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Elsinoë species are phytopathogenic fungi that cause serious scab diseases on economically important plants. The disease symptoms arise from the effects of a group of phytotoxins known as elsinochromes, produced via a type-I polyketide synthase (PKS) biosynthetic pathway. The elsinochrome gene cluster was first annotated in Elsinoë fawcettii where the main type-I PKS gene was characterized as EfPKS1. A later study showed that this gene and the associated cluster had not been correctly annotated, and that EfPKS1 was actually the anchor gene of the melanin biosynthetic pathway. A new type-I PKS gene EfETB1 associated with elsinochrome production was also identified. The aim of this study was to identify all type-I PKS genes in the genomes of seven Elsinoë species with the goal of independently verifying the PKS containing clusters for both melanin and elsinochrome production. A total of six type-I PKS classes were identified, although there was variation between the species in the number and type of classes present. Genes similar to the E. fawcettii EfPKS1 and EfETB1 type-I PKS genes were associated with melanin and elsinochrome production respectively in all species. The complete melanin and elsinochrome PKS containing clusters were subsequently annotated in all the species with high levels of synteny across Elsinoë species. This study provides a genus-level overview of type-I PKS distribution in Elsinoë species, including an additional line of support for the annotation of the melanin and elsinochrome PKS containing clusters in these important plant pathogens., (© 2024. The Author(s).)

The social amoeba Polysphondylium violaceum uses chemoattractants different from those of Dictyoctelium discoideum for cell aggregation. However, the detailed mechanisms in P. violaceum remain unknown. We have previously reported that the polyketide synthase StlA is involved in inducing aggregation in this species. To elucidate the mechanism of StlA-induced aggregation in P. violaceum, we analyzed the phenotype of P. violaceum stlA- (Pv-stlA-) mutants in more detail. Unlike our previous results, the mutant cells did not exhibit proper chemotaxis toward glorin. Defective aggregation was not restored by glorin pulses, 8Br-cAMP, or deletion of the homologue of PufA that is a translational repressor of protein kinase A, whereas mutant cells grown in the presence of 4-methyl-5-pentylbenzene-1,3-diol (MPBD), the putative Pv-StlA product, aggregated normally without it after starvation. Furthermore, the early developmental marker gene, dscA, was downregulated in the mutant cells. Our data thus suggested that StlA is required for the transition from growth to development in P. violaceum., (© The Author(s) 2024. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

Bacterial trans -acyltransferase polyketide synthases ( trans -AT PKSs) are among the most complex enzymes, which are responsible for generating a wide range of natural products, identified as trans -AT polyketides. These polyketides have received significant attention in drug development due to their structural diversity and potent bioactivities. With approximately 300 synthesized molecules discovered so far, trans -AT PKSs are found widespread in bacteria. Their biosynthesis pathways exhibit considerable genetic diversity, leading to the emergence of numerous enzymes with novel mechanisms, serving as a valuable resource for genetic engineering aimed at modifying small molecules' structures and creating new engineered enzymes. Despite the systematic discussions on trans -AT polyketides and their biosynthesis in earlier studies, the continuous advancements in tools, methods, compound identification, and biosynthetic pathways require a fresh update on accumulated knowledge. This review seeks to provide a comprehensive discussion for the 27 types of trans -AT polyketides discovered within the last seven years, detailing their sources, structures, biological activities, and biosynthetic pathways. By reviewing this new knowledge, a more profound understanding of the trans -AT polyketide family can be achieved.

Mycobacteria are known for their complex cell wall, which comprises layers of peptidoglycan, polysaccharides and unusual fatty acids known as mycolic acids that form their unique outer membrane. Polyketide synthase 13 (Pks13) of Mycobacterium tuberculosis , the bacterial organism causing tuberculosis, catalyses the last step of mycolic acid synthesis prior to export to and assembly in the cell wall. Due to its essentiality, Pks13 is a target for several novel anti-tubercular inhibitors, but its 3D structure and catalytic reaction mechanism remain to be fully elucidated. Here, we report the molecular structure of the catalytic core domains of M. tuberculosis Pks13 (Mt-Pks13), determined by transmission cryo-electron microscopy (cryoEM) to a resolution of 3.4 Å. We observed a homodimeric assembly comprising the ketoacyl synthase (KS) domain at the centre, mediating dimerization, and the acyltransferase (AT) domains protruding in opposite directions from the central KS domain dimer. In addition to the KS-AT di-domains, the cryoEM map includes features not covered by the di-domain structural model that we predicted to contain a dimeric domain similar to dehydratases, yet likely lacking catalytic function. Analytical ultracentrifugation data indicate a pH-dependent equilibrium between monomeric and dimeric assembly states, while comparison with the previously determined structures of M. smegmatis Pks13 indicates architectural flexibility. Combining the experimentally determined structure with modelling in AlphaFold2 suggests a structural scaffold with a relatively stable dimeric core, which combines with considerable conformational flexibility to facilitate the successive steps of the Claisen-type condensation reaction catalysed by Pks13.

Type III polyketide synthases (PKSs) catalyze the formation of a variety of polyketide natural products with remarkable structural diversity and biological activities. Despite significant progress in structural and mechanistic studies of type III PKSs in bacteria, fungi, and plants, research on type III PKSs in cyanobacteria is lacking. Here, we report structural and mechanistic insights into CylI, a type III PKS that catalyzes the formation of the alkylresorcinol intermediate in cylindrocyclophane biosynthesis. The crystal structure of apo-CylI reveals a distinct arrangement of structural elements that are proximal to the active site. We further solved the crystal structures of CylI in complexes with two substrate analogues at resolutions of 1.9 Å. The complex structures indicate that N259 is the key residue that determines the substrate preference of CylI. We also solved the crystal structure of CylI complexed with the alkylresorcinol product at a resolution of 2.0 Å. Structural analysis and mutagenesis experiments suggested that S170 functions as a key residue that determines cyclization specificity. On the basis of this result, a double mutant was engineered to completely switch the cyclization of CylI from aldol condensation to lactonization. This work elucidates the molecular basis of type III PKS in cyanobacteria and lays the foundation for engineering CylI-like enzymes to generate new products., (© 2024 The Protein Society.)

Modular polyketide synthases (PKSs) are capable of synthesizing diverse natural products with fascinating bioactivities. Canonical enoyl-CoA hydratases (ECHs) are components of the β-branching cassette that modifies the polyketide chain by adding a β-methyl branch. Herein, it is demonstrated that the deletion of an atypical ECH Q domain (featuring a Q 280 residue) of Art21, a didomain protein contains an ECH Q domain and a thioesterase (TE) domain, reprograms the polyketide assembly line from synthesizing tetracyclic aurantinins (ARTs) to bicyclic auritriacids (ATAs) with much lower antibacterial activities. Genes encoding the ECH Q -TE didomain proteins distribute in many PKS gene clusters from different bacteria. Significantly, the ART PKS machinery can be directed to make ARTs, ATAs, or both of them by employing appropriate ECH Q -TE proteins, implying a great potential for using this reprogramming strategy in polyketide structure diversification., (© 2024 The Author(s). Advanced Science published by Wiley‐VCH GmbH.)

