration mechanism that could be exploited for polyketide structural diversity by combinatorial biosynthesis and synthetic biology.     

A Long-Range Acting Dehydratase Domain as the Missing Link for C17-Dehydration in Iso-Migrastatin Biosynthesis

The dehydratase domains (DHs) of the iso-migrastatin (iso-MGS) polyketide synthase (PKS) were investigated by systematic inactivation of the DHs in module-6, -9, -10 of MgsF (i.e., DH6, DH9, DH10) and module-11 of MgsG (i.e., DH11) in vivo, followed by structural characterization of the metabolites accumulated by the mutants, and biochemical characterization of DH10 in vitro, using polyketide substrate mimics with varying chain lengths. These studies allowed us to assign the functions for all four DHs, identifying DH10 as the dedicated dehydratase that catalyzes the dehydration of the C17 hydroxy group during iso-MGS biosynthesis. In contrast to canonical DHs that catalyze dehydration of the β-hydroxy groups of the nascent polyketide intermediates, DH10 acts in a long-range manner that is unprecedented for type I PKSs, a novel dehydration mechanism that could be exploited for polyketide structural diversity by combinatorial biosynthesis and synthetic biology.     

An Unusual Dehydratase Acting on Glycerate and a Ketoreducatse Stereoselectively Reducing α‐Ketone in Polyketide Starter Unit Biosynthesis

Polyketide synthases (PKSs) usually employ a ketoreductase (KR) to catalyze the reduction of a β‐keto group, followed by a dehydratase (DH) that drives the dehydration to form a double bond between the α‐ and β‐carbon atoms. Herein, a DH*‐KR* involved in FR901464 biosynthesis was characterized: DH* acts on glyceryl‐S‐acyl carrier protein (ACP) to yield ACP‐linked pyruvate; subsequently KR* reduces α‐ketone that yields L ‐lactyl‐S‐ACP as starter unit for polyketide biosynthesis. Genetic and biochemical evidence was found to support a similar pathway that is involved in the biosynthesis of lankacidins. These results not only identified new PKS domains acting on different substrates, but also provided additional options for engineering the PKS starter pathway or biocatalysis.     

Characterization of the alnumycin gene cluster reveals unusual gene products for pyran ring formation and dioxan biosynthesis

Alnumycin is closely related to the benzoisochromanequinone (BIQ) polyketides such as actinorhodin. Exceptional structural features include differences in aglycone tailoring that result in the unique alnumycin chromophore and the existence of an unusual 4-hydroxymethyl-5-hydroxy-1,3-dioxan moiety. Cloning and sequencing of the alnumycin gene cluster from Streptomyces sp. CM020 revealed expected biosynthesis genes for polyketide assembly, but several genes encoding subsequent tailoring enzymes were highly atypical. Heterologous expression studies confirmed that all of the genes required for alnumycin biosynthesis resided within the sequenced clone. Inactivation of genes aln4 and aln5 showed that the mechanism of pyran ring formation differs from actinorhodin and granaticin pathways. Further inactivation studies identified two genes, alnA and alnB, involved in the synthesis and attachment of the dioxan moiety, and resulted in the production of the polyketide prealnumycin.     

Rational Control of Polyketide Extender Units by Structure‐Based Engineering of a Crotonyl‐CoA Carboxylase/Reductase in Antimycin Biosynthesis

Bioengineering of natural product biosynthesis is a powerful approach to expand the structural diversity of bioactive molecules. However, in polyketide biosynthesis, the modification of polyketide extender units, which form the carbon skeletons, has remained challenging. Herein, we report the rational control of polyketide extender units by the structure‐based engineering of a crotonyl‐CoA carboxylase/reductase (CCR), in the biosynthesis of antimycin. Site‐directed mutagenesis of the CCR enzyme AntE, guided by the crystal structure solved at 1.5 Å resolution, expanded its substrate scope to afford indolylmethylmalonyl‐CoA by the V350G mutation. The mutant A182L selectively catalyzed carboxylation over the regular reduction. Furthermore, the combinatorial biosynthesis of heterocycle‐ and substituted arene‐bearing antimycins was achieved by an engineered Streptomyces strain bearing AntE^V350G. These findings deepen our understanding of the molecular mechanisms of the CCRs, which will serve as versatile biocatalysts for the manipulation of building blocks, and set the stage for the rational design of polyketide biosynthesis.     

