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A metabolome-guided screening approach in the novel myxobacterium Corallococcus sp. MCy9072 resulted in the isolation of the unprecedented natural product myxofacycline A, which features a rare isoxazole substructure. Identification and genomic investigation of additional producers alongside targeted gene inactivation experiments and heterologous expression of the corresponding biosynthetic gene cluster in the host Myxococcus xanthus DK1622 confirmed a noncanonical megaenzyme complex as the biosynthetic origin of myxofacycline A. Induced expression of the respective genes led to significantly increased production titers enabling the identification of six further members of the myxofacycline natural product family. Whereas myxofacyclines A-D display an isoxazole substructure, intriguingly myxofacyclines E and F were found to contain 4-pyrimidinole, a heterocycle unprecedented in natural products. Lastly, myxofacycline G features another rare 1,2-dihydropyrol-3-one moiety. In addition to a full structure elucidation, we report the underlying biosynthetic machinery and present a rationale for the formation of all myxofacyclines. Unexpectedly, an extraordinary polyketide synthase-nonribosomal peptide synthetase hybrid was found to produce all three types of heterocycle in these natural products.

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.

The standalone metallo-β-lactamase-type thioesterase (MβL-TE), belongs to the group V nonreducing polyketide synthase agene cluster, catalyzes the rate-limiting step of product releasing. Our work first investigated on the orthologous MβL-TEs from different origins to determine which nonconserved amino acid residues are important to the hydrolysis efficiency. A series of chimeric MβL-TEs were constructed by fragment swapping and site-directed mutagenesis, in vivo enzymatic assay showed that two nonconserved residues A19 and E75 (numbering in HyTE) were critical to the catalytic performance. Protein structure modeling suggested that these two residues are located in different areas of HyTE. A19 is on the entrance to the active sites, whereas E75 resides in the linker between the two β strands which hold the metal-binding sites. Combining with computational simulations and comparative enzymatic assay, different screening criteria were set up for selecting the variants on the two noncatalytic and nonconserved key residues to improve the catalytic activity. The rational design on A19 and E75 gave five candidates in total, two (A19F and E75Q) of which were thus found significantly improved the enzymatic performance of HyTE. The double-point mutant was constructed to further improve the activity, which was increased by 28.4-fold on product accumulation comparing to the wild-type HyTE. This study provides a novel approach for engineering on nonconserved residues to optimize enzymatic performance.

Nonribosomal peptide synthetase and polyketide synthase systems are home to complex enzymology and produce compounds of great therapeutic value. Despite this, they have continued to be difficult to characterize due to their substrates remaining enzyme-bound by a thioester bond. Here, we have developed a strategy to directly trap and characterize the thioester-bound enzyme intermediates and applied the strategy to the azinomycin biosynthetic pathway. The approach was initially applied in vitro to evaluate its efficacy and subsequently moved to an in situ system, where a protein of interest was isolated from the native organism to avoid needing to supply substrates. When the nonribosomal peptide synthetase AziA3 was isolated from Streptomyces sahachiroi, the capture strategy revealed AziA3 functions in the late stages of epoxide moiety formation of the azinomycins. The strategy was further validated in vitro with a nonribosomal peptide synthetase involved in colibactin biosynthesis. In the long term, this method will be utilized to characterize thioester-bound metabolites within not only the azinomycin biosynthetic pathway but also other cryptic metabolite pathways.

Expression of a nonribosomal peptide synthetase-nonreducing polyketide synthase hybrid gene pcr10109 from Penicillium crustosum PRB-2 in Aspergillus nidulans led to the accumulation of 4-hydroxy-6-(4-hydroxyphenyl)-α-pyrone (1). Adding para-hydroxybenzoic acid into the medium in which the overexpressing mutant is growing increased the product yield up to 5-fold. This strategy could be helpful for heterologous gene expression experiments requiring special substrates for product formation.

Although swarming motility and biofilms are opposed collective behaviors, both contribute to bacterial survival and host colonization. Pseudovibrio bacteria have attracted attention because they are part of the microbiome of healthy marine sponges. Two-thirds of Pseudovibrio genomes contain a member of a nonribosomal peptide synthetase-polyketide synthase gene cluster family, which is also found sporadically in Pseudomonas pathogens of insects and plants. After developing reverse genetics for Pseudovibrio, we isolated heptapeptides with an ureido linkage and related nonadepsipeptides we termed pseudovibriamides A and B, respectively. A combination of genetics and imaging mass spectrometry experiments showed heptapetides were excreted, promoting motility and reducing biofilm formation. In contrast to lipopeptides widely known to affect motility/biofilms, pseudovibriamides are not surfactants. Our results expand current knowledge on metabolites mediating bacterial collective behavior.

