Polyketide Assembly Research Articles

Multifactorial Control of Iteration Events in a Modular Polyketide Assembly Line

Multifactorial Control of Iteration Events in a Modular Polyketide Assembly Line

Biosynthesis of the Respiratory Toxin Bongkrekic Acid in the Pathogenic Bacterium Burkholderia gladioli

Bongkrekic acid (BA), an infamous respiratory toxin of the pathogenic bacterium Burkholderia gladioli, causes lethal intoxications when tempe bongkrek is produced with contaminated Rhizopus oligosporus cultures. Genome sequencing of B. gladioli pathovar cocovenenans unveiled the genetic basis for BA biosynthesis, and pointed to a homologous bon gene cluster in a B. gladioli strain from an infected rice plant. For functional genetics in B. gladioli λ Red recombination was established. Dissection of the modular type I polyketide synthase (a trans-AT PKS) provided insights into complex polyketide assembly. Isoprenoid-like β-branching events and a six-electron oxidation of a methyl group to a carboxylic acid give rise to the unique branched tricarboxylic fatty acid. The role of the cytochrome P450 monooxygenase, BonL, was proven by structural elucidation of deoxybongkrekic acid from a mutant.

Symbiotic Cooperation in the Biosynthesis of a Phytotoxin

Natural products play a key role in symbiotic interactions between microorganisms and higher organisms, covering all kingdoms of life. The function of these secondary metabolites may range from signaling compounds in mutualism to virulence factors and antibiotics in parasitic relationships. In many cases the interactions involve multiple partners and thus the biogenetic basis of chemical mediators can be quite complex. This complexity is well exemplified by the unparalleled tripartite relationship among the rice-seedling-blight fungus Rhizopus microsporus, its host plant Oryza sativa and endosymbiotic bacteria that reside in the fungal cytosol. The bacterial symbionts (Burkholderia species) produce a phytotoxin complex to assist the phytopathogenic fungus in colonizing rice seedlings. In turn the bacteria profit from a safe niche and access to nutrients released from the decaying plant. Initially, the macrolide rhizoxin (1, Figure 1) and various congeners such as WF-1360F (2) were isolated from cultures of R. microsporus van Tieghem var. chinensis and identified as the causative agent of rice seedling blight. Rhizoxin efficiently inhibits eukaryotic cell proliferation by binding to b-tubulin and thus blocking the formation of the mitotic spindle. Notably, the pure compound alone evokes the typical symptoms of seedling root swelling. Only recently, through detection, isolation, and cultivation of the endosymbionts we could unequivocally prove that actually associated bacteria are the true producers of the toxin complex. The importance of this metabolic capability has been underlined by the finding that fungal reproduction depends entirely on the presence of the bacterial symbionts. Survival of the toxinogenic symbiosis is warranted by the strict sporulation control and exclusive dispersal of spores harboring endosymbionts. Moreover, the unusual mutualism has been fine-tuned through symbiosis factors such as a type-III secretion system and a novel lipopolysaccharide O-antigen that sets the symbionts into a “stealth mode” by decorating the outer membrane of the endosymbionts. The host, on the other hand, acquired resistance towards rhizoxin by mutation of the b-tubulin. Because of its ecological and medicinal relevance as an antimitotic agent, the biosynthesis of rhizoxin has been studied. Cloning, sequencing, and molecular analyses of the rhizoxin (rhi) biosynthetic gene cluster in the genome of Burkholderia rhizoxinica revealed the molecular basis for a complex polyketide assembly line required for the biosynthesis of the virulence factor. Whereas the biosynthesis of the macrolide backbone has been decoded by mutational analyses, polyketide tailoring mechanisms and the biological role of the bis(epoxidation) have remained elusive. Herein we elucidate the dual epoxidation of rhizoxin and its impact on rice seedling blight and report an unprecedented case for symbiotic cooperation in the biosynthesis of an ecologically relevant natural product. In a broader survey on rhizoxin-positive Rhizopus species we discovered that the unusual bacterial–fungal association is not restricted to a single isolate but has spread worldwide. We have identified eight related Burkholderia–Rhizopus associations from geographically highly different regions on five continents; these findings underlign the ecological imporFigure 1. A) Structures of rhizoxin and congeners. B) Phylogenetic relationship of Rhizopus microsporus strains; structures of the corresponding metabolites indicate which strains can produce bisepoxides. The numbers on top of the branches indicate the clade probability values; the scale on the left site relates the length of a branch to the distance (number of changes that have taken place along a branch).