Valsa pyri , the causal agent of pear canker disease, typically induces cankers on the bark of infected trees and even leads to tree mortality. Secondary metabolites produced by pathogenic fungi play a crucial role in the pathogenic process. In this study, secondary metabolic regulator VpLaeA was identified in V. pyri . VpLaeA was found to strongly affect the pathogenicity, fruiting body formation, and toxicity of secondary metabolites of V. pyri . Additionally, VpLaeA was found to be required for the response of V. pyri to some abiotic stresses. Transcriptome data analysis revealed that many of differentially expressed genes were involved in the secondary metabolite biosynthesis. Among them, about one third of secondary metabolite biosynthesis core genes were regulated by VpLaeA at different periods. Seven differentially expressed secondary metabolite biosynthesis core genes ( VpPKS9 , VpPKS10 , VpPKS33 , VpNRPS6 , VpNRPS7 , VpNRPS16 , and VpNRPS17 ) were selected for knockout. Two modular polyketide synthase genes ( VpPKS10 and VpPKS33 ) that were closely related to the virulence of V. pyri from the above seven genes were identified. Notably, VpPKS10 and VpPKS33 also affected the production of fruiting body of V. pyri but did not participate in the resistance of V. pyri to abiotic stresses. Overall, this study demonstrates the multifaceted biological functions of VpLaeA in V. pyri and identifies two toxicity-associated polyketide synthase genes in Valsa species fungi for the first time., Competing Interests: The author(s) declare no conflict of interest.

Three decades of studies on the multifunctional 6-deoxyerythronolide B synthase have laid a foundation for understanding the chemistry and evolution of polyketide antibiotic biosynthesis by a large family of versatile enzymatic assembly lines. Recent progress in applying chemical and structural biology tools to this prototypical assembly-line polyketide synthase (PKS) and related systems has highlighted several features of their catalytic cycles and associated protein dynamics. There is compelling evidence that multiple mechanisms have evolved in this enzyme family to channel growing polyketide chains along uniquely defined sequences of 10-100 active sites, each of which is used only once in the overall catalytic cycle of an assembly-line PKS. Looking forward, one anticipates major advances in our understanding of the mechanisms by which the free energy of a repetitive Claisen-like reaction is harnessed to guide the growing polyketide chain along the assembly line in a manner that is kinetically robust yet evolutionarily adaptable.

Utilizing bioinformatics tools, this study expands our understanding of secondary metabolism in Botrytis cinerea , identifying novel genes within polyketide synthase (PKS), non-ribosomal peptide synthetase (NRPS), sesquiterpene cyclase (STC), diterpene cyclase (DTC), and dimethylallyltryptophan synthase (DMATS) families. These findings enrich the genetic framework associated with B. cinerea 's pathogenicity and ecological adaptation, offering insights into uncharted metabolic pathways. Significantly, the discovery of previously unannotated genes provides new molecular targets for developing targeted antifungal strategies, promising to enhance crop protection and advance our understanding of fungal biochemistry. This research not only broadens the scope of known secondary metabolites but also opens avenues for future exploration into B. cinerea 's biosynthetic capabilities, potentially leading to novel antifungal compounds. Our work underscores the importance of integrating bioinformatics and genomics for fungal research, paving the way for sustainable agricultural practices by pinpointing precise molecular interventions against B. cinerea . This study sets a foundation for further investigations into the fungus's secondary metabolism, with implications for biotechnology and crop disease management.

Enzymatic modifications of small molecules are a common phenomenon in natural product biosynthesis, leading to the production of diverse bioactive compounds. In polyketide biosynthesis, modifications commonly take place after the completion of the polyketide backbone assembly by the polyketide synthases and the mature products are released from the acyl-carrier protein (ACP). However, exceptions to this rule appear to be widespread, as on-line hydroxylation, methyl transfer, and cyclization during polyketide assembly process are common, particularly in trans-AT PKS systems. Many of these modifications are catalyzed by specific domains within the modular PKS systems. However, several of the on-line modifications are catalyzed by stand-alone proteins. Those include the on-line Baeyer-Villiger oxidation, α-hydroxylation, halogenation, epoxidation, and methyl esterification during polyketide assembly, dehydrogenation of ACP-bound short fatty acids by acyl-CoA dehydrogenase-like enzymes, and glycosylation of ACP-bound intermediates by discrete glycosyltransferase enzymes. This review article highlights some of these trans-acting proteins that catalyze enzymatic modifications of ACP-bound small molecules in natural product biosynthesis., (© 2024 Wiley‐VCH GmbH.)

Octacosamicin A is an antifungal metabolite featuring a linear polyene-polyol chain flanked by N-hydroxyguanidine and glycine moieties. We report here that sub-inhibitory concentrations of streptomycin elicited the production of octacosamicin A in Amycolatopsis azurea DSM 43854 T . We identified the biosynthetic gene cluster (oca BGC) that encodes a modular polyketide synthase (PKS) system for assembling the polyene-polyol chain of octacosamicin A. Our analysis suggested that the N-hydroxyguanidine unit originates from a 4-guanidinobutyryl-CoA starter unit, while the PKS incorporates an α-hydroxyketone moiety using a (2R)-hydroxymalonyl-CoA extender unit. The modular PKS system contains a non-canonical terminal module that lacks thioesterase (TE) and acyl carrier protein (ACP) domains, indicating the biosynthesis is likely to employ an unconventional and cryptic off-loading mechanism that attaches glycine to the polyene-polyol chain via an intermolecular amidation reaction., (© 2023 Wiley-VCH GmbH.)

Tuberculosis is one of the deadly infectious diseases that has resurfaced in multiple/ extensively resistant variants (MDR/XDR), threatening humankind. Today's world has a higher prevalence of tuberculosis (TB) than it has ever had throughout human history. Due to severe adverse effects, the marketed medications are not entirely effective in these forms. So, developing new drugs with a promising target is an immense necessity. Pks13 has emerged as a promising target for the mycobacterium. The concluding step of mycolic acid production involved Pks13, a crucial enzyme that helps form the precursor of mycolic acid via the Claisen-condensation reaction. It has five domains at the active site for targeting the enzyme and is used to test chemical entities for their antitubercular activity. Benzofurans, thiophenes, coumestans, N-phenyl indoles, and β lactones are the ligands that inhibit the Pks13 enzyme, showing potential antitubercular properties., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

The chemical scaffolds of numerous therapeutics are polyketide natural products, many formed by bacterial modular polyketide synthases (PKS). The large and flexible dimeric PKS modules have distinct extension and reducing regions. Structures are known for all individual enzyme domains and several extension regions. Here, we report the structure of the full reducing region from a modular PKS, the ketoreductase (KR), dehydratase (DH), and enoylreductase (ER) domains of module 5 of the juvenimicin PKS. The modular PKS-reducing region has a different architecture than the homologous fatty acid synthase (FAS) and iterative PKS systems in its arrangement of domains and dimer interface. The structure reveals a critical role for linker peptides in the domain interfaces, leading to discovery of key differences in KR domains dependent on module composition. Finally, our studies provide insight into the mechanism underlying modular PKS intermediate shuttling by carrier protein (ACP) domains., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Assembly line polyketide synthases (PKSs) are a large family of multifunctional enzymes responsible for synthesizing many medicinally relevant natural products with remarkable structural variety and biological activity. The decrease in cost of genomic sequencing paired with development of computational tools like antiSMASH presents an opportunity to survey the vast diversity of assembly line PKS. Mining the genomic data in the National Center for Biotechnology Information database, our updated catalogue (https://orphanpkscatalog2022.stanford.edu/catalog) presented in this article revealed 8799 non-redundant assembly line polyketide synthase clusters across 4083 species, representing a threefold increase over the past 4 years. Additionally, 95% of the clusters are 'orphan clusters' for which natural products are neither chemically nor biologically characterized. Our analysis indicates that the diversity of assembly line PKSs remains vastly under-explored and also highlights the promise of a genomics-driven approach to natural product discovery.