In recent years, there has been an increasing demand for the replacement of synthetic food colorants with naturally derived alternatives. Filamentous fungi are prolific producers of secondary metabolites including polyketide-derived pigments, many of which have not been fully characterized yet. During our ongoing investigations of black aspergilli, we noticed that Aspergillus homomorphus turned yellow when cultivated on malt extract agar plates. Chemical discovery guided by UV and MS led to the isolation of two novel yellow natural products, and their structures were elucidated as aromatic α-pyrones homopyrones A (1) and B (2) by HRMS and NMR. Combined investigations including retro-biosynthesis, genome mining, and gene deletions successfully linked both compounds to their related biosynthetic gene clusters. This demonstrated that homopyrones are biosynthesized by using cinnamoyl-CoA as the starter unit, followed by extension with three malonyl-CoA units, and lactonization to give the core hybrid backbone structure. The polyketide synthase AhpA includes a C-methylation domain, which however seems to be promiscuous since only 2 is C-methylated. Altogether, the homopyrones represent a rare case of hybrid phenylpropanoid- and polyketide-derived natural products in filamentous fungi. KEY POINTS: • Homopyrones represent a rare type of fungal polyketides synthesized from cinnamic-CoA. • CRISPR/Cas9 technology has been firstly applied in Aspergillus homomorphus.

Mupirocin is a clinically important antibiotic produced by Pseudomonas fluorescens NCIMB 10586 that is assembled by a complex trans-AT polyketide synthase. The polyketide fragment, monic acid, is esterified by a 9-hydroxynonanoic acid (9HN) side chain which is essential for biological activity. The ester side chain assembly is initialised from a 3-hydroxypropionate (3HP) starter unit attached to the acyl carrier protein (ACP) MacpD, but the fate of this species is unknown. Herein we report the application of NMR spectroscopy, mass spectrometry, chemical probes and in vitro assays to establish the remaining steps of 9HN biosynthesis. These investigations reveal a complex interplay between a novel iterative or "stuttering" KS-AT didomain (MmpF), the multidomain module MmpB and multiple ACPs. This work has important implications for understanding the late-stage biosynthetic steps of mupirocin and will be important for future engineering of related trans-AT biosynthetic pathways (e.g. thiomarinol). ispartof: ANGEWANDTE CHEMIE-INTERNATIONAL EDITION vol:61 issue:50 ispartof: location:Germany status: published

The loops of modular polyketide synthases (PKSs) serve diverse functions but are largely uncharacterized. They frequently contain amino acid repeats resulting from genetic events such as slipped-strand mispairing. Determining the tolerance of loops to amino acid changes would aid in understanding and engineering these multidomain molecule factories. Here, tandem repeats in the DNA encoding 949 modules within 129 cis-acyltransferase PKSs were catalogued, and the locations of the corresponding amino acids within the module were identified. The most frequently inserted interdomain loop corresponds with the updated module boundary immediately downstream of the ketosynthase (KS), while the loops bordering the dehydratase (DH) are nearly intolerant to such insertions. From the 949 modules, no repetitive sequence loop insertions are located within ACP, and only 2 reside within KS, indicating the sensitivity of these domains to alteration.

Polyketide synthase-terpenoid synthase (PKS-TPS) hybrid pathways for biosynthesis of unique sesquiterpenyl epoxy-cyclohexenoids (SECs) have been found to be widely distributed in plant pathogenic fungi. However, the natural and ecological functions of these pathways and their metabolites still remain cryptic. In this study, the whole PKS-TPS hybrid pathway in the predominant nematode-trapping fungus Arthrobotrys oligospora was first proposed according to all the intermediates and their derivatives from all the A. oligospora mutants with a deficiency in each gene involved in SEC biosynthesis. Most mutants displayed significantly increased trap formation which was correlated with alteration of the ammonia level. Further analysis revealed that the main metabolites involved in ammonia metabolism were largely increased in most mutants. However, significantly retarded colonization in soil were observed in most mutants compared to the wild-type strain due to significantly decreased antibacterial activities. Our results suggested that A. oligospora used the PKS-TPS hybrid pathway for fungal soil colonization via decreasing fungal nematode-capturing ability. This also provided solid evidence that boosting fungal colonization in soil was the secondary metabolite whose biosynthesis depended on a PKS-TPS hybrid pathway.

