Biosynthesis of polyketides by trans-AT polyketide synthases

Helfrich, Eric J. N.; Piel, Jörn

doi:10.1039/c5np00125k

Cited by 346 publications

(454 citation statements)

References 594 publications

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“…Previous studies have inactivated the terminal off-loading thioesterase (TE) domains of NRPS–PKS pathways to build up these intermediates and promote their hydrolysis and detection. 21 However, in the colibactin pathway, an atypical editing-type TE, ClbQ, promotes promiscuous off-loading of most of the precolibactin assembly intermediates, as observed in the multidomain signatures here and in other recent studies. 11h We speculate that these assembly line derailment products unusually enhanced by ClbQ could have functional cellular roles, as has been suggested from our previous in vitro assays.…”

Section: Discussionsupporting

confidence: 71%

Domain-Targeted Metabolomics Delineates the Heterocycle Assembly Steps of Colibactin Biosynthesis

et al. 2017

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Modular polyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs) comprise giant multidomain enzymes responsible for the “assembly line” biosynthesis of many genetically encoded small molecules. Site-directed mutagenesis, protein biochemical, and structural studies have focused on elucidating the catalytic mechanisms of individual multidomain proteins and protein domains within these megasynthases. However, probing their functions at the cellular level typically has invoked the complete deletion (or overexpression) of multidomain-encoding genes or combinations of genes and comparing those mutants with a control pathway. Here we describe a “domain-targeted” metabolomic strategy that combines genome editing with pathway analysis to probe the functions of individual PKS and NRPS catalytic domains at the cellular metabolic level. We apply the approach to the bacterial colibactin pathway, a genotoxic NRPS–PKS hybrid pathway found in certain Escherichia coli. The pathway produces precolibactins, which are converted to colibactins by a dedicated peptidase, ClbP. Domain-targeted metabolomics enabled the characterization of “multidomain signatures”, or functional readouts of NRPS–PKS domain contributions to the pathway-dependent metabolome. These multidomain signatures provided experimental support for individual domain contributions to colibactin biosynthesis and delineated the assembly line timing events of colibactin heterocycle formation. The analysis also led to the structural characterization of two reactive precolibactin metabolites. We demonstrate the fate of these reactive intermediates in the presence and absence of ClbP, which dictates the formation of distinct product groups resulting from alternative cyclization cascades. In the presence of the peptidase, the reactive intermediates are converted to a known genotoxic scaffold, providing metabolic support of our mechanistic model for colibactin-induced genotoxicity. Domain-targeted metabolomics could be more widely used to characterize NRPS–PKS pathways with unprecedented genetic and metabolic precision.

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Section: Discussionsupporting

confidence: 71%

Domain-Targeted Metabolomics Delineates the Heterocycle Assembly Steps of Colibactin Biosynthesis

et al. 2017

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“…With the exception of PKSs that utilise trans ‐acting acyl transferases,5 a type I PKS generally contains a minimum of three domains: a ketosynthase (KS), an acyl transferase (AT) and an acyl carrier protein (ACP). Optional ketoreductase (KR), dehydratase (DH) and enoyl reductase (ER) domains define the degree of β‐carbon processing after each round of chain elongation 6.…”

Section: Introductionmentioning

confidence: 99%

Discovery of Unusual Biaryl Polyketides by Activation of a Silent Streptomyces venezuelae Biosynthetic Gene Cluster

et al. 2016

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Comparative transcriptional profiling of a ΔbldM mutant of Streptomyces venezuelae with its unmodified progenitor revealed that the expression of a cryptic biosynthetic gene cluster containing both type I and type III polyketide synthase genes is activated in the mutant. The 29.5 kb gene cluster, which was predicted to encode an unusual biaryl metabolite, which we named venemycin, and potentially halogenated derivatives, contains 16 genes including one—vemR—that encodes a transcriptional activator of the large ATP‐binding LuxR‐like (LAL) family. Constitutive expression of vemR in the ΔbldM mutant led to the production of sufficient venemycin for structural characterisation, confirming its unusual biaryl structure. Co‐expression of the venemycin biosynthetic gene cluster and vemR in the heterologous host Streptomyces coelicolor also resulted in venemycin production. Although the gene cluster encodes two halogenases and a flavin reductase, constitutive expression of all three genes led to the accumulation only of a monohalogenated venemycin derivative, both in the native producer and the heterologous host. A competition experiment in which equimolar quantities of sodium chloride and sodium bromide were fed to the venemycin‐producing strains resulted in the preferential incorporation of bromine, thus suggesting that bromide is the preferred substrate for one or both halogenases.