Interrogation of Global Active Site Occupancy of a Fungal Iterative Polyketide Synthase Reveals Strategies for Maintaining Biosynthetic Fidelity

Nonreducing iterative polyketide synthases (NR-PKSs) are responsible for assembling the core of fungal aromatic natural products with diverse biological properties. Despite recent advances in the field, many mechanistic details of polyketide assembly by these megasynthases remain unknown. To expand our understanding of substrate loading, polyketide elongation, cyclization, and product release, active site occupancy and product output were explored by Fourier transform mass spectrometry using the norsolorinic acid anthrone-producing polyketide synthase, PksA, from the aflatoxin biosynthetic pathway in Aspergillus parasiticus. Here we report the simultaneous observation of covalent intermediates from all catalytic domains of PksA from in vitro reconstitution reactions. The data provide snapshots of iterative catalysis and reveal an underappreciated editing function for the C-terminal thioesterase domain beyond its recently established synthetic role in Claisen/Dieckmann cyclization and product release. The specificity of thioesterase catalyzed hydrolysis was explored using biosynthetically relevant protein-bound and small molecule acyl substrates and demonstrated activity against hexanoyl and acetyl, but not malonyl. Processivity of polyketide extension was supported by the inability of a nonhydrolyzable malonyl analog to trap products of intermediate chain lengths and by the detection of only fully extended species observed covalently bound to, and as the predominant products released by, PksA. High occupancy of the malonyl transacylase domain and fast relative rate of malonyl transfer compared to starter unit transfer indicate that rapid loading of extension units onto the carrier domain facilitates efficient chain extension in a manner kinetically favorable to ultimate product formation.

Cloning and Characterization of the Polyether Salinomycin Biosynthesis Gene Cluster of Streptomyces albus XM211

Salinomycin is widely used in animal husbandry as a food additive due to its antibacterial and anticoccidial activities. However, its biosynthesis had only been studied by feeding experiments with isotope-labeled precursors. A strategy with degenerate primers based on the polyether-specific epoxidase sequences was successfully developed to clone the salinomycin gene cluster. Using this strategy, a putative epoxidase gene, slnC, was cloned from the salinomycin producer Streptomyces albus XM211. The targeted replacement of slnC and subsequent trans-complementation proved its involvement in salinomycin biosynthesis. A 127-kb DNA region containing slnC was sequenced, including genes for polyketide assembly and release, oxidative cyclization, modification, export, and regulation. In order to gain insight into the salinomycin biosynthesis mechanism, 13 gene replacements and deletions were conducted. Including slnC, 7 genes were identified as essential for salinomycin biosynthesis and putatively responsible for polyketide chain release, oxidative cyclization, modification, and regulation. Moreover, 6 genes were found to be relevant to salinomycin biosynthesis and possibly involved in precursor supply, removal of aberrant extender units, and regulation. Sequence analysis and a series of gene replacements suggest a proposed pathway for the biosynthesis of salinomycin. The information presented here expands the understanding of polyether biosynthesis mechanisms and paves the way for targeted engineering of salinomycin activity and productivity.