Notwithstanding the "one-module-one-elongation-cycle" paradigm of assembly line polyketide synthases (PKSs), some PKSs harbor modules that iteratively elongate their substrates through a defined number of cycles. While some insights into module iteration, also referred to as "stuttering", have been derived through in vivo and in vitro analysis of a few PKS modules, a general understanding of the mechanistic principles underlying module iteration remains elusive. This report serves as the first interrogation of a stuttering module from a trans-AT subfamily PKS that is also naturally split across two polypeptides. Previous work has shown that Module 5 of the NOCAP ( noc ardiosis a ssociated p olyketide) synthase iterates precisely three times in the biosynthesis of its polyketide product, resulting in an all-trans-configured triene moiety in the polyketide product. Here, we describe the intrinsic catalytic properties of this NOCAP synthase module. Through complementary experiments in vitro and in E. coli, the "split-and-stuttering" module was shown to catalyze up to five elongation cycles, although its dehydratase domain ceased to function after three cycles. Unexpectedly, the central olefinic group of this truncated product had a cis configuration. Our findings set the stage for further in-depth analysis of a structurally and functionally unusual PKS module with contextual biosynthetic plasticity.

Cytochalasins are a group of fungal secondary metabolites with diverse structures and bioactivities, including chaetoglobosin A production. Chaetoglobosin A is produced by Chaetomium globosum and has potential antifungal activity. Bioinformatics analysis of the chaetoglobosin A gene cluster (che) showed it that consists of nine open reading frames, including those encoding polyketide synthases (PKSs), PKS extender units, post-PKS modifications, and proposed regulators. Here, the role of the CgcheR regulator was investigated using gene disruption experiments. The CgcheR disruptant (ΔCgcheR) completely abolished the production of chaetoglobosin A, which was restored by the introduction of a copy of the wild-type CgcheR gene, suggesting that CgcheR is involved in chaetoglobosin A biosynthesis. A transcriptional analysis of the CgcheR disruptant indicated that CgCheR activates the transcription of chaetoglobosin biosynthetic genes in a pathway-specific manner. Furthermore, constitutive overexpression of CgcheR significantly improved the production of chaetoglobosin A from 52 to 260 mg/L. Surprisingly, CgcheR also played a critical role in sporulation; the CgcheR disruptant lost the ability to produce spores, suggesting that the regulator modulates cellular development. Our results not only shed light on the regulation of chaetoglobosin A biosynthesis, but also indicate a relationship between secondary metabolism and fungal morphogenesis.

Statins are effective cholesterol-lowering drugs. Lovastatin, one of the precursors of statins, is formed from dihydromonacolin L (DML), which is synthesized by lovastatin nonaketide synthase (LovB), with the assistance of a separate trans-acting enoyl reductase (LovC). A full DML synthesis comprises 8 polyketide synthetic cycles with about 35 steps. The assembling of the LovB–LovC complex, and the structural basis for the iterative and yet permutative functions of the megasynthase have remained a mystery. Here, we present the cryo-EM structures of the LovB–LovC complex at 3.60 Å and the core LovB at 2.91 Å resolution. The domain organization of LovB is an X-shaped face-to-face dimer containing eight connected domains. The binding of LovC laterally to the malonyl-acetyl transferase domain allows the completion of a L-shaped catalytic chamber consisting of six active domains. This architecture and the structural details of the megasynthase provide the basis for the processing of the intermediates by the individual catalytic domains. The detailed architectural model provides structural insights that may enable the re-engineering of the megasynthase for the generation of new statins., Biosynthesis of the statin precursor lovastatin depends on the LovB–LovC megasynthase complex. Here, the authors present cryoEM structures of LovB–LovC and core LovB, providing structural insights into the catalytic cycle underlying lovastatin production.

The prominent protein producing workhorse Trichoderma reesei secretes a typical yellow pigment that is synthesized by a gene cluster including two polyketide synthase encoding genes sor1 and sor2. Two transcription factors (YPR1 and YPR2) that are encoded in the same cluster have been shown to regulate the expression of the sor genes. However, the physiological relevance of the yellow pigment synthesis in T. reesei is not completely clear. In this study, a yellow pigment hyper-producer OEypr1 and three yellow pigment non-producers, OEypr1-sor1, Δypr1, and OEypr2, were constructed. Their phenotypic features in mycelial growth, conidiation, cell wall integrity, stress tolerance, and cellulase production were determined. Whereas hyperproduction of the yellow pigment caused significant defects in all the physiological aspects tested, the non-producers showed similar colony growth, but improved conidiation, maintenance of cell wall integrity, and stress tolerance compared to the control strain. Moreover, in contrast to the severely compromised extracellular cellobiohydrolase production in the yellow pigment hyperproducer, loss of the yellow pigment hardly affected induced cellulase gene expression. Our results demonstrate that interfering with the yellow pigment synthesis constitutes an engineering strategy to endow T. reesei with preferred features for industrial application.