Microbial polyketide synthase (PKS) genes encode the biosynthesis of many biomedically or otherwise commercially important natural products. Despite extensive discovery efforts, metagenomic analyses suggest that only a small fraction of nature's polyketide biosynthetic potential has been realized. Much of this potential originates from type I PKSs (T1PKSs), which can be further delineated based on their domain organization and the structural features of the compounds they encode. Notably, phylogenetic relationships among ketosynthase (KS) domains provide an effective method to classify the larger and more complex T1PKS genes in which they occur. Increased access to large metagenomic data sets from diverse habitats provides opportunities to assess T1PKS biosynthetic diversity and distributions through their smaller and more tractable KS domain sequences. Here, we used the web tool NaPDoS2 to detect and classify over 35,000 type I KS domains from 137 metagenomic data sets reported from eight diverse, globally distributed biomes. We found biome-specific separation with soils enriched in KSs from modular cis -acetyltransferase (AT) and hybrid cis -AT KSs relative to other biomes and marine sediments enriched in KSs associated with polyunsaturated fatty acid and enediyne biosynthesis. We linked the phylum Actinobacteria to soil-derived enediyne and cis -AT KSs while marine-derived KSs associated with enediyne and monomodular PKSs were linked to phyla from which the compounds produced by these biosynthetic enzymes have not been reported. These KSs were phylogenetically distinct from those associated with experimentally characterized PKSs suggesting they may be associated with novel structures or enzyme functions. Finally, we employed our metagenome-extracted KS domains to evaluate the PCR primers commonly used to amplify type I KSs and identified modifications that could increase the KS sequence diversity recovered from amplicon libraries. IMPORTANCE Polyketides are a crucial source of medicines, agrichemicals, and other commercial products. Advances in our understanding of polyketide biosynthesis, coupled with the increased availability of metagenomic sequence data, provide new opportunities to assess polyketide biosynthetic potential across biomes. Here, we used the web tool NaPDoS2 to assess type I polyketide synthase (PKS) diversity and distributions by detecting and classifying ketosynthase (KS) domains across 137 metagenomes. We show that biomes are differentially enriched in type I KS domains, providing a roadmap for future biodiscovery strategies. Furthermore, KS phylogenies reveal biome-specific clades that do not include biochemically characterized PKSs, highlighting the biosynthetic potential of poorly explored environments. The large metagenome-derived KS data set allowed us to identify regions of commonly used type I KS PCR primers that could be modified to capture a larger extent of environmental KS diversity. These results facilitate both the search for novel polyketides and our understanding of the biogeographical distribution of PKSs across Earth's major biomes., Competing Interests: The authors declare no conflict of interest.

The genotoxin colibactin produced by Escherichia coli is involved in the development of colorectal cancers. This secondary metabolite is synthesized by a multi-protein machinery, mainly composed of non-ribosomal peptide synthetase (NRPS)/polyketide synthase (PKS) enzymes. In order to decipher the function of a PKS-NRPS hybrid enzyme implicated in a key step of colibactin biosynthesis, we conducted an extensive structural characterization of the ClbK megaenzyme. Here we present the crystal structure of the complete trans-AT PKS module of ClbK showing structural specificities of hybrid enzymes. In addition, we report the SAXS solution structure of the full-length ClbK hybrid that reveals a dimeric organization as well as several catalytic chambers. These results provide a structural framework for the transfer of a colibactin precursor through a PKS-NRPS hybrid enzyme and can pave the way for re-engineering PKS-NRPS hybrid megaenzymes to generate diverse metabolites with many applications., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

The biosynthesis of antitumor oxazole-containing conglobatin is directed by a multienzyme assembly line of nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS), in which an uncanonical iterative-acting C-terminal thioesterase domain, Cong-TE, ligated two fully elongated chains/conglobatin monomers on the terminal acylcarrier protein and subsequently cyclized the resulting dimer to a C 2 -symmetric macrodiolide. Screening of the conglobatin producer for secondary metabolites led to the discovery of two new compounds conglactones A ( 1 ) and B ( 2 ), possessing inhibitory activities to phytopathogenic microorganisms and cancer cells, respectively. The compounds 1 and 2 feature the ester bond-linked hybrid structures consisting of an aromatic polyketide benwamycin I ( 3 ) and one (for 1 )/two (for 2 ) molecules of the conglobatin monomer ( 5 ). Genetic mutational analysis revealed that the production of 1 and 2 was correlated with the biosynthetic pathways of 3 and 5 . Biochemical analysis indicated that 1 and 2 were produced by Cong-TE from 3 and an N -acetylcysteamine thioester form of 5 ( 7 ). Furthermore, the substrate compatibility of Cong-TE was demonstrated by enzymatically generating a bunch of ester products from 7 and 43 exotic alcohols. This property of Cong-TE was further validated by producing 36 hybrid esters in the fermentation of conglobatin producer fed with nonindigenous alcohols. This work shows a prospect of developing Cong-TE for green synthesis of valuable oxazole-containing esters, thus complementing the environmentally unfriendly chemosynthesis strategies.

Nature serves as a rich source of molecules with immense chemical diversity. Aptly named, these 'natural products' boast a wide variety of environmental, medicinal and industrial applications. Type II polyketides, in particular, confer substantial medicinal benefits, including antibacterial, antifungal, anticancer and anti-inflammatory properties. These molecules are produced by enzyme assemblies known as type II polyketide synthases (PKSs), which use domains such as the ketosynthase chain-length factor and acyl carrier protein to produce polyketides with varying lengths, cyclization patterns and oxidation states. In this work, we use a novel bioinformatic workflow to identify biosynthetic gene clusters (BGCs) that code for the core type II PKS enzymes. This method does not rely on annotation and thus was able to unearth previously 'hidden' type II PKS BGCs. This work led us to identify over 6000 putative type II PKS BGCs spanning a diverse set of microbial phyla, nearly double those found in most recent studies. Notably, many of these newly identified BGCs were found in non-actinobacteria, which are relatively underexplored as sources of type II polyketides. Results from this work lay an important foundation for future bioprospecting and engineering efforts that will enable sustainable access to diverse and structurally complex molecules with medicinally relevant properties.

