Polyketide Synthesis Research Articles

Starter unit flexibility for engineered product synthesis by the nonreducing polyketide synthase PksA.

Nonreducing polyketide synthases (NR-PKSs) are unique among PKSs in their domain structure, notably including a starter unit:acyl-carrier protein (ACP) transacylase (SAT) domain that selects an acyl group as the primer for biosynthesis, most commonly acetyl-CoA from central metabolism. This clan of mega-enzymes resembles fatty acid synthases (FASs) by sharing both their central chain elongation steps and their capacity for iterative catalysis. In this mode of synthesis, catalytic domains involved in chain extension exhibit substrate plasticity to accommodate growing chains as small as two carbons to 20 or more. PksA is the NR-PKS central to the biosynthesis of the mycotoxin aflatoxin B1 whose SAT domain accepts an unusual hexanoyl starter from a dedicated yeast-like FAS. Explored in this paper is the ability of PksA to utilize a selection of potential starter units as substrates to initiate and sustain extension and cyclization to on-target, programmed polyketide synthesis. Most of these starter units were successfully accepted and properly processed by PksA to achieve biosynthesis of the predicted naphthopyrone product. Analysis of the on-target and derailment products revealed trends of tolerance by individual PksA domains to alternative starter units. In addition, natural and un-natural variants of the active site cysteine were examined and found to be capable of biosynthesis, suggesting possible direct loading of starter units onto the β-ketoacyl synthase (KS) domain. In light of the data assembled here, the predictable synthesis of unnatural products by NR-PKSs is more fully defined.

Reaction design, discovery, and development as a foundation to function-oriented synthesis.

Convergent C-C bond-forming reactions define the fabric of organic synthesis and, when applied in complex molecule synthesis, can have a profound impact on efficiency by decreasing the longest linear sequence of transformations required to convert simple starting materials to complex targets. Despite their well-appreciated strategic significance, campaigns in natural product synthesis typically embrace only a small suite of reactivity to achieve such bond construction (i.e., nucleophilic addition to polarized π-bonds, nucleophilic substitution, cycloaddition, and metal-catalyzed "cross-coupling"), therefore limiting the sites at which convergent coupling chemistry can be strategically employed. In our opinion, it is far too often that triumphs in the field are defined by chemical sequences that do not address the challenges associated with discovery, development, and production of natural product-inspired agents. We speculated that advancing an area of chemical reactivity not represented in the few well-established strategies for convergent C-C bond formation may lead to powerful new retrosynthetic relationships that could simplify approaches to the syntheses of a variety of different classes of natural products. Our studies ultimately embraced the pursuit of strategies to control the course of metallacycle-mediated "cross-coupling" between substrates containing sites of simple π-unsaturation (ubiquitous functionality in organic chemistry including alkenes, alkynes, allenes, aldehydes, and imines, among others). In just eight years since our initial publication in this area, we have defined over 20 stereoselective intermolecular C-C bond-forming reactions that provide access to structural motifs of relevance for the synthesis of polyketides, fatty acids, alkaloids, and terpenes, while doing so in a direct and stereoselective fashion. These achievements continue to serve as the foundation of my group's activity in natural product and function-oriented synthesis, where our achievements in reaction development are challenged in the context of complex targets. Among our early efforts, we achieved the most concise synthesis of a benzoquinone ansamycin ever described (macbecin I), and moved beyond this achievement to explore the role of our chemistry in function-oriented synthesis targeting the discovery of natural product-inspired Hsp90 inhibitors. These later efforts have led to the discovery of a uniquely selective benzoquinone ansamycin-inspired Hsp90 inhibitor that lacks the problematic quinone present in the natural series. This achievement was made possible by a concise chemical synthesis pathway that had at its core the application of metallacycle-mediated cross-coupling chemistry.