2-Alkenyl-tetrahydropyrans belong to a rare class of natural products that exhibit broad antifungal activities. Their structural instability and rareness in nature have restrained their discovery and drug development. In this study, the heterologous expression of a single highly reducing polyketide synthase (HR-PKS, App1) from Trichoderma applanatum in Aspergillus nidulans leads to the formation of seven 2-alkenyl-tetrahydropyran derivatives including one known compound virensol C (1) and six new compounds (2–7). However, introducing App1 into Saccharomyces cerevisiae resulted in the identification of additional two 2-alkenyl-tetrahydropyrans lacking the hydroxyl or methoxyl group at the C-2 position (8 and 9). The structures of the isolated compounds were elucidated by extensive spectroscopic analysis using NMR and HR-ESI-MS.

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.

NFAT-133 is a Streptomyces-derived aromatic polyketide compound with immunosuppressive, antidiabetic, and antitrypanosomal activities. It inhibits transcription mediated by nuclear factor of activated T cells (NFAT), leading to the suppression of interleukin-2 expression and T cell proliferation. It also activates the AMPK pathway in L6 myotubes and increases glucose uptake. In addition to NFAT-133, a number of its congeners, e.g., panowamycins and benwamycins, have been identified. However, little is known about their modes of formation in the producing organisms. Through genome sequencing of Streptomyces pactum ATCC 27456, gene inactivation, and genetic complementation experiments, the biosynthetic gene cluster of NFAT-133 and its congeners has been identified. The cluster contains a highly disordered genetic organization of type I modular polyketide synthase genes with several genes that are necessary for the formation of the aromatic core unit and tailoring processes. In addition, a number of new analogs of NFAT-133 were isolated and their chemical structures elucidated. It is suggested that the heptaketide NFAT-133 is derived from an octaketide intermediate, TM-123. The current study shows yet another unusual biosynthetic pathway involving a noncanonical polyketide synthase assembly line to produce a group of small molecules with valuable bioactivities.

Aspergillus carbonarius is the major producer of ochratoxin A (OTA) among Aspergillus species, but the contribution of this secondary metabolite to fungal virulence has not been assessed. We characterized the functions and addressed the roles of three factors in the regulation of OTA synthesis and pathogenicity in A. carbonarius: LaeA, a transcriptional factor regulating the production of secondary metabolites; polyketide synthase, required for OTA biosynthesis; and glucose oxidase (GOX), regulating gluconic acid (GLA) accumulation and acidification of the host tissue during fungal growth. Deletion of laeA in A. carbonarius resulted in significantly reduced OTA production in colonized nectarines and grapes. The ∆laeA mutant was unable to efficiently acidify the colonized tissue, as a direct result of diminished GLA production, leading to attenuated virulence in infected fruit compared to the wild type (WT). The designed Acpks‐knockout mutant resulted in complete inhibition of OTA production in vitro and in colonized fruit. Interestingly, physiological analysis revealed that the colonization pattern of the ∆Acpks mutant was similar to that of the WT strain, with high production of GLA in the colonized tissue, suggesting that OTA accumulation does not contribute to A. carbonarius pathogenicity. Disruption of the Acgox gene inactivated GLA production in A. carbonarius, and this mutant showed attenuated virulence in infected fruit compared to the WT strain. These data identify the global regulator LaeA and GOX as critical factors modulating A. carbonarius pathogenicity by controlling transcription of genes important for fungal secondary metabolism and infection., LaeA and GOX are critical factors modulating A. carbonarius pathogenicity in fruits by controlling transcription of genes important for fungal secondary metabolism and infection; OTA does not contribute to A. carbonarius virulence.

As a unique structural moiety in natural products, cinnamoyl lipids (CLs), are proposed to be assembled by unusual type II polyketide synthases (PKSs). Herein, we demonstrate that the assembly of the CL compounds youssoufenes is accomplished by a PKS system that uniquely harbors three phylogenetically different ketosynthase/chain length factor (KS/CLF) complexes (YsfB/C, YsfD/E, and YsfJ/K). Through in vivo gene inactivation and in vitro reconstitution, as well as an intracellular tagged carrier-protein tracking (ITCT) strategy developed in this study, we successfully elucidated the isomerase-dependent ACP-tethered polyunsaturated chain elongation process. The three KS/CLFs were revealed to modularly assemble different parts of the youssoufene skeleton, during which benzene ring closure happens right after the formation of an ACP-tethered C18 polyene. Of note, the ITCT strategy could significantly contribute to the elucidation of other carrier-protein-dependent biosynthetic machineries.