It is well known that polyunsaturated fatty acids (PUFAs) in Schizochytrium sp. are mainly synthesized via the polyketide synthase (PKS) pathway. However, the specific mechanism of PKS in fatty acid synthesis is still unclear. In this work, the functions of ORFA, ORFB, ORFC, and their individual functional domain genes on fatty acid synthesis were investigated through heterologous expression in Yarrowia lipolytica . The results showed that the expression of ORFA, ORFB, ORFC, and their individual functional domains all led to the increase of the very long-chain PUFA content (mainly eicosapentaenoic acid). Furthermore, the transcriptomic analysis showed that except for the 3-ketoacyl-ACP synthase (KS) domain of ORFB, the expression of an individual functional domain, including malonyl-CoA: ACP acyltransferase, 3-hydroxyacyl-ACP dehydratase (DH), 3-ketoacyl-ACP reductase, and KS domains of ORFA, acyltransferase domains of ORFB, and two DH domains of ORFC resulted in upregulation of the tricarboxylic acid cycle and pentose phosphate pathway, downregulation of the triacylglycerol biosynthesis, fatty acid synthesis pathway, and β-oxidation in Yarrowia lipolytica . These results provide a theoretical basis for revealing the function of PKS in fatty acid synthesis in Y. lipolytica and elucidate the possible mechanism for PUFA biosynthesis.

Background: During the disease cycle, plant pathogenic fungi exhibit a morphological transition between hyphal growth (the phase of active infection) and the production of long-term survival structures that remain dormant during "overwintering." Verticillium dahliae is a major plant pathogen that produces heavily melanized microsclerotia (MS) that survive in the soil for 14 or more years. These MS are multicellular structures produced during the necrotrophic phase of the disease cycle. Polyketide synthases (PKSs) are responsible for catalyzing production of many secondary metabolites including melanin. While MS contribute to long-term survival, hyphal growth is key for infection and virulence, but the signaling mechanisms by which the pathogen maintains hyphal growth are unclear., Results: We analyzed the VdPKSs that contain at least one conserved domain potentially involved in secondary metabolism (SM), and screened the effect of VdPKS deletions in the virulent strain AT13. Among the five VdPKSs whose deletion affected virulence on cotton, we found that VdPKS9 acted epistatically to the VdPKS1-associated melanin pathway to promote hyphal growth. The decreased hyphal growth in VdPKS9 mutants was accompanied by the up-regulation of melanin biosynthesis and MS formation. Overexpression of VdPKS9 transformed melanized hyphal-type (MH-type) into the albinistic hyaline hyphal-type (AH-type), and VdPKS9 was upregulated in the AH-type population, which also exhibited higher virulence than the MH-type., Conclusions: We show that VdPKS9 is a powerful negative regulator of both melanin biosynthesis and MS formation in V. dahliae. These findings provide insight into the mechanism of how plant pathogens promote their virulence by the maintenance of vegetative hyphal growth during infection and colonization of plant hosts, and may provide novel targets for the control of melanin-producing filamentous fungi., (© 2022. The Author(s).)

The enzymes that comprise type II polyketide synthases (PKSs) are powerful biocatalysts that, once well-understood and strategically applied, could enable cost-effective and sustainable access to a range of pharmaceutically relevant molecules. Progress toward this goal hinges on gaining ample access to materials for in vitro characterizations and structural analysis of the components of these synthases. A central component of PKSs is the acyl carrier protein (ACP), which serves as a hub during the biosynthesis of type II polyketides. Herein, we share methods for accessing type II PKS ACPs via heterologous expression in E. coli . We also share how the installation of reactive and site-specific spectroscopic probes can be leveraged to study the conformational dynamics and interactions of type II PKS ACPs., (© 2022. The Author(s).)

Highly reducing polyketide synthases (HR-PKSs) produce structurally diverse polyketides (PKs). The PK diversity is constructed by a variety of factors, including the β-keto processing, chain length, methylation pattern, and relative and absolute configurations of the substituents. We examined the stereochemical course of the PK processing for the synthesis of polyhydroxy PKs such as phialotides, phomenoic acid, and ACR-toxin. Heterologous expression of a HR-PKS gene, a trans-acting enoylreductase gene, and a truncated non-ribosomal peptide synthetase gene resulted in the formation of a linear PK with multiple stereogenic centers. The absolute configurations of the stereogenic centers were determined by chemical degradation followed by comparison of the degradation products with synthetic standards. A stereochemical rule was proposed to explain the absolute configurations of other reduced PKs and highlights an error in the absolute configurations of a reported structure. The present work demonstrates that focused functional analysis of functionally related HR-PKSs leads to a better understanding of the stereochemical course., (© 2021 Wiley-VCH GmbH.)

Assembly-line polyketide synthases (PKSs) are large and complex enzymatic machineries with a multimodular architecture, typically encoded in bacterial genomes by biosynthetic gene clusters. Their modularity has led to an astounding diversity of biosynthesized molecules, many with medical relevance. Thus, understanding the mechanisms that drive PKS evolution is fundamental for both functional prediction of natural PKSs as well as for the engineering of novel PKSs. Here, we describe a repetitive genetic element in assembly-line PKS genes which appears to play a role in accelerating the diversification of closely related biosynthetic clusters. We named this element GRINS: genetic repeats of intense nucleotide skews. GRINS appear to recode PKS protein regions with a biased nucleotide composition and to promote gene conversion. GRINS are present in a large number of assembly-line PKS gene clusters and are particularly widespread in the actinobacterial genus Streptomyces While the molecular mechanisms associated with GRINS appearance, dissemination, and maintenance are unknown, the presence of GRINS in a broad range of bacterial phyla and gene families indicates that these genetic elements could play a fundamental role in protein evolution., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

Polyketide synthase (PKS) and non-ribosomal peptide synthetase (NRPS) multienzymes produce numerous high value metabolites. The protein subunits which constitute these megasynth(et)ases must undergo ordered self-assembly to ensure correct organisation of catalytic domains for the biosynthesis of a given natural product. Short amino acid regions at the N- and C-termini of each subunit, termed docking domains (DDs), often occur in complementary pairs, which interact to facilitate substrate transfer and maintain pathway fidelity. This review details all structurally characterised examples of NRPS and PKS DDs to date and summarises efforts to utilise DDs for the engineering of biosynthetic pathways., (© The Author(s) 2021. Published by Oxford University Press on behalf of Society of Industrial Microbiology and Biotechnology.)