Biosynthesis and function of secondary metabolites

Natural products have long been used by humans owing to their beneficial effects. Indulgences such as coffee and tea, with their moderate stimulatory properties, have a long cultural tradition and are today some of the most important agricultural products worldwide. Other drugs have much stronger impacts on the central nervous system. For example, coca was originally used by the Inca civilisation because of its strong stimulatory effects, which result in feelings of happiness and euphoria. Opium was used by many ancient cultures as an efficient analgesic, but this also illustrates one of the first examples of drug abuse as it was used by warriors as an anxiolytic when girding their loins. Besides their pleasant traits these highly active drugs have adverse side effects, including most importantly an extremely high potential to cause addiction in combination with physical and mental prostration upon long-term abuse. Therefore, the production, importation, trading, and possession of these drugs are today under strict control by public authorities. Another historically interesting example of drug misuse is given by the death penalty imposed on Socrates in 399 B.C., carried out by the forced ingestion of the proverbial cup of hemlock. These traditional and historical examples of the usage of bioactive natural products all originated in the ancient cultures, without any knowledge about the underlying chemistry. This situation started to change at the beginning of the 19th century, when Friedrich Wilhelm Adam Serturner (1783–1841) first isolated morphine from opium, naming it after Morpheus, the Greek god of dreams and demonstrating its activity in self-tests, as was typical practice during these early times of chemistry. A likewise important discovery was made by Alexander Fleming (1881–1955), who isolated the antibiotic penicillin from the mould Penicillium notatum in a groundbreaking contribution, which was awarded the Nobel Prize in 1945. The discovery of new bioactive natural products is still a fascinating field in organic chemistry as demonstrated by the recent paradigms of the anticancer drug epothilon, the immunosuppressant rapamycin, or the proteasome inhibitor salinosporamide, to name but a few of hundreds of possible examples. Finding new secondary metabolites is a prerequisite for the development of novel pharmaceuticals, and this is an especially urgent task in the case of antibiotics due to the rapid spreading of bacterial resistances and the emergence of multiresistant pathogenic strains, which poses severe clinical problems in the treatment of infectious diseases. This Thematic Series on the biosynthesis and function of secondary metabolites deals with the discovery of new biologically active compounds from all kinds of sources, including plants, bacteria, and fungi, and also with their biogenesis. Biosynthetic aspects are closely related to functional investigations, because a deep understanding of metabolic pathways to natural products, not only on a chemical, but also on a genetic and enzymatic level, allows for the expression of whole biosynthetic gene clusters in heterologous hosts. This technique can make interesting, new secondary metabolites available from unculturable microorganisms, or may be used to optimise their availability by fermentation, for further research and also for production in the pharmaceutical industry. Especially fascinating is the intrinsic logic of the polyketide and nonribosomal peptide biosynthetic machineries, which is strongly correlated with the logic of fatty acid biosynthesis as part of the primary metabolism. Insights into the mechanisms of modular polyketide and nonribosomal peptide assembly lines open up the possibility for direct modifications, e.g., of oxidation states of the natural product’s carbon backbone by simple domain knockouts within the responsible megasynthases, or the introduction of a variety of alternative biosynthetic starters by mutasynthesis approaches, thus leading to new variants of known metabolites, which may have improved properties for therapeutic use. Another interesting aspect is the usage of enzymes in chemical transformations, which can provide synthetic organic chemists with an efficient access route to typically chiral building blocks that may otherwise be difficult to obtain. It is my great pleasure to thank all of the contributors to this Thematic Series, the staff of the Beilstein-Institut, and my esteemed colleague and teacher Prof. Henning Hopf (Braunschweig) for opening the opportunity to guest-edit the present issue. Enjoy reading it! Jeroen S. Dickschat Braunschweig, October 2011

ChemInform Abstract: A Comprehensive Overview on Genomically Directed Assembly of Aromatic Polyketides and Macrolide Lactones Using Fungal Megasynthases

AbstractReview: 31 refs.

Policing starter unit selection of the enterocin type II polyketide synthase by the type II thioesterase EncL

Enterocin is an atypical type II polyketide synthase (PKS) product from the marine actinomycete ‘Streptomyces maritimus’. The enterocin biosynthesis gene cluster (enc) codes for proteins involved in the assembly and attachment of the rare benzoate primer that initiates polyketide assembly with the addition of seven malonate molecules and culminates in a Favorskii-like rearrangement of the linear poly-β-ketone to give its distinctive non-aromatic, caged core structure. Fundamental to enterocin biosynthesis, which utilizes a single acyl carrier protein (ACP), EncC, for both priming with benzoate and elongating with malonate, involves maintaining the correct balance of acyl-EncC substrates for efficient polyketide assembly. Here, we report the characterization of EncL as a type II thioesterase that functions to edit starter unit (mis)priming of EncC. We performed a series of in vivo mutational studies, heterologous expression experiments, in vitro reconstitution studies, and Fourier-transform mass spectrometry-monitored competitive enzyme assays that together support the proposed selective hydrolase activity of EncL toward misprimed acetyl-ACP over benzoyl-ACP to facilitate benzoyl priming of the enterocin PKS complex. While this system resembles the R1128 PKS that also utilizes an editing thioesterase (ZhuC) to purge acetate molecules from its initiation module ACP in favor of alkylacyl groups, the enterocin system is distinct in its usage of a single ACP for both priming and elongating reactions with different substrates.