The colinearity of canonical modular polyketide synthases, which creates a direct link between multienzyme structure and the chemical structure of the biosynthetic end-product, has become a cornerstone of knowledge-based genome mining. Here we report genetic and enzymatic evidence for the remarkable role of an enoylreductase in the polyketide synthase for azalomycin F biosynthesis. This internal enoylreductase domain, previously identified as acting only in the second of two chain extension cycles on an initial iterative module, is shown here also to catalyse enoylreduction in trans within the next module. The mechanism for this rare deviation from colinearity appears to involve direct cross-modular interaction of the reductase with the longer acyl chain, rather than backtransfer of the substrate into the iterative module, suggesting an additional and surprising plasticity in natural PKS assembly-line catalysis.

A gene cluster encoding a cryptic trans‐acyl transferase polyketide synthase (PKS) was identified in the genomes of Burkholderia gladioli BCC0238 and BCC1622, both isolated from the lungs of cystic fibrosis patients. Bioinfomatics analyses indicated the PKS assembles a novel member of the glutarimide class of antibiotics, hitherto only isolated from Streptomyces species. Screening of a range of growth parameters led to the identification of gladiostatin, the metabolic product of the PKS. NMR spectroscopic analysis revealed that gladiostatin, which has promising activity against several human cancer cell lines and inhibits tumor cell migration, contains an unusual 2‐acyl‐4‐hydroxy‐3‐methylbutenolide in addition to the glutarimide pharmacophore. An AfsA‐like domain at the C‐terminus of the PKS was shown to catalyze condensation of 3‐ketothioesters with dihydroxyacetone phosphate, thus indicating it plays a key role in polyketide chain release and butenolide formation., Genome mining enabled the discovery of gladiostatin, a novel glutarimide antibiotic from the cystic fibrosis associated bacterium Burkholderia gladioli. Gladiostatin contains an unusual 2‐acyl‐3‐hydroxy‐4‐methylbutenolide and exhibits promising activity against human cancer cells. The polyketide synthase responsible for its biosynthesis employs a novel mechanism for chain release, resulting in formation of a phosphorylated butenolide intermediate.

Polyketides are a large group of natural biomolecules that are normally produced by bacteria, fungi and plants. These molecules have clinical importance due to their anti-cancer, anti-microbial, anti-oxidant and anti-inflammatory properties. Polyketides are biosynthesized from units of acyl-CoA by different polyketide synthases (PKSs), which display wide diversity of functional domains and mechanisms of action between fungi and bacteria. Co-culture of different micro-organisms can produce novel products distinctive from those produced during single cultures. This study compared the new polyketides produced in such co-culture systems and discusses aspects of the cultivation systems, product structures and identification techniques. Current results indicate that the formation of new polyketides may be the result of activation of previously silent PKSs genes induced during co-culture. This review indicated a potential way to produce pure therapeutic polyketides by microbial fermentation and a potential way to develop functional foods and agricultural products using co-co-culture of different micro-organisms. It also pointed out a new perspective for studies on the process of functional foods, especially those involving multiple micro-organisms.

Anaerobic bacteria have only recently been recognized as a source of antibiotics; yet, the metabolic potential of Negativicutes (Gram‐negative staining Firmicutes) such as the oak‐associated Dendrosporobacter quercicolus has remained unknown. Genome mining of D. quercicolus and phylogenetic analyses revealed a gene cluster for a type II polyketide synthase (PKS) complex that belongs to the most ancestral enzyme systems of this type. Metabolic profiling, NMR analyses, and stable‐isotope labeling led to the discovery of a new family of anthraquinone‐type polyphenols, the dendrubins, which are diversified by acylation, methylation, and dimerization. Dendrubin A and B were identified as strong antibiotics against a range of clinically relevant, human‐pathogenic mycobacteria., A path of polyketide ancestry: Genome mining revealed an ancestral type II polyketide synthase (PKS) system of the oak‐associated anaerobic bacterium Dendrosporobacter quercicolus, a member of the underexplored class of Negativicutes. Metabolic profiling led to the discovery of a new family of aromatic polyketides (dendrubins) that exhibit potent activities against a range of clinically relevant pathogenic mycobacteria (see figure).