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“…1B) (7). The bry pathway for bryostatin production is what is known as a trans-acetyltransferase (AT)-type polyketide synthase (PKS) pathway (8,12). PKSs are related to fatty acid synthases, and they construct a molecule in a similar fashion from two-car-bon units derived from malonate (13), using multiple enzymatic domains in a typical PKS protein.…”

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confidence: 99%

Lack of Overt Genome Reduction in the Bryostatin-Producing Bryozoan Symbiont “Candidatus Endobugula sertula”

Miller

Vanee

Fong

et al. 2016

Appl Environ Microbiol

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The uncultured bacterial symbiont "Candidatus Endobugula sertula" is known to produce cytotoxic compounds called bryostatins, which protect the larvae of its host, Bugula neritina. The symbiont has never been successfully cultured, and it was thought that its genome might be significantly reduced. Here, we took a shotgun metagenomics and metatranscriptomics approach to assemble and characterize the genome of "Ca. Endobugula sertula." We found that it had specific metabolic deficiencies in the biosynthesis of certain amino acids but few other signs of genome degradation, such as small size, abundant pseudogenes, and low coding density. We also identified homologs to genes associated with insect pathogenesis in other gammaproteobacteria, and these genes may be involved in host-symbiont interactions and vertical transmission. Metatranscriptomics revealed that these genes were highly expressed in a reproductive host, along with bry genes for the biosynthesis of bryostatins. We identified two new putative bry genes fragmented from the main bry operon, accounting for previously missing enzymatic functions in the pathway. We also determined that a gene previously assigned to the pathway, bryS, is not expressed in reproductive tissue, suggesting that it is not involved in the production of bryostatins. Our findings suggest that "Ca. Endobugula sertula" may be able to live outside the host if its metabolic deficiencies are alleviated by medium components, which is consistent with recent findings that it may be possible for "Ca. Endobugula sertula" to be transmitted horizontally. IMPORTANCEThe bryostatins are potent protein kinase C activators that have been evaluated in clinical trials for a number of indications, including cancer and Alzheimer's disease. There is, therefore, considerable interest in securing a renewable supply of these compounds, which is currently only possible through aquaculture of Bugula neritina and total chemical synthesis. However, these approaches are labor-intensive and low-yielding and thus preclude the use of bryostatins as a viable therapeutic agent. Our genome assembly and transcriptome analysis for "Ca. Endobugula sertula" shed light on the metabolism of this symbiont, potentially aiding isolation and culturing efforts. Our identification of additional bry genes may also facilitate efforts to express the complete pathway heterologously. Marine invertebrates, such as tunicates and sponges, are known to harbor symbiotic communities of bacteria. Because these animals are sessile and have limited physical defenses, their microbiomes are thought to serve defensive functions, and bacterial symbionts in these invertebrates have been implicated in the production of many bioactive small molecules (1, 2). Such small molecules can possess therapeutically relevant activities, so there is great interest in studying the biosynthetic potential and symbiotic functions of these defensive microorganisms (1, 2). However, most symbionts (and most environmental bacteria in general [1,3,4]) are difficul...

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Biosynthesis of polyketides by trans-AT polyketide synthases

Cited by 346 publications

References 594 publications

Domain-Targeted Metabolomics Delineates the Heterocycle Assembly Steps of Colibactin Biosynthesis

Domain-Targeted Metabolomics Delineates the Heterocycle Assembly Steps of Colibactin Biosynthesis

Discovery of Unusual Biaryl Polyketides by Activation of a Silent Streptomyces venezuelae Biosynthetic Gene Cluster

Lack of Overt Genome Reduction in the Bryostatin-Producing Bryozoan Symbiont “Candidatus Endobugula sertula”

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