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.     

Cloning, sequencing and analysis of the enterocin biosynthesis gene cluster from the marine isolate 'Streptomyces maritimus': evidence for the derailment of an aromatic polyketide synthase

BACKGROUND: Polycyclic aromatic polyketides, such as the tetracyclines and anthracyclines, are synthesized by bacterial aromatic polyketide synthases (PKSs). Such PKSs contain a single set of iteratively used individual proteins for the construction of a highly labile poly-beta-carbonyl intermediate that is cyclized by associated enzymes to the core aromatic polyketide. A unique polyketide biosynthetic pathway recently identified in the marine strain 'Streptomyces maritimus' deviates from the normal aromatic PKS model in the generation of a diverse series of chiral, non-aromatic polyketides. RESULTS: A 21.3 kb gene cluster encoding the biosynthesis of the enterocin and wailupemycin family of polyketides from 'S. maritimus' has been cloned and sequenced. The biosynthesis of these structurally diverse polyketides is encoded on a 20 open reading frames gene set containing a centrally located aromatic PKS. The architecture of this novel type II gene set differs from all other aromatic PKS clusters by the absence of cyclase and aromatase encoding genes and the presence of genes encoding the biosynthesis and attachment of the unique benzoyl-CoA starter unit. In addition to the previously reported heterologous expression of the gene set, in vitro and in vivo expression studies with the cytochrome P-450 EncR and the ketoreductase EncD, respectively, support the involvement of the cloned genes in enterocin biosynthesis. CONCLUSIONS: The enterocin biosynthesis gene cluster represents the most versatile type II PKS system investigated to date. A large series of divergent metabolites are naturally generated from the single biochemical pathway, which has several metabolic options for creating structural diversity. The absence of cyclase and aromatase gene products and the involvement of an oxygenase-catalyzed Favorskii-like rearrangement provide insight into the observed spontaneity of this pathway. This system provides the foundation for engineering hybrid expression sets in the generation of structurally novel compounds for use in drug discovery.     

Structure and Biosynthesis of Isatropolones,Bioactive Amine‐Scavenging Fluorescent Natural Products from Streptomyces Gö66

The natural products isatropolone A–C ( 1 – 3 ) were reisolated from Streptomyces Gö66, with 1 and 3 showing potent activity against Leishmania donovani. They contain a rare tropolone ring derived from a type II polyketide biosynthesis pathway. Their biosynthesis was elucidated by labeling experiments, analysis of the biosynthesis gene cluster, its partial heterologous expression, and structural characterization of various intermediates. Owing to their 1,5‐diketone moiety, they can react with ammonia, amines, lysine, and lysine‐containing peptides and proteins, which results in the formation of a covalent bond and subsequent pyridine ring formation. Their fluorescence properties change upon amine binding, enabling the simple visualization of reacted amines including proteins.     

Chemoenzymatic Synthesis of Spinosyn A

Following the biosynthesis of polyketide backbones by polyketide synthases (PKSs), post‐PKS modifications result in a significantly elevated level of structural complexity that renders the chemical synthesis of these natural products challenging. We report herein a total synthesis of the widely used polyketide insecticide spinosyn A by exploiting the prowess of both chemical and enzymatic methods. As more polyketide biosynthetic pathways are characterized, this chemoenzymatic approach is expected to become readily adaptable to streamlining the synthesis of other complex polyketides with more elaborate post‐PKS modifications.     