Transcriptomic characterisation and genomic glimps into the toxigenic dinoflagellate Azadinium spinosum, with emphasis on polykeitde synthase genes

BackgroundUnicellular dinoflagellates are an important group of primary producers within the marine plankton community. Many of these species are capable of forming harmful algae blooms (HABs) and of producing potent phycotoxins, thereby causing deleterious impacts on their environment and posing a threat to human health. The recently discovered toxigenic dinoflagellate Azadinium spinosum is known to produce azaspiracid toxins. These toxins are most likely produced by polyketide synthases (PKS). Recently, PKS I-like transcripts have been identified in a number of dinoflagellate species. Despite the global distribution of A. spinosum, little is known about molecular features. In this study, we investigate the genomic and transcriptomic features of A. spinosum with a focus on polyketide synthesis and PKS evolution.ResultsWe identify orphan and homologous genes by comparing the transcriptome data of A. spinosum with a diverse set of 18 other dinoflagellates, five further species out of the Rhizaria Alveolate Stramelopile (RAS)-group, and one representative from the Plantae. The number of orphan genes in the analysed dinoflagellate species averaged 27%. In contrast, within the A. spinosum transcriptome, we discovered 12,661 orphan transcripts (18%). The dinoflagellates toxins known as azaspiracids (AZAs) are structurally polyethers; we therefore analyse the transcriptome of A. spinosum with respect to polyketide synthases (PKSs), the primary biosynthetic enzymes in polyketide synthesis. We find all the genes thought to be potentially essential for polyketide toxin synthesis to be expressed in A. spinosum, whose PKS transcripts fall into the dinoflagellate sub-clade in PKS evolution.ConclusionsOverall, we demonstrate that the number of orphan genes in the A. spinosum genome is relatively small compared to other dinoflagellate species. In addition, all PKS domains needed to produce the azaspiracid carbon backbone are present in A. spinosum. Our study underscores the extraordinary evolution of such gene clusters and, in particular, supports the proposed structural and functional paradigm for PKS Type I genes in dinoflagellates.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-014-1205-6) contains supplementary material, which is available to authorized users.

Complete Genome Sequence of Bacillus amyloliquefaciens subsp. plantarum CC178, a Phyllosphere Bacterium Antagonistic to Plant Pathogenic Fungi.

Bacillus amyloliquefaciens subsp. plantarum strain CC178 is a phyllosphere bacterium with antagonistic activity against a wide range of plant fungal pathogens. The genome of strain CC178 is 3,916,828 bp in size and harbors 3,972 genes. Six giant gene clusters are dedicated to the nonribosomal synthesis of antimicrobial polypeptides and polyketides.

Endogenous cross-talk of fungal metabolites.

Non-ribosomal peptide (NRP) synthesis in fungi requires a ready supply of proteogenic and non-proteogenic amino acids which are subsequently incorporated into the nascent NRP via a thiotemplate mechanism catalyzed by NRP synthetases. Substrate amino acids can be modified prior to or during incorporation into the NRP, or following incorporation into an early stage amino acid-containing biosynthetic intermediate. These post-incorporation modifications involve a range of additional enzymatic activities including but not exclusively, monooxygenases, methyltransferases, epimerases, oxidoreductases, and glutathione S-transferases which are essential to effect biosynthesis of the final NRP. Likewise, polyketide biosynthesis is directly by polyketide synthase megaenzymes and cluster-encoded ancillary decorating enzymes. Additionally, a suite of additional primary metabolites, for example: coenzyme A (CoA), acetyl CoA, S-adenosylmethionine, glutathione (GSH), NADPH, malonyl CoA, and molecular oxygen, amongst others are required for NRP and polyketide synthesis (PKS). Clearly these processes must involve exquisite orchestration to facilitate the simultaneous biosynthesis of different types of NRPs, polyketides, and related metabolites requiring identical or similar biosynthetic precursors or co-factors. Moreover, the near identical structures of many natural products within a given family (e.g., ergot alkaloids), along with localization to similar regions within fungi (e.g., conidia) suggests that cross-talk may exist, in terms of biosynthesis and functionality. Finally, we speculate if certain biosynthetic steps involved in NRP and PKS play a role in cellular protection or environmental adaptation, and wonder if these enzymatic reactions are of equivalent importance to the actual biosynthesis of the final metabolite.