Fungal polyketide synthases play important and differential roles in synthesizing secondary metabolites and regulating several cell events, including asexual development, environmental adaptation, and pathogenicity. This study shows the important functions of a highly reducing polyketide synthase, Pks11, in Beauveria bassiana, a filamentous fungal insect pathogen used worldwide for pest biocontrol. The deletion of pks11 led to severe defects in conidial yields on different media and a decrease of 36.27% in the mean thickness of conidial cell wall under normal conditions. Compared with the wild-type, Δpks11 showed higher tolerance to oxidation and increased sensitivity to high temperature during colony growth. Moreover, the lack of pks11 caused a decrease in conidial germination after exposure to UV radiation but did not affect the virulence of B. bassiana against Galleria mellonella larvae via typical cuticle infection. These findings concurred with the alteration in the transcript levels of some phenotype-related genes. These data suggested that pks11 played vital roles in the asexual development, cell wall integrity, and fungal responses to oxidation, high temperature, and UV irradiation of B. bassiana., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Background: Polyketide synthases (PKSs) include ketone synthase (KS), acyltransferase (AT) and acyl carrier protein (ACP) domains to catalyse the elongation of polyketide chains. Some PKSs also contain ketoreductase (KR), dehydratase (DH) and enoylreductase (ER) domains as modification domains. Insertion, deletion or substitution of the catalytic domains may lead to the production of novel polyketide derivatives or to the accumulation of desired products. Epothilones are 16-membered macrolides that have been used as anticancer drugs. The substrate promiscuity of the module 4 AT domain of the epothilone PKS (EPOAT4) results in production of epothilone mixtures; substitution of this domain may change the ratios of epothilones. In addition, there are two dormant domains in module 9 of the epothilone PKS. Removing these redundant domains to generate a simpler and more efficient assembly line is a desirable goal., Results: The substitution of module 4 drastically diminished the activity of epothilone PKS. However, with careful design of the KS-AT linker and the post-AT linker, replacing EPOAT4 with EPOAT2, EPOAT6, EPOAT7 or EPOAT8 (specifically incorporating methylmalonyl-CoA (MMCoA)) significantly increased the ratio of epothilone D (4) to epothilone C (3) (the highest ratio of 4:3 = 4.6:1), whereas the ratio of 4:3 in the parental strain Schlegelella brevitalea 104-1 was 1.4:1. We also obtained three strains by swapping EPOAT4 with EPOAT3, EPOAT5, or EPOAT9, which specifically incorporate malonyl-CoA (MCoA). These strains produced only epothilone C, and the yield was increased by a factor of 1.8 compared to that of parental strain 104-1. Furthermore, mutations of five residues in the AT domain identified Ser310 as the critical factor for MMCoA recognition in EPOAT4. Then, the mutation of His308 to valine or tyrosine combined with the mutation of Phe310 to serine further altered the product ratios. At the same time, we successfully deleted the inactive module 9 DH and ER domains and fused the ΨKR domain with the KR domain through an ~ 25-residue linker to generate a productive and simplified epothilone PKS., Conclusions: These results suggested that the substitution and deletion of catalytic domains effectively produces desirable compounds and that selection of the linkers between domains is crucial for maintaining intact PKS catalytic activity.

Bacterial modular type I polyketide synthases (PKSs) are complex multidomain assembly line proteins that produce a range of pharmaceutically relevant molecules with a high degree of stereochemical control. Due to their colinear properties, they have been considerable targets for rational biosynthetic pathway engineering. Among the domains harbored within these complex assembly lines, ketoreductase (KR) domains have been extensively studied with the goal of altering their stereoselectivity by site-directed mutagenesis, as they confer much of the stereochemical complexity present in pharmaceutically active reduced polyketide scaffolds. Here we review all efforts to date to perform site-directed mutagenesis on PKS KRs, most of which have been done in the context of excised KR domains on model diffusible substrates such as β-keto N-acetyl cysteamine thioesters. We also discuss the challenges around translating the findings of these studies to alter stereocontrol in the context of a complex multidomain enzymatic assembly line., (© 2020 Wiley-VCH GmbH.)

Eicosapentaenoic acid (EPA) is an essential nutritional supplement for human health. The most prominent dietary source of EPA is fish oil, which is unsustainable because of the decline in fishery resources and serious environmental pollution. Alternatively, a heterologous polyketide synthase pathway for EPA biosynthesis was assembled in Thraustochytrid Aurantiochytrium . A 2A peptide-based facile assembly platform that can achieve multigene expression as a polycistron was first established. The platform was then applied to express the EPA biosynthetic gene cluster from Shewanella japonica in Aurantiochytrium . In the shake flask fermentation, the lipid and PUFA yields of the mutant were increased by 26.9 and 36.0%, respectively, and led to about 5-fold increase of the EPA yield. The final EPA titer reached 2.7 g/L in fed-batch fermentation. This study provides a novel metabolic engineering strategy to regulate the EPA ratio in microalgal oil for human nutritional supplementation.

Heterocysts are the specialized cells for nitrogen fixation in some filamentous cyanobacteria. To protect the oxygen labile nitrogen fixing enzyme, nitrogenase, heterocysts keep their inner environment microoxic by developing layers of barrier on the outside of their outer membranes. Heterocyst specific glycolipids (Hgls) are constituents of the layer of barrier and amphipathic compounds, synthesized from a very long chain fatty alcohol as a hydrophobic tail and a sugar as a polar head. In the model heterocystous cyanobacterium Anabaena sp. PCC 7120, Hgls are made of fatty alcohol with 26 carbons and a glucose, linked by an ether bond in alpha configuration. The fatty alcohol is synthesized via reactions of a polyketide synthase, HglE A . In Anabaena sp. PCC 7120, another polyketide synthase HglE2 shared more than 50% identity in an amino acid sequence with HglE A and is expected to be involved in Hgls synthesis. However, no direct evidence has been reported. Here, we experimentally show that HglE A is the contributor of Hgls synthesis, and that HglE2 is not involved in the development of the heterocyst specific glycolipid layer.

In the present study, we characterized the potential toxin genes for polyketide synthase (PKS) and saxitoxin (STX) biosynthesis using the transcriptomes of two non-STX producing dinoflagellates Amphidinium carterae and Prorocentrum micans. RNA sequencing revealed 94 and 166 PKS contigs in A. carterae and P. micans, respectively. We first detected type III PKS, which was closely related to bacteria. In addition, dozens of homologs of 20 STX biosynthesis genes were identified. Interestingly, the core STX-synthesizing genes sxtA and sxtB were only found in P. micans, whereas sxtD was detected in A. carterae alone. Bioinformatic analysis showed that the first two core genes (sxtA and sxtG) had a low sequence similarity (37.0-67.6%) and different domain organization compared to those of other toxigenic dinoflagellates, such as Alexandrium pacificum. These might result in the breakdown of the initial reactions in STX production and ultimately the loss of the ability to synthesize the toxins in both dinoflagellates. Our findings suggest that toxin-related PKS and sxt genes are commonly found in non-STX producing dinoflagellates. In addition to their involvement in the synthesis of toxins, our result indicates that genes may also have other molecular metabolic functions., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Major phenotypic innovations in social amoeba evolution occurred at the transition between the Polysphondylia and group 4 Dictyostelia, which comprise the model organism Dictyostelium discoideum, such as the formation of a new structure, the basal disk. Basal disk differentiation and robust stalk formation require the morphogen DIF-1, synthesized by the polyketide synthase StlB, the des-methyl-DIF-1 methyltransferase DmtA, and the chlorinase ChlA, which are conserved throughout Dictyostelia. To understand how the basal disk and other innovations evolved in group 4, we sequenced and annotated the Polysphondylium violaceum (Pvio) genome, performed cell type-specific transcriptomics to identify cell-type marker genes, and developed transformation and gene knock-out procedures for Pvio. We used the novel methods to delete the Pvio stlB gene. The Pvio stlB- mutants formed misshapen curly sorogens with thick and irregular stalks. As fruiting body formation continued, the upper stalks became more regular, but structures contained 40% less spores. The stlB- sorogens overexpressed a stalk gene and underexpressed a (pre)spore gene. Normal fruiting body formation and sporulation were restored in Pvio stlB- by including DIF-1 in the supporting agar. These data indicate that, although conserved, stlB and its product(s) acquired both a novel role in the group 4 Dictyostelia and a role opposite to that in its sister group., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Type I polyketide synthases (T1PKSs) are one of the most extensively studied PKSs, which can act either iteratively or via an assembly-line mechanism. Domains in the T1PKSs can readily be predicted by computational tools based on their highly conserved sequences. However, to distinguish between iterative and noniterative at the module level remains an overwhelming challenge, which may account for the seemingly biased distribution of T1PKSs in fungi and bacteria: small iterative monomodular T1PKSs that are responsible for the enormously diverse fungal natural products exist almost exclusively in fungi. Here we report the discovery of iterative T1PKSs that are unexpectedly both abundant and widespread in Streptomyces Seven of 11 systematically selected T1PKS monomodules from monomodular T1PKS biosynthetic gene clusters (BGCs) were experimentally confirmed to be iteratively acting, synthesizing diverse branched/nonbranched linear intermediates, and two of them produced bioactive allenic polyketides and citreodiols as end products, respectively. This study indicates the huge potential of iterative T1PKS BGCs from streptomycetes in the discovery of novel polyketides., Competing Interests: The authors declare no competing interest.