Cloning, sequencing and analysis of the enterocin biosynthesis gene cluster from the marine isolate ‘Streptomyces maritimus’: evidence for the derailment of an aromatic polyketide synthase

Background: Polycyclic aromatic polyketides, such as the tetracyclines and anthracyclines, are synthesized by bacterial aromatic polyketide synthases (PKSs). Such PKSs contain a single set of iteratively used individual proteins for the construction of a highly labile poly-β-carbonyl intermediate that is cyclized by associated enzymes to the core aromatic polyketide. A unique polyketide biosynthetic pathway recently identified in the marine strain ‘Streptomyces maritimus’ deviates from the normal aromatic PKS model in the generation of a diverse series of chiral, non-aromatic polyketides.Results: A 21.3 kb gene cluster encoding the biosynthesis of the enterocin and wailupemycin family of polyketides from ‘S. maritimus’ has been cloned and sequenced. The biosynthesis of these structurally diverse polyketides is encoded on a 20 open reading frames gene set containing a centrally located aromatic PKS. The architecture of this novel type II gene set differs from all other aromatic PKS clusters by the absence of cyclase and aromatase encoding genes and the presence of genes encoding the biosynthesis and attachment of the unique benzoyl-CoA starter unit. In addition to the previously reported heterologous expression of the gene set, in vitro and in vivo expression studies with the cytochrome P-450 EncR and the ketoreductase EncD, respectively, support the involvement of the cloned genes in enterocin biosynthesis.Conclusions: The enterocin biosynthesis gene cluster represents the most versatile type II PKS system investigated to date. A large series of divergent metabolites are naturally generated from the single biochemical pathway, which has several metabolic options for creating structural diversity. The absence of cyclase and aromatase gene products and the involvement of an oxygenase-catalyzed Favorskii-like rearrangement provide insight into the observed spontaneity of this pathway. This system provides the foundation for engineering hybrid expression sets in the generation of structurally novel compounds for use in drug discovery.     

Enzymology of Pyran Ring A Formation in Salinomycin Biosynthesis

Tetrahydropyran rings are a common feature of complex polyketide natural products, but much remains to be learned about the enzymology of their formation. The enzyme SalBIII from the salinomycin biosynthetic pathway resembles other polyether epoxide hydrolases/cyclases of the MonB family, but SalBIII plays no role in the conventional cascade of ring opening/closing. Mutation in the salBIII gene gave a metabolite in which ring A is not formed. Using this metabolite in vitro as a substrate analogue, SalBIII has been shown to form pyran ring A. We have determined the X‐ray crystal structure of SalBIII, and structure‐guided mutagenesis of putative active‐site residues has identified Asp38 and Asp104 as an essential catalytic dyad. The demonstrated pyran synthase activity of SalBIII further extends the impressive catalytic versatility of α+β barrel fold proteins.     

A new mode of stereochemical control revealed by analysis of the biosynthesis of dihydrogranaticin in Streptomyces violaceoruber Tü22.