Structure and biosynthesis of the marine streptomycete ansamycin ansalactam A and its distinctive branched chain polyketide extender unit.

Reported is the structure and biosynthesis of ansalactam A, an ansamycin class polyketide produced by an unusual modification of the polyketide pathway. This new metabolite, produced by a marine sediment-derived bacterium of the genus Streptomyces , possesses a novel spiro γ-lactam moiety and a distinctive isobutyryl polyketide fragment observed for the first time in this class of natural products. The structure of ansalactam A was defined by spectroscopic methods including X-ray crystallographic analysis. Biosynthetic studies with stable isotopes further led to the discovery of a new, branched chain polyketide synthase extender unit derived from (E)-4-methyl-2-pentenoic acid for polyketide assembly observed for the first time in this class of natural products.

A comprehensive overview on genomically directed assembly of aromatic polyketides and macrolide lactones using fungal megasynthases

Fungal polyketide synthases (PKSs) catalyze a carbon-carbon bond forming reaction in an iterative manner using a variety of acyl-CoA molecules as substrates when biosynthesizing complex polyketides. Although most members from this class of natural products exhibit notable biological activities, often they are naturally produced in trace levels or cultivation of the analyte-producing organism is less than feasible. Appropriately, to contend with the former challenge, one must identify any translational bottleneck and perform functional analysis of the associated enzymes. In recent years, many gene clusters purportedly responsible for biosynthesizing polyketides have been identified and cataloged from a variety of fungal genomes including genes coding for iterative PKSs, particulary bikaverin, zearalenone and hypothemycin biosynthetic enzymes. Mounting appreciation of these highly specific codons and their translational consequence will afford scientists the ability to anticipate the fungal metabolite by correlating an organism's genomic cluster to an appropriate biosynthetic system. It was observed in recent reports, the successful production of these recombinant enzymes using an Escherichia coli expression system which in turn conferred the anticipated metabolite in vitro. This review will focus on iterative PKSs responsible for biosynthesizing bikaverin, zearalenone and hypothemycin, and expand on befitting enzymatic reaction mechanisms and development of a highly versatile system that could potentially generate biologically active compounds.

ChemInform Abstract: Polyketide Assembly by Alkene—Alkyne Reductive Cross‐Coupling: Spiroketals Through the Union of Homoallylic Alcohols.

AbstractA reaction sequence involving regioselective formal hydroalkynylation, reductive ene‐yne cross‐coupling, and oxidative cyclization is developed to prepare stereochemically dense spiroketals following similar pathways.

Polyketide assembly by alkene-alkyne reductive cross-coupling: spiroketals through the union of homoallylic alcohols.

A reaction sequence composed of regioselective formal hydroalkynylation, reductive ene-yne cross-coupling, and oxidative cyclization has been developed to prepare stereochemically dense spiroketals. Overall, the chemistry defines a three-component coupling process for the union of two homoallylic alcohol-based substrates with trimethylsilylacetylene.

Functionally Distinct Modules Operate Two Consecutive α,β→β,γ Double‐Bond Shifts in the Rhizoxin Polyketide Assembly Line

Verrückte Bindungen: Die biochemische Analyse des Rhizoxin-Biosynthesewegs zeigte, dass die Dieneinheit nicht im Ganzen, sondern über zwei enzymatische Operationen verschoben wird. Die erste Verschiebung resultiert aus einer formalen β,γ-Dehydratisierung in Modul 7; die zweite Doppelbindung wird zunächst durch Modul 8 erzeugt und dann durch ein „Verschiebemodul“ mit einer neuartigen DH*-Domäne verschoben. ACP = Acyl-Carrier-Protein, DH* = Dehydratase-ähnliche Verschiebedomäne. Detailed facts of importance to specialist readers are published as ”Supporting Information”. Such documents are peer-reviewed, but not copy-edited or typeset. They are made available as submitted by the authors. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Functionally Distinct Modules Operate Two Consecutive α,β→β,γ Double‐Bond Shifts in the Rhizoxin Polyketide Assembly Line

Functionally Distinct Modules Operate Two Consecutive α,β→β,γ Double‐Bond Shifts in the Rhizoxin Polyketide Assembly Line

Synthesis and biochemical analysis of complex chain-elongation intermediates for interrogation of molecular specificity in the erythromycin and pikromycin polyketide synthases.