First Stereoselective Synthesis of the Cytotoxic Polyketide (4R)‐1‐(3,5‐Dihydroxyphenyl)‐4‐hydroxypentan‐2‐one

AbstractThe first stereoselective synthesis of the cytotoxic polyketide (4R)‐1‐(3,5‐dihydroxyphenyl)‐4‐hydroxypentan‐2‐one (1) was achieved from readily available propylene oxide and 3,5‐dimethoxybenzyl alcohol. The synthesis involves Jacobsen's hydrolytic kinetic resolution (HKR) and Grignard reaction as key steps.

Probing Protein Interactions of Cyanylated Acyl Carrier Proteins using Vibrational Spectroscopy

Acyl carrier proteins (ACPs) are highly conserved protein domains involved in the synthesis of polyketides, natural products essential to pharmaceutical drug development. ACPs are part of a multicomponent modular system known as polyketide synthases (PKSs). Understanding these systems permits the manipulation of PKSs to produce “unnatural” natural compounds essential for combating health issues. ACPs interact with PKS domains through a flexible 4′phosphopantetheine (4′-Ppant) prosthetic arm that undergoes important and fast conformational changes difficult to track with NMR and X-ray crystallography. We installed a thiocyanate (-SCN) probe on the terminal thiol of a 4′ Ppant arm to observe these conformations on a picosecond time scale. The method enables the identification of factors affecting 4′ Ppant arm movement. We observed the effects on 6-deoxyerythronolide B synthase (DEBS) and fatty acid synthase (FAS) ACP conformation when titrated with their respective catalytic ketosynthase (KS) partners. Vibrational spectroscopy reveals that dynamic protein-protein interactions as well as substrate chain length perturb the conformation and location of the 4′ Ppant arm.

A chiral phosphoric acid catalyst for asymmetric construction of 1,3-dioxanes.

A novel method of enantioselective 1,3-dioxane construction via a hemiacetalization/intramolecular oxy-Michael addition cascade by a chiral phosphoric acid catalyst was developed. The product was successfully transformed into an optically active 1,3-polyol motif, indicating that the proposed reaction can provide useful chiral building blocks for the de novo synthesis of polyketides.

Methylation-dependent acyl transfer between polyketide synthase and nonribosomal peptide synthetase modules in fungal natural product biosynthesis.

Biochemical studies of purified and dissected fungal polyketide synthase and nonribosomal peptide synthetase (PKS-NRPS) hybrid enzymes involved in biosynthesis of pseurotin and aspyridone indicate that one α-methylation step during polyketide synthesis is a prerequisite and a key checkpoint for chain transfer between PKS and NRPS modules. In the absence of the resulting γ-methyl feature, the completed polyketide intermediate is offloaded as an α-pyrone instead of being aminoacylated by the NRPS domain. These examples illustrate that precisely timed tailoring domain activities play critical roles in the overall programming of the iterative PKS (and NRPS) functions.

Total synthesis of (±)-hippolachnin A.

The first total synthesis of the marine polyketide (±)-hippolachnin A has been achieved in nine linear steps and an overall yield of 9%. Rapid access to the oxacyclobutapentalene core structure was secured by strategic application of an ene cyclization.

Total Synthesis of (±)‐Hippolachnin A

AbstractThe first total synthesis of the marine polyketide (±)‐hippolachnin A has been achieved in nine linear steps and an overall yield of 9 %. Rapid access to the oxacyclobutapentalene core structure was secured by strategic application of an ene cyclization.