Lichen-forming fungi are known to produce a large number of secondary metabolites. Some metabolites are deposited in the cortical layer of the lichen thallus where they exert important ecological functions, such as UV filtering. The fact that closely related lineages of lichen-forming fungi can differ in cortical chemistry suggests that natural product biosynthesis in lichens can evolve independent from phylogenetic constraints. Usnic acid is one of the major cortical pigments in lichens. Here we used a comparative genomic approach on 46 lichen-forming fungal species of the Lecanoromycetes to elucidate the biosynthetic gene content and evolution of the gene cluster putatively responsible for the biosynthesis of usnic acid. Whole-genome sequences were gathered from taxa belonging to different orders and families of Lecanoromycetes, where Parmeliaceae is the most well-represented taxon, and analyzed with a variety of genomic tools. The highest number of biosynthetic gene clusters was found in Evernia prunastri, Pannoparmelia angustata, and Parmotrema austrosinense, respectively, and lowest in Canoparmelia nairobiensis, Bulbothrix sensibilis, and Hypotrachyna scytodes. We found that all studied species producing usnic acid contain the putative usnic acid biosynthetic gene cluster, whereas the cluster was absent in all genomes of species lacking usnic acid. The absence of the gene cluster was supported by an additional unsuccessful search for ß-ketoacylsynthase, the most conserved domain of the gene cluster, in the genomes of species lacking usnic acid. The domain architecture of this PKS cluster—homologous to the already known usnic acid PKS cluster (MPAS) and CYT450 (MPAO)—varies within the studied species, whereas the gene arrangement is highly similar in closely related taxa. We hypothesize that the ancestor of these lichen-forming fungi contained the putative usnic acid producing PKS cluster and that the gene cluster was lost repeatedly during the evolution of these groups. Our study provides insight into the genomic adaptations to the evolutionary success of these lichen-forming fungal species and sets a baseline for further exploration of biosynthetic gene content and its evolutionary significance.

The conspicuous bright golden to orange-reddish coloration of species of the basidiomycete genus Laetiporus is a hallmark feature of their fruiting bodies, known among mushroom hunters as the “chicken of the woods”. This report describes the identification of an eight-domain mono-modular highly reducing polyketide synthase as sole enzyme necessary for laetiporic acid biosynthesis. Heterologous pathway reconstitution in both Aspergillus nidulans and Aspergillus niger verified that LpaA functions as a multi-chain length polyene synthase, which produces a cocktail of laetiporic acids with a methyl-branched C26–C32 main chain. Laetiporic acids show a marked antifungal activity on Aspergillus protoplasts. Given the multiple products of a single biosynthesis enzyme, our work underscores the diversity-oriented character of basidiomycete natural product biosynthesis.

Modular polyketide synthases (PKSs) produce complex, bioactive secondary metabolites in assembly line-like multistep reactions. Longstanding efforts to produce novel, biologically active compounds by recombining intact modules to new modular PKSs have mostly resulted in poorly active chimeras and decreased product yields. Recent findings demonstrate that the low efficiencies of modular chimeric PKSs also result from rate limitations in the transfer of the growing polyketide chain across the noncognate module:module interface and further processing of the non-native polyketide substrate by the ketosynthase (KS) domain. In this study, we aim at disclosing and understanding the low efficiency of chimeric modular PKSs and at establishing guidelines for modular PKSs engineering. To do so, we work with a bimodular PKS testbed and systematically vary substrate specificity, substrate identity, and domain:domain interfaces of the KS involved reactions. We observe that KS domains employed in our chimeric bimodular PKSs are bottlenecks with regards to both substrate specificity as well as interaction with the acyl carrier protein (ACP). Overall, our systematic study can explain in quantitative terms why early oversimplified engineering strategies based on the plain shuffling of modules mostly failed and why more recent approaches show improved success rates. We moreover identify two mutations of the KS domain that significantly increased turnover rates in chimeric systems and interpret this finding in mechanistic detail.

The indolizidine alkaloid swainsonine (SW) is a deadly mycotoxin to livestock that can be produced by different plant-associated fungi, including the endophytic entomopathogenic fungi Metarhizium species. The SW biosynthetic gene cluster has been identified but the genetic mechanism of SW biosynthesis remains obscure. To unveil the SW biosynthetic pathway, we performed gene deletions in M. robertsii, heterologous expression of a core biosynthetic gene, substrate feedings, mass spectrometry, and bioassay analyses in this study. It was unveiled that SW is produced via a multibranched pathway by the hybrid nonribosomal peptide-polyketide synthase (NRPS-PKS) gene cluster in M. robertsii. The precursor pipecolic acid can be converted from lysine by both the SW biosynthetic cluster and the unclustered genes such as lysine cyclodeaminase. The hybrid NRPS-PKS enzyme produces three intermediates with and without domain skipping. Intriguingly, the biosynthetic process is coupled with the cis to trans nonenzymatic epimerization of C1-OH for both hydroxyl- and dihydroxyl-indolizidine intermediates. We also found that SW production was dispensable for fungal colonization of plants and infection of insect hosts. Functional characterization of the SW biosynthetic genes in this study may benefit the safe use of Metarhizium fungi as insect biocontrol agents and the management of livestock pastures from SW contamination by genetic manipulation of the toxin-producing fungi.