The proteins of trans -acyltransferase modular polyketide synthases (PKSs) self-organize into assembly lines, enabling the multienzyme biosynthesis of complex organic molecules. Docking domains comprised of ∼25 residues at the C- and N-termini of these polypeptides ( C DDs and N DDs) help drive this association through the formation of four-helix bundles. Molecular connectors like these are desired in synthetic contexts, such as artificial biocatalytic systems and biomaterials, to orthogonally join proteins. Here, the ability of six C DD/ N DD pairs to link non-PKS proteins is examined using green fluorescent protein (GFP) variants. As observed through size-exclusion chromatography and Förster resonance energy transfer (FRET), matched but not mismatched pairs of Venus+ C DD and N DD+mTurquoise2 fusion proteins associate with low micromolar affinities.

Intermediates in aromatic amino acid biosynthesis can serve as substrates for the synthesis of bioactive compounds. In this study we used two intermediates in the shikimate pathway of Escherichia coli , chorismate and anthranilate, to synthesize three bioactive compounds: 4-hydroxycoumarin (4-HC), 2,4-dihydroxyquinoline (DHQ), and 4-hydroxy-1-methyl-2(1H)-quinolone (NMQ). We introduced genes for the synthesis of salicylic acid from chorismate to supply the substrate for 4-HC and the gene encoding N -methyltransferase for the synthesis of N -methylanthranilate from anthranilate. Polyketide synthases and coenzyme (Co)A ligases were tested to determine the optimal combination of genes for the synthesis of each compound. We also tested several constructs and identified the best one for increasing levels of endogenous substrates for chorismate, anthranilate, and malonyl-CoA. With the use of these strategies, 255.4 mg/L 4-HC, 753.7 mg/L DHQ, and 17.5 mg/L NMQ were synthesized. This work provides a basis for the synthesis of diverse coumarin and quinoline derivatives with potential medical applications.

We investigated potential biosynthetic pathways of long chain alkenols (LCAs), long chain alkyl diols (LCDs), and long chain hydroxy fatty acids (LCHFAs) in Nannochloropsis oceanica and Nannochloropsis gaditana, by combining culturing experiments with genomic and transcriptomic analyses. Incubation of Nannochloropsis spp. in the dark for 1 week led to significant increases in the cellular concentrations of LCAs and LCDs in both species. Consistently, 13C-labelled substrate experiments confirmed that both LCA and LCD were actively produced in the dark from C14-18 fatty acids by either condensation or elongation/hydroxylation, although no enzymatic evidence was found for the former pathway. Nannochloropsis spp. did, however, contain (i) multiple polyketide synthases (PKSs) including one type (PKS-Clade II) that might catalyze incomplete fatty acid elongations leading to the formation of 3-OH-fatty acids, (ii) 3-hydroxyacyl dehydratases (HADs), which can possibly form Δ2/Δ3 monounsaturated fatty acids, and (iii) fatty acid elongases (FAEs) that could elongate 3-OH-fatty acids and Δ2/Δ3 monounsaturated fatty acids to longer products. The enzymes responsible for reduction of the long chain fatty acids to LCDs and LCAs are, however, unclear. A putative wax ester synthase/acyl coenzyme A (acyl-CoA): diacylglycerol acyltransferase is likely to be involved in the esterification of LCAs and LCDs in the cell wall. Our data thus provide useful insights in predicting the biosynthetic pathways of LCAs and LCDs in phytoplankton suggesting a key role of FAE and PKS enzymes., (� The Author(s) 2019. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Deflecting biomineralized crystals attached to vestibular hair cells are necessary for maintaining balance. Zebrafish (Danio rerio) are useful organisms to study these biomineralized crystals called otoliths, as many required genes are homologous to human otoconial development. We sought to identify and characterize the causative gene in a trio of homozygous recessive mutants, no content (nco) and corkscrew (csr), and vanished (vns), which fail to develop otoliths during early ear development. We show that nco, csr, and vns have potentially deleterious mutations in polyketide synthase (pks1), a multi-modular protein that has been previously implicated in biomineralization events in chordates and echinoderms. We found that Otoconin-90 (Oc90) expression within the otocyst is diffuse in nco and csr; therefore, it is not sufficient for otolith biomineralization in zebrafish. Similarly, normal localization of Otogelin, a protein required for otolith tethering in the otolithic membrane, is not sufficient for Oc90 attachment. Furthermore, eNOS signaling and Endothelin-1 signaling were the most up- and down-regulated pathways during otolith agenesis in nco, respectively. Our results demonstrate distinct processes for otolith nucleation and biomineralization in vertebrates and will be a starting point for models that are independent of Oc90-mediated seeding. This study will serve as a basis for investigating the role of eNOS signaling and Endothelin-1 signaling during otolith formation., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Modularity is a fundamental property of megasynthases such as polyketide synthases (PKSs). In this study, we exploit the close resemblance between PKSs and animal fatty acid synthase (FAS) to re-engineer animal FAS to probe the modularity of the FAS/PKS family. Guided by sequence and structural information, we truncate and dissect animal FAS into its components, and reassemble them to generate new PKS-like modules as well as bimodular constructs. The novel re-engineered modules resemble all four common types of PKSs and demonstrate that this approach can be a powerful tool to deliver products with higher catalytic efficiency. Our data exemplify the inherent plasticity and robustness of the overall FAS/PKS fold, and open new avenues to explore FAS-based biosynthetic pathways for custom compound design., (© 2018 The Protein Society.)

Polyketides are a structurally and functionally diverse family of bioactive natural products that have proven to be a rich source of pharmaceutical and agrochemical lead compounds. Many polyketides are biosynthesized by large multifunctional megaenzymes termed type I modular polyketide synthases (PKSs). These systems possess a distinctive assembly line-like architecture, comprising a series of linearly arranged, multidomain extension modules, housed in sequence within giant polypeptide chains. Due to their inherently modular structures, PKSs represent attractive targets for reengineering, enabling access to functionally optimized "nonnatural" natural products. In this chapter we describe methods for the molecular cloning, recombinant over-expression, and purification of intact PKS modules and multimodular PKS polypeptides. The usefulness of these methods is demonstrated by applying them to the study of the abyssomicin C PKS, a >1MDa multimodular synthase responsible for the biosynthesis of a polyketide antimicrobial lead compound., (© 2019 Elsevier Inc. All rights reserved.)