A class of Streptomyces aromatic polyketide antibiotics, the benzoisochromanequinones, all shows trans stereochemistry at C-3 and C-15 in the pyran ring. The opposite stereochemical control found in actinorhodin (3S, 15R, ACT) from S. coelicolor A3(2) and dihydrogranaticin (3R, 15S, DHGRA) from S. violaceoruber Tü22 was studied by functional expression of the potentially relevant ketoreductase genes, actIII, actVI-ORF1, gra-ORF5, and gra-ORF6. A common bicyclic intermediate was postulated to undergo stereospecific reduction to provide either the 3-(S) or the 3-(R) configuration of an advanced intermediate, 4-dihydro-9-hydroxy-1-methyl-10-oxo-3-H-naphtho[2,3-c]pyran-3-acetic acid (DNPA). Combinations of the four ketoreductase genes were coexpressed with the early biosynthetic genes encoding a type II minimal polyketide synthase, aromatase, and cyclase. gra-ORF6 was essential to produce (R)-DNPA in DHGRA biosynthesis. Out of the various recombinants carrying the relevant ketoreductases, the set of gra-ORF5 and -ORF6 under translational coupling (on pIK191) led to the most efficient production of (R)-DNPA as a single product, implying a possible unique cooperative function whereby gra-ORF6 might encode a "guiding" protein to control the regio- and stereochemical course of reduction at C-3 catalyzed by the gra-ORF5 protein. Updated BLAST-based database analysis suggested that the gra-ORF6 product, a putative short-chain dehydrogenase, has virtually no sequence homology with the actVI-ORF1 protein, which was previously shown to determine the 3-(S) configuration of DNPA in ACT biosynthesis. This demonstrates an example of opposite stereochemical control in antibiotic biosynthesis, providing a key branch point to afford diverse chiral metabolic pools.     

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.     

Enantioselective Total Synthesis of the Putative Biosynthetic Intermediate Ambruticin J

The family of anti-fungal natural products known as the ambruticins are structurally distinguished by a pair of pyran rings adorning a divinylcyclopropane core. Previous characterization of their biosynthesis, including the expression of a genetically modified producing organism, revealed that the polyketide synthase pathway proceeds via a diol intermediate, known as ambruticin J. Herein, we report the first enantioselective total synthesis of the putative PKS product, ambruticin J, according to a triply convergent synthetic route featuring a Suzuki-Miyaura cross-coupling and a Julia-Kocienski olefination for fragment assembly. This synthesis takes advantage of synthetic methodology previously developed by our laboratory for the stereoselective generation of the trisubstituted cyclopropyl linchpin.     

Polyketide double bond biosynthesis. Mechanistic analysis of the dehydratase-containing module 2 of the picromycin/methymycin polyketide synthase

Picromycin/methymycin synthase (PICS) is a modular polyketide synthase (PKS) that is responsible for the biosynthesis of both 10-deoxymethynolide (1) and narbonolide (2), the parent 12- and 14-membered aglycone precursors of the macrolide antibiotics methymycin and picromycin, respectively. PICS module 2 is a dehydratase (DH)-containing module that catalyzes the formation of the unsaturated triketide intermediate using malonyl-CoA as the chain extension substrate. Recombinant PICS module 2+TE, with the PICS thioesterase domain appended to the C-terminus to allow release of polyketide products, was expressed in Escherichia coli. Purified PICS module 2+TE converted malonyl-CoA and 4, the N-acetylcysteamine thioester of (2S,3R)-2-methyl-3-hydroxypentanoic acid, to a 1:2 mixture of the triketide acid (4S,5R)-4-methyl-5-hydroxy-2-heptenoic acid (5) and (3S,4S,5R)-3,5-dihydroxy-4-methyl-n-heptanoic acid-delta-lactone (10) with a combined kcat of 0.6 min(-1). The triketide lactone 10 is formed by thioesterase-catalyzed cyclization of the corresponding d-3-hydroxyacyl-SACP intermediate, a reaction which competes with dehydration catalyzed by the dehydratase domain. PICS module 2+TE showed a strong preference for the syn-diketide-SNAC 4, with a 20-fold greater kcat/K(m) than the anti-(2S,3S)-diketide-SNAC 14, and a 40-fold advantage over the syn-(2R,3S)-diketide-SNAC 13. PICS module 2(DH(0))+TE, with an inactivated DH domain, produced exclusively 10, while three PICS module 2(KR(0))+TE mutants, with inactivated KR domains, produced exclusively or predominantly the unreduced triketide ketolactone, (4S,5R)-3-oxo-4-methyl-5-hydroxy-n-heptanoic acid-delta-lactone (7). These studies establish for the first time the structure and stereochemistry of the intermediates of a polyketide chain elongation cycle catalyzed by a DH-containing module, while confirming the importance of key active site residues in both KR and DH domains.     