The 6-deoxyerythronolide B synthase (DEBS) and pikromycin (Pik) polyketide synthase (PKS) are unique multifunctional enzyme systems that are responsible for the biosynthesis of the erythromycin and pikromycin 14-membered ring aglycones, respectively. Together, these natural product biosynthetic systems provide excellent platforms to examine the fundamental structural and catalytic elements that govern polyketide assembly, processing, and macrocyclization. In these studies, the native pentaketide intermediate for DEBS was synthesized and employed for in vitro chemoenzymatic synthesis of macrolactone products in engineered monomodules Ery5, Ery5-TE, and Ery6. A comparative analysis was performed with the corresponding Pik module 5 (PikAIII) and module 6 (PikAIV), dissecting key similarities and differences between these highly related PKSs. The data revealed that individual modules in the DEBS and Pik PKSs possess distinctive molecular selectivity profiles and suggest that substrate recognition has evolved unique characteristics in each system.

The Biosynthetic Logic of Polyketide Diversity

Molecular Lego: Polyketides represent a highly diverse group of natural products with structurally intriguing carbon skeletons (see picture) which are assembled from simple acyl building blocks. A combination of chemical, biochemical, and genetics studies have provided exciting new insights into the programming of polyketide assembly and the sophisticated enzymatic machineries involved. This review highlights recent developments in the field.Polyketides constitute one of the major classes of natural products. Many of these compounds or derivatives thereof have become important therapeutics for clinical use; in contrast, various polyketides are infamous food-spoiling toxins or virulence factors. What is particularly remarkable about this heterogeneous group of compounds comprising of polyethers, polyenes, polyphenols, macrolides, and enediynes is that they are mainly derived from one of the simplest building blocks available in nature: acetic acid. Investigations at the chemical, genetic, and biochemical levels have shed light on the biosynthetic programs that lead to the large structural diversity of polyketides .This review highlights recently unveiled biosynthetic mechanisms to generate highly diverse and complex molecules.

Polyketide assembly lines of uncultivated sponge symbionts from structure-based gene targeting

There is increasing evidence that uncultivated bacterial symbionts are the true producers of numerous bioactive compounds isolated from marine sponges. The localization and heterologous expression of biosynthetic genes could clarify this issue and provide sustainable supplies for a wide range of pharmaceuticals. However, identification of genes in the usually highly complex symbiont communities remains a challenging task. For polyketides, one of the most important groups of sponge-derived drug candidates, we have developed a general strategy that allows one to rapidly access biosynthetic gene clusters based on chemical moieties. Using this method, we targeted polyketide synthase genes from two different sponge metagenomes. We have obtained from a sponge-bacterial association a complete pathway for the rare and potent antitumor agent psymberin from Psammocinia aff. bulbosa. The data support the symbiont hypothesis and provide insights into natural product evolution in previously inaccessible bacteria.

Biosynthesis of polyketide synthase extender units

This review covers the biosynthesis of extender units that are utilized for the assembly of polyketides by polyketide synthases. The metabolic origins of each of the currently known polyketide synthase extender units are covered.

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.

Exploiting the mosaic structure of trans-acyltransferase polyketide synthases for natural product discovery and pathway dissection

Modular polyketide synthases (PKSs) are giant bacterial enzymes that synthesize many polyketides of therapeutic value. In contrast to PKSs that provide acyltransferase (AT) activities in cis, trans-AT PKSs lack integrated AT domains and exhibit unusual enzymatic features with poorly understood functions in polyketide assembly. This has retarded insight into the assembly of products such as mupirocin, leinamycin and bryostatin 1. We show that trans-AT PKSs evolved in a fundamentally different fashion from cis-AT systems, through horizontal recruitment and assembly of substrate-specific ketosynthase (KS) domains. The insights obtained from analysis of these KS mosaics will facilitate both the discovery of novel polyketides by genome mining, as we demonstrate for the thailandamides of Burkholderia thailandensis, and the extraction of chemical information from short trans-AT PCR products, as we show using metagenomic DNA of marine sponges. Our data also suggest new strategies for dissecting polyketide biosynthetic pathways and engineering polyketide assembly.