Malonyl-CoA is an important central metabolite for the production of diverse valuable chemicals including natural products, but its intracellular availability is often limited due to the competition with essential cellular metabolism. Several malonyl-CoA biosensors have been developed for high-throughput screening of targets increasing the malonyl-CoA pool. However, they are limited for use only in Escherichia coli and Saccharomyces cerevisiae and require multiple signal transduction steps. Here we report development of a colorimetric malonyl-CoA biosensor applicable in three industrially important bacteria: E. coli , Pseudomonas putida , and Corynebacterium glutamicum RppA, a type III polyketide synthase producing red-colored flaviolin, was repurposed as a malonyl-CoA biosensor in E. coli Strains with enhanced malonyl-CoA accumulation were identifiable by the colorimetric screening of cells showing increased red color. Other type III polyketide synthases could also be repurposed as malonyl-CoA biosensors. For target screening, a 1,858 synthetic small regulatory RNA library was constructed and applied to find 14 knockdown gene targets that generally enhanced malonyl-CoA level in E. coli These knockdown targets were applied to produce two polyketide (6-methylsalicylic acid and aloesone) and two phenylpropanoid (resveratrol and naringenin) compounds. Knocking down these genes alone or in combination, and also in multiple different E. coli strains for two polyketide cases, allowed rapid development of engineered strains capable of enhanced production of 6-methylsalicylic acid, aloesone, resveratrol, and naringenin to 440.3, 30.9, 51.8, and 103.8 mg/L, respectively. The malonyl-CoA biosensor developed here is a simple tool generally applicable to metabolic engineering of microorganisms to achieve enhanced production of malonyl-CoA-derived chemicals., Competing Interests: Conflict of interest statement: S.Y.L., D.Y., and S.M.Y. declare that the sRNA technology described here is patent filed including, but not limited to, KR 10-1575587, US 9388417, EP 13735942.8, CN 201380012767.X, KR 10-1690780, KR 10-1750855, US 15317939, CN 201480081132.X for potential commercialization. Additionally, S.Y.L. and D.Y. have conflict of interest as the RppA biosensor technology is of commercial interest and is patent filed including, but not limited to KR 10-2018-0066323.

Streptomyces cinnamonensis A495 is a variant of the monensin producer which instead of the native polyether antibiotic gives rise to antibiotic and anti-tumour shunt-product premonensin. Through the supplementation of the fermentation medium with suitable precursors, premonensin can be derivatized via the incorporation of new-to-nature extender units into the biosynthetic machinery. Polyketide extender units require activation, typically in form of coenzyme A-thioesters. These are membrane impermeable and thus in the past an artificial mimic was employed. Here, we show the use and preliminary characterization of a highly substrate promiscuous new enzyme for the endogenous thioester formation in a Streptomyces strain. These intracellularly activated alternative extender units are significantly better incorporated into premonensin than the synthetically activated counterparts., Significance and Impact of the Study: Polyketide natural products are of enormous relevance in medicine. The hit-rate in finding active compounds for the potential treatment of various diseases among this substance family of microbial origin is high. However, most polyketides require derivatization to render them suitable for the application. Of relevance in this field is the incorporation of artificial substances into the biogenesis of polyketides, hampered by both the microbial metabolism and the complexity of the enzymes involved. This manuscript describes the straightforward and selective biosynthetic incorporation of synthetic substances into a reduced polyketide and showcases a promising new enzyme to aid this purpose., (© 2018 The Society for Applied Microbiology.)

Polyketides are secondary metabolites with diverse biological activities. Polyketide synthases (PKS) are often encoded from genes clustered in the same genomic region. Functional analyses and genomic studies show that most fungi are capable of producing a repertoire of polyketides. We considered the potential of Ceratocystidaceae for producing polyketides using a comparative genomics approach. Our aims were to identify the putative polyketide biosynthesis gene clusters, to characterize them and predict the types of polyketide compounds they might produce. We used sequences from nineteen species in the genera, Ceratocystis, Endoconidiophora, Davidsoniella, Huntiella, Thielaviopsis and Bretziella, to identify and characterize PKS gene clusters, by employing a range of bioinformatics and phylogenetic tools. We showed that the genomes contained putative clusters containing a non-reducing type I PKS and a type III PKS. Phylogenetic analyses suggested that these genes were already present in the ancestor of the Ceratocystidaceae. By contrast, the various reducing type I PKS-containing clusters identified in these genomes appeared to have distinct evolutionary origins. Although one of the identified clusters potentially allows for the production of melanin, their functional characterization will undoubtedly reveal many novel and important compounds implicated in the biology of the Ceratocystidaceae., (Copyright © 2018 British Mycological Society. Published by Elsevier Ltd. All rights reserved.)

Polyketides are a large family of structurally complex natural products including compounds with important bioactivities. Polyketides are biosynthesized by polyketide synthases (PKSs), multienzyme complexes derived evolutionarily from fatty acid synthases (FASs). The focus of this review is to critically compare the properties of FASs with iterative aromatic PKSs, including type II PKSs and fungal type I nonreducing PKSs whose chemical logic is distinct from that of modular PKSs. This review focuses on structural and enzymological studies that reveal both similarities and striking differences between FASs and aromatic PKSs. The potential application of FAS and aromatic PKS structures for bioengineering future drugs and biofuels is highlighted.

The structural diversity and complexity of marine natural products have made them a rich and productive source of new bioactive molecules for drug development. The identification of these new compounds has led to extensive study of the protein constituents of the biosynthetic pathways from the producing microbes. Essential processes in the dissection of biosynthesis have been the elucidation of catalytic functions and the determination of 3D structures for enzymes of the polyketide synthases and nonribosomal peptide synthetases that carry out individual reactions. The size and complexity of these proteins present numerous difficulties in the process of going from gene to structure. Here, we review the problems that may be encountered at the various steps of this process and discuss some of the solutions devised in our and other labs for the cloning, production, purification, and structure solution of complex proteins using Escherichia coli as a heterologous host., (© 2018 Elsevier Inc. All rights reserved.)

Dinoflagellates are prolific producers of polyketide compounds, many of which are potent toxins with adverse impacts on human and marine animal health. To identify polyketide synthase (PKS) genes in the brevetoxin-producing dinoflagellate, Karenia brevis, we assembled a transcriptome from 595 million Illumina reads, sampled under different growth conditions. The assembly included 125,687 transcripts greater than 300 nt in length, with over half having >100× coverage. We found 121 transcripts encoding Type I ketosynthase (KS) domains, of which 99 encoded single KS domains, while 22 contained multiple KS domains arranged in 1-3 protein modules. Phylogenetic analysis placed all single domain and a majority of multidomain KSs within a monophyletic clade of protist PKSs. In contrast with the highly amplified single-domain KSs, only eight single-domain ketoreductase transcripts were found in the assembly, suggesting that they are more evolutionarily conserved. The multidomain PKSs were dominated by trans-acyltransferase architectures, which were recently shown to be prevalent in other algal protists. Karenia brevis also expressed several hybrid nonribosomal peptide synthetase (NRPS)/PKS sequences, including a burA-like sequence previously reported in a wide variety of dinoflagellates. This contrasts with a similarly deep transcriptome of Gambierdiscus polynesiensis, which lacked NRPS/PKS other than the burA-like transcript, and may reflect the presence of amide-containing polyketides in K. brevis and their absence from G. polynesiensis. In concert with other recent transcriptome analyses, this study provides evidence for both single domain and multidomain PKSs in the synthesis of polyketide compounds in dinoflagellates., (© 2017 The Authors Journal of Phycology published by Wiley Periodicals, Inc. on behalf of Phycological Society of America.)