A Single Module Type I Polyketide Synthase Directs de Novo Macrolactone Biogenesis during Galbonolide Biosynthesis in Streptomyces galbus

Galbonolide (GAL) A and B are antifungal macrolactone polyketides produced by Streptomyces galbus. During their polyketide chain assembly, GAL-A and -B incorporate methoxymalonate and methylmalonate, respectively, in the fourth chain extension step. The methoxymalonyl-acyl carrier protein biosynthesis locus (galG to K) is specifically involved in GAL-A biosynthesis, and this locus is neighbored by a gene cluster composed of galA-E. GalA-C constitute a single module, highly reducing type I polyketide synthase (PKS). GalD and GalE are cytochrome P450 and Rieske domain protein, respectively. Gene knock-out experiments verified that galB, -C, and -D are essential for GAL biosynthesis. A galD mutant accumulated a GAL-C that lacked two hydroxyl groups and a double bond when compared with GAL-B. A [U-(13)C]propionate feeding experiment indicated that no rare precursor other than methoxymalonate was incorporated during GAL biogenesis. A search of the S. galbus genome for a modular type I PKS system, the type that was expected to direct GAL biosynthesis, resulted in the identification of only one modular type I PKS gene cluster. Homology analysis indicated that this PKS gene cluster is the locus for vicenistatin biosynthesis. This cluster was previously reported in Streptomyces halstedii. A gene deletion of the vinP2 ortholog clearly demonstrated that this modular type I PKS system is not involved in GAL biosynthesis. Therefore, we propose that GalA-C direct macrolactone polyketide formation for GAL. Our studies provide a glimpse into a novel biochemical strategy used for polyketide synthesis; that is, the iterative assembly of propionates with highly programmed β-keto group modifications.

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.

Chemoenzymatic Synthesis of Spinosyn A

AbstractFollowing 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.

Bacilysin from Bacillus amyloliquefaciens FZB42 has specific bactericidal activity against harmful algal bloom species.

Harmful algal blooms, caused by massive and exceptional overgrowth of microalgae and cyanobacteria, are a serious environmental problem worldwide : In the present study, we looked for Bacillus strains with sufficiently strong anticyanobacterial activity to be used as biocontrol agents. Among 24 strains, Bacillus amyloliquefaciens FZB42 showed the strongest bactericidal activity against Microcystis aeruginosa, with a kill rate of 98.78%. The synthesis of the anticyanobacterial substance did not depend on Sfp, an enzyme that catalyzes a necessary processing step in the nonribosomal synthesis of lipopeptides and polyketides, but was associated with the aro gene cluster that is involved in the synthesis of the sfp-independent antibiotic bacilysin. Disruption of bacB, the gene in the cluster responsible for synthesizing bacilysin, or supplementation with the antagonist N-acetylglucosamine abolished the inhibitory effect, but this was restored when bacilysin synthesis was complemented. Bacilysin caused apparent changes in the algal cell wall and cell organelle membranes, and this resulted in cell lysis. Meanwhile, there was downregulated expression of glmS, psbA1, mcyB, and ftsZ-genes involved in peptidoglycan synthesis, photosynthesis, microcystin synthesis, and cell division, respectively. In addition, bacilysin suppressed the growth of other harmful algal species. In summary, bacilysin produced by B. amyloliquefaciens FZB42 has anticyanobacterial activity and thus could be developed as a biocontrol agent to mitigate the effects of harmful algal blooms.

Proteomic insight into the effect of ethanol on citrinin biosynthesis pathway in Monascus purpureus NTU 568

Monascus species produce several potent bioactive metabolites through polyketide secondary metabolic pathways; however, little is known of the regulation of metabolic processes in this organism. Therefore, we investigated the effect of extreme growth conditions on the production of secondary metabolites by Monascus purpureus strain NTU 568 and identified pathways that plausibly regulate the polyketide pathways by using proteomic analysis. Citrinin, a type of antibiotic, is synthesized through the polyketide pathway in M. purpureus NTU 568. We hypothesized that production of citrinin might be inhibited by ethanol. When M. purpureus NTU 568 was cultured in the medium containing 4% ethanol, the secretion of secondary metabolites was inhibited, but the dry cell weight was increased. We also found that branched chain amino acid degradation and the expression level of aldehyde dehydrogenase (ALDH) were downregulated, but proteins related to the heat shock response were induced. Furthermore, polyketide synthesis-related proteins, fatty acid synthase, epoxide hydrolase, and proteins involved in the shikimate secondary metabolic pathway were inhibited by ethanol treatment. These results suggested that reduced production of polyketide metabolites resulted not only from the expression levels of proteins in the polyketide synthesis pathway but also from reduction in the concentration of primary metabolism-generated molecules (e.g., acetyl-CoA, fatty acids) that are used as substrates for polyketide syntheses. This study provides insights into the polyketide secondary metabolism of Monascus as well as into improvement of the production of bioactive secondary metabolites in Monascus species.