Many microbial secondary metabolites are produced by multienzyme complexes comprising nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs). The ketosynthase (KS) domains of polyketide synthase normally catalyze the decarboxylative Claisen condensation of acyl and malonyl blocks to extend the polyketide chain. However, the terminal KS domain in tenuazonic acid synthetase 1 (TAS1) from the fungus Pyricularia oryzae conducts substrate cyclization. Here, we report on the unique features of the KS domain in TAS1. We observed that this domain is monomeric, not dimeric as is typical for KSs. Analysis of a 1.68-Å resolution crystal structure suggests that the substrate cyclization is triggered via proton abstraction from the active methylene moiety in the substrate by a catalytic His-322 residue. Additionally, we show that TAS1 KS promiscuously accepts aminoacyl substrates and that this promiscuity can be increased by a single amino acid substitution in the substrate-binding pocket of the enzyme. These findings provide insight into a KS domain that accepts the amino acid–containing substrate in an NRPS–PKS hybrid enzyme and provide hints to the substrate cyclization mechanism performed by the KS domain in the biosynthesis of the mycotoxin tenuazonic acid.

Caves are considered to be extreme and challenging environments. It is believed that the ability of microorganisms to produce secondary metabolites enhances their survivability and adaptiveness in the energy-starved cave environment. Unfortunately, information on the genetic potential for the production of secondary metabolites, such as polyketides and nonribosomal peptides, is limited. In the present study, we aimed to identify and characterize genes responsible for the production of secondary metabolites in the microbial community of one of the deepest caves in the world, Krubera-Voronja Cave (43.4184 N 40.3083 E, Western Caucasus). The analysed sample materials included sediments, drinkable water from underground camps, soil and clay from the cave walls, speleothems and coloured spots from the cave walls. The type II polyketide synthases (PKSs) ketosynthases α and β and the adenylation domains of nonribosomal peptide synthetases (NRPSs) were investigated using a metagenomic approach. Taxonomic diversity analysis showed that most PKS sequences could be attributed to Actinobacteria followed by unclassified bacteria and Acidobacteria, while the NRPS sequences were more taxonomically diverse and could be assigned to Proteobacteria, Actinobacteria, Cyanobacteria, Firmicutes, Chloroflexi, etc. Only three putative metabolites could be predicted: an angucycline group polyketide, a massetolide A-like cyclic lipopeptide and a surfactin-like lipopeptide. The absolute majority of PKS and NRPS sequences showed low similarity with the sequences of the reference biosynthetic pathways, suggesting that these sequences could be involved in the production of novel secondary metabolites.

Complex polyketides are typically associated with microbial metabolism. Here, we report that animals also make complex, microbe-like polyketides. We show there is a widespread branch of fatty acid synthase- (FAS)-like polyketide synthase (PKS) proteins, which sacoglossan animals use to synthesize complex products. The purified sacogolassan protein EcPKS1 uses only methylmalonyl-CoA as a substrate, otherwise unknown in animal lipid metabolism. Sacoglossans are sea slugs, some of which eat algae, digesting the cells but maintaining functional chloroplasts. Here, we provide evidence that polyketides support this unusual photosynthetic partnership. The FAS-like PKS family represents an uncharacterized branch of polyketide and fatty acid metabolism, encoding a large diversity of biomedically relevant animal enzymes and chemicals awaiting discovery. The biochemical characterization of an intact animal polyketide biosynthetic enzyme opens the door to understanding the immense untapped metabolic potential of metazoans., Complex polyketides are usually produced by microbes, whereas the origin of polyketides found in animals remained unknown. This study shows that sacoglossan animals, such as sea slugs, employ fatty acid synthase-like proteins to produce microbe-like polyketides.

Pathogenic bacteria of the Burkholderia pseudomallei group cause severe infectious diseases such as glanders and melioidosis. Malleicyprols were identified as important bacterial virulence factors, yet the biosynthetic origin of their cyclopropanol warhead has remained enigmatic. By a combination of mutational analysis and metabolomics we found that sulfonium acids, dimethylsulfoniumpropionate (DMSP) and gonyol, known as osmolytes and as crucial components in the global organosulfur cycle, are key intermediates en route to the cyclopropanol unit. Functional genetics and in vitro analyses uncover a specialized pathway to DMSP involving a rare prokaryotic SET‐domain methyltransferase for a cryptic methylation, and show that DMSP is loaded onto the NRPS‐PKS hybrid assembly line by an adenylation domain dedicated to zwitterionic starter units. Then, the megasynthase transforms DMSP into gonyol, as demonstrated by heterologous pathway reconstitution in E. coli., Sulfur cycle meets pathogen. Sulfonium acids known from global sulfur cycles were elucidated as the biosynthetic origin of the cyclopropanol warhead of malleicyprols, virulence factors of human‐pathogenic bacteria. The pathway involves a specialized S‐methyl transferase to form DMSP, which is activated by a zwitterion‐specific adenylation domain and transformed into the key intermediate gonyol.