The type I polyketide geldanamycin is a potent anti-tumor reagent. Its biosynthesis includes three steps: the biosynthesis of precursors, such as 3-amino-5-hydroxybenzoic acid (AHBA), the polyketide synthase (PKS) chain extension, and the post-PKS modifications. According to the genomic and transcriptomic analysis, the PKS chain extension was deduced to be the rate-limiting step for geldanamycin production in Streptomyces hygroscopicus XM201. In order to improve the expression of PKS genes, a strong endogenous promoter 5063p was obtained based on the transcriptomic analysis and XylE enzymatic assay. By replacing the native PKS promoter gdmA1p with 5063p, the expression of the PKS genes during geldanamycin fermentation was increased by 4-141-folds, and the geldanamycin yield was increased by 39%. Interestingly, AHBA feeding experiment showed that the supply of AHBA in turn become a new rate-limiting factor for geldanamycin production. Further combined overexpression of the 6-gene AHBA biosynthetic cassette and PKS genes increased the yield of geldanamycin by 88%, from 773 mg L -1 of the wild-type to 1450 mg L -1 in the derived strain. Our results suggested that improved expression of all PKS genes in a particular biosynthetic gene cluster is important for the yield increase of the corresponding polyketide natural product., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Parrot feathers contain red, orange, and yellow polyene pigments called psittacofulvins. Budgerigars are parrots that have been extensively bred for plumage traits during the last century, but the underlying genes are unknown. Here we use genome-wide association mapping and gene-expression analysis to map the Mendelian blue locus, which abolishes yellow pigmentation in the budgerigar. We find that the blue trait maps to a single amino acid substitution (R644W) in an uncharacterized polyketide synthase (MuPKS). When we expressed MuPKS heterologously in yeast, yellow pigments accumulated. Mass spectrometry confirmed that these yellow pigments match those found in feathers. The R644W substitution abolished MuPKS activity. Furthermore, gene-expression data from feathers of different bird species suggest that parrots acquired their colors through regulatory changes that drive high expression of MuPKS in feather epithelia. Our data also help formulate biochemical models that may explain natural color variation in parrots. VIDEO ABSTRACT., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The fungus Slafractonia leguminicola, the causal agent of blackpatch disease of legumes produces two mycotoxins slaframine and swainsonine, causing slobbers' symptoms and locoism of grazing animals, respectively. The genetics of this important fungus is poorly understood. This work aimed to develop a genetic transformation system and evaluate the efficacy of RNA interference (RNAi) in S. leguminicola. In this study, S. leguminicola was transformed using a PEG-mediated method with a fungal construct that carries a hygromycin resistance cassette. To assess the use of RNAi, a silencing construct pSilentPKS1-AS was constructed which includes inverted repeat transgenes of the polyketide synthase gene (pks1) that is involved in melanin biosynthesis. Transformation of S. leguminicola with the IRT pks1 vector decreased pks1 transcripts levels 82-92% in knockdown mutants when compared with the wild type and was accompanied with a reduction in melanin and swainsonine production. These results demonstrate that RNAi can be a useful tool for studying gene function in S. leguminicola.

Fungal secondary metabolites have many important biological roles and some, like the toxic polyketide aflatoxin, have been intensively studied at the genetic level. Complete sets of polyketide synthase (PKS) genes can now be identified in fungal pathogens by whole genome sequencing and studied in order to predict the biosynthetic potential of those fungi. The pine needle pathogen Dothistroma septosporum is predicted to have only three functional PKS genes, a small number for a hemibiotrophic fungus. One of these genes is required for production of dothistromin, a polyketide virulence factor related to aflatoxin, whose biosynthetic genes are dispersed across one chromosome rather than being clustered. Here we evaluated the evolution of the other two genes, and their predicted gene clusters, using phylogenetic and population analyses. DsPks1 and its gene cluster are quite conserved amongst related fungi, whilst DsPks2 appears to be novel. The DsPks1 protein was predicted to be required for dihydroxynaphthalene (DHN) melanin biosynthesis but functional analysis of DsPks1 mutants showed that D. septosporum produced mainly dihydroxyphenylalanine (DOPA) melanin, which is produced by a PKS-independent pathway. Although the secondary metabolites made by these two PKS genes are not known, comparisons between strains of D. septosporum from different regions of the world revealed that both PKS core genes are under negative selection and we suggest they may have important cryptic roles in planta., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Product template (PT) domains from fungal nonreducing polyketide synthases (NR-PKSs) are responsible for controlling the aldol cyclizations of poly-β-ketone intermediates assembled during the catalytic cycle. Our ability to understand the high regioselective control that PT domains exert is hindered by the inaccessibility of intrinsically unstable poly-β-ketones for in vitro studies. We describe here the crystallographic application of "atom replacement" mimetics in which isoxazole rings linked by thioethers mimic the alternating sites of carbonyls in the poly-β-ketone intermediates. We report the 1.8-Å cocrystal structure of the PksA PT domain from aflatoxin biosynthesis with a heptaketide mimetic tethered to a stably modified 4'-phosphopantetheine, which provides important empirical evidence for a previously proposed mechanism of PT-catalyzed cyclization. Key observations support the proposed deprotonation at C4 of the nascent polyketide by the catalytic His1345 and the role of a protein-coordinated water network to selectively activate the C9 carbonyl for nucleophilic addition. The importance of the 4'-phosphate at the distal end of the pantetheine arm is demonstrated to both facilitate delivery of the heptaketide mimetic deep into the PT active site and anchor one end of this linear array to precisely meter C4 into close proximity to the catalytic His1345. Additional structural features, docking simulations, and mutational experiments characterize protein-substrate mimic interactions, which likely play roles in orienting and stabilizing interactions during the native multistep catalytic cycle. These findings afford a view of a polyketide "atom-replaced" mimetic in a NR-PKS active site that could prove general for other PKS domains., Competing Interests: The authors declare no conflict of interest.

Dehydration reactions play a crucial role in the de novo biosynthesis of fatty acids and a wide range of pharmacologically active polyketide natural products with strong emphasis on human medicine. The type I polyketide synthase PpsC from Mycobacterium tuberculosis catalyzes key biosynthetic steps of lipid virulence factors phthiocerol dimycocerosates and phenolic glycolipids. Given the insolubility of the natural C 28 -C 30 fatty acyl substrate of the PpsC dehydratase (DH) domain, we investigated its structure-function relationships in the presence of shorter surrogate substrates. Since most enzymes belonging to the (R)-specific enoyl hydratase/hydroxyacyl dehydratase family conduct the reverse hydration reaction in vitro, we have determined the X-ray structures of the PpsC DH domain, both unliganded (apo) and in complex with trans-but-2-enoyl-CoA or trans-dodec-2-enoyl-CoA derivatives. This study provides for the first time a snapshot of dehydratase-ligand interactions following a hydration reaction. Our structural analysis allowed us to identify residues essential for substrate binding and activity. The structural comparison of the two complexes also sheds light on the need for long acyl chains for this dehydratase to carry out its function, consistent with both its in vitro catalytic behavior and the physiological role of the PpsC enzyme., (Copyright © 2017. Published by Elsevier Ltd.)