Cloning and elucidation of the FR901464 gene cluster revealing a complex acyltransferase-less polyketide synthase using glycerate as starter units

FR901464, an antitumor natural product, represents a new class of potent anticancer small molecules targeting spliceosome and inhibiting both splicing and nuclear retention of pre-mRNA. Herein we describe the biosynthetic gene cluster of FR901464, identified by degenerate primer PCR amplification of a gene encoding the 3-hydroxy-3-methylglutaryl-CoA synthase (HCS) postulated to be involved in the biosynthesis of a β-branched polyketide from Pseudomonas sp. No. 2663. This cluster consists of twenty open reading frames (ORFs) and was localized to 93-kb DNA segment, and its involvement in FR901464 biosynthesis was confirmed by gene inactivation and complementation. FR901464 is biosynthesized by a hybrid polyketide synthase (PKS)/nonribosomal peptide synthetase (NRPS), HCS, and acyltransferases (AT)-less system. The PKS/NRPS modules feature unusual domain organization including multiple domain redundancy, inactivation, and tandem. Biochemical characterization of a glyceryl transferase and an acyl carrier protein (ACP) in the start module revealed that it incorporates D-1,3-bisphosphoglycerate, which is dephosphorylated and transferred to ACP as the starter unit. Furthermore, an oxidative Baeyer-Villiger reaction followed by chain release was postulated to form a pyran moiety. On the basis of in silico analysis and genetic and biochemical evidances, a biosynthetic pathway for FR901464 was proposed, which sets the stage to further investigate the complex PKS biochemically and engineer the biosynthetic machinery for the production of novel analogues.     

Production of New Cladosporin Analogues by Reconstitution of the Polyketide Synthases Responsible for the Biosynthesis of this Antimalarial Agent

The antimalarial agent cladosporin is a nanomolar inhibitor of the Plasmodium falciparum lysyl‐tRNA synthetase, and exhibits activity against both blood‐ and liver‐stage infection. Cladosporin can be isolated from the fungus Cladosporium cladosporioides, where it is biosynthesized by a highly reducing (HR) and a non‐reducing (NR) iterative type I polyketide synthase (PKS) pair. Genome sequencing of the host organism and subsequent heterologous expression of these enzymes in Saccharomyces cerevisiae produced cladosporin, confirming the identity of the putative gene cluster. Incorporation of a pentaketide intermediate analogue indicated a 5+3 assembly by the HR PKS Cla2 and the NR PKS Cla3 during cladosporin biosynthesis. Advanced‐intermediate analogues were synthesized and incorporated by Cla3 to furnish new cladosporin analogues. A putative lysyl‐tRNA synthetase resistance gene was identified in the cladosporin gene cluster. Analysis of the active site emphasizes key structural features thought to be important in resistance to cladosporin.     

Enzymology of Anthraquinone‐γ‐Pyrone Ring Formation in Complex Aromatic Polyketide Biosynthesis

Aromatic‐fused γ‐pyrones are structural features of many bioactive natural products and valid scaffolds for medicinal chemistry. However, the enzymology of their formation has not been completely established. Now it is demonstrated that TxnO9, a CalC‐like protein belonging to a START family, functions as an unexpected anthraquinone‐γ‐pyrone synthase involved in the biosynthesis of antitumor antibiotic trioxacarcin A (TXN‐A). Structural analysis by NMR identified a likely substrate/product‐binding mode and putative key active sites of TxnO9, which allowed an enzymatic mechanism to be proposed. Moreover, a subset of uncharacterized homologous proteins bearing an unexamined Lys‐Thr dyad exhibit the same function. Therefore, the functional assignment and mechanistic investigation of this γ‐pyrone synthase elucidated an undescribed step in TXN‐A biosynthesis, and the discovery of this new branch of polyketide heterocyclases expands the functions of the START superfamily.