Evolution of ketosynthase domains of polyketide synthase genes in the Cladonia chlorophaea species complex (Cladoniaceae)

Lichen-forming fungi synthesize a diversity of polyketides, but only a few non-reducing polyketide synthase (PKS) genes from a lichen-forming fungus have been linked with a specific polyketide. While it is a challenge to link the large number of PKS paralogs in fungi with specific products, it might be expected that the PKS paralogs from closely related species would be similar because of recent evolutionary divergence. The objectives of this study were to reconstruct a PKS gene phylogeny of the Cladonia chlorophaea species complex based on the ketosynthase domain, a species phylogeny of the complex, and to explore the presence of PKS gene paralogs among members of the species complex. DNA was isolated from 51 individuals of C. chlorophaea and allies to screen for the presence of 13 PKS paralogs. A 128 sequence PKS gene phylogeny using deduced amino acid sequences estimated from the 13 PKS paralogs and sequences subjected to BLASTx comparisons showed losses of each of two PKS domains (reducing and methylation). This research provided insight into the evolution of PKS genes in the C. chlorophaea group, species evolution in the group, and it identified potential directions for further investigation of polyketide synthesis in the C. chlorophaea species complex.

Improving polyketide and fatty acid synthesis by engineering of the yeast acetyl-CoA carboxylase

Polyketides and fatty acids are important in the production of pharmaceuticals, industrial chemicals, and biofuels. The synthesis of the malonyl-CoA building block, catalyzed by acetyl-CoA carboxylase (Acc1), is considered a limiting step to achieving high titers of polyketides and fatty acids in Saccharomyces cerevisiae. Acc1 is deactivated by AMP-activated serine/threonine protein kinase (Snf1) when glucose is depleted. To prevent this deactivation, the enzyme was aligned with the Rattus norvegicus (rat) Acc1 to identify a critical amino acid (Ser-1157) for phosphorylation and deactivation. Introduction of a S1157A mutation into Acc1 resulted in 9-fold higher specific activity following glucose depletion. The enzyme was tested in yeast engineered to produce the polyketide 6-methylsalisylic acid (6-MSA). Both 6-MSA and native fatty acid levels increased by 3-fold. Utilization of this modified Acc1 enzyme will also be beneficial for other products built from malonyl-CoA.

ChemInform Abstract: Sequential Catalysis for Stereoselective Synthesis of Complex Polyketides

AbstractReview: 111 refs.

Biocatalysts for natural product biosynthesis.

Natural products are important sources of pharmaceuticals, in part owing to their diverse biological activities. Enzymes from natural product biosynthetic pathways have become attractive candidates as biocatalysts for modifying the structures and bioactivities of these complex compounds. Numerous enzymes have been harvested to generate innovative scaffolds, large-scale synthesis of chiral building blocks, and semisynthesis of medicinally relevant natural product derivatives. This review discusses recent examples from three areas: (a) polyketide catalytic domain engineering geared toward synthesis of new polyketides, (b) engineering of tailoring enzymes (other than oxidative enzymes) as biocatalysts, and (c) in vitro total synthesis of natural products using purified enzyme components. With the availability of exponentially increasing genomic information and new genome mining tools, many new and powerful biocatalysts tailored for pharmaceutical synthesis will likely emerge from secondary metabolism.