Aims Red yeast rice (RYR), produced by inoculating Monascus strains to steamed rice, contains many kinds of physiologically bioactive compounds, among which monacolin K can be used as an antihypercholesterolaemic agent. However, RYR can be polluted by the mycotoxin citrinin, which has nephrotoxic and hepatotoxic activities. To avoid the risk of citrinin contamination in Monascus fermented products, it is important to screen for Monascus strains that produce no or low citrinin. Methods and results Five autochthonous Monascus strains with high-yield monacolin K and undetectable citrinin were obtained using high-performance liquid chromatography (HPLC). All five strains were identified as Monascus ruber based on Genealogical Concordance Phylogenetic Species Recognition criteria. Polymerase chain reaction revealed that citrinin polyketide synthase (pksCT) gene was found in these strains, but transcriptional regulator (ctnA) was not found. Conclusions Five strains are potential strains for producing high-quality RYR. The distribution of the pksCT gene was not restricted to Monascus purpureus and Monascus sanguineus, and M. ruber strains were diverse in pksCT and ctnA genes. Significance and impact of the study The integration of citrinin HPLC analysis and pksCT and ctnA genes amplification could provide a complementary approach in valuable Monascus strains screening.

Polyketide synthase (PKS) engineering is an attractive method to generate new molecules such as commodity, fine and specialty chemicals. A significant challenge in PKS design is engineering a partially reductive module to produce a saturated β-carbon through a reductive loop exchange. In this work, we sought to establish that chemoinformatics, a field traditionally used in drug discovery, could provide a viable strategy to reductive loop exchanges. We first introduced a set of donor reductive loops of diverse genetic origin and chemical substrate structures into the first extension module of the lipomycin PKS (LipPKS1). Product titers of these engineered unimodular PKSs correlated with atom pair chemical similarity between the substrate of the donor reductive loops and recipient LipPKS1, reaching a titer of 165 mg/L of short chain fatty acids produced by Streptomyces albus J1074 harboring these engineered PKSs. Expanding this method to larger intermediates requiring bimodular communication, we introduced reductive loops of divergent chemosimilarity into LipPKS2 and determined triketide lactone production. Collectively, we observed a statistically significant correlation between atom pair chemosimilarity and production, establishing a new chemoinformatic method that may aid in the engineering of PKSs to produce desired, unnatural products.

Aromatic polyketides have attractive biological activities and pharmacological properties. Different from other polyketides, aromatic polyketides are characterized by their polycyclic aromatic structure. The biosynthesis of aromatic polyketides is usually accomplished by the type II polyketide synthases (PKSs), which produce highly diverse polyketide chains by sequential condensation of the starter units with extender units, followed by reduction, cyclization, aromatization and tailoring reactions. Recently, significant progress has been made in characterization and engineering of type II PKSs to produce novel products and improve product titers. In this review, we briefly summarize the architectural organizations and genetic contributions of PKS genes to provide insight into the biosynthetic process. We then review the most recent progress in engineered biosynthesis of aromatic polyketides, with emphasis on generating novel molecular structures. We also discuss the current challenges and future perspectives in the rational engineering of type II PKSs for large scale production of aromatic polyketides.

Neoantimycins are anticancer compounds of 15-membered ring antimycin-type depsipeptides. They are biosynthesized by a hybrid multimodular protein complex of nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS), typically from the starting precursor 3-formamidosalicylate. Examining fermentation extracts of Streptomyces conglobatus, here we discovered four new neoantimycin analogs, unantimycins B-E, in which 3-formamidosalicylates are replaced by an unusual 3-hydroxybenzoate (3-HBA) moiety. Unantimycins B-E exhibited levels of anticancer activities similar to those of the chemotherapeutic drug cisplatin in human lung cancer, colorectal cancer, and melanoma cells. Notably, they mostly displayed no significant toxicity toward noncancerous cells, unlike the serious toxicities generally reported for antimycin-type natural products. Using site-directed mutagenesis and heterologous expression, we found that unantimycin productions are correlated with the activity of a chorismatase homolog, the nat-hyg5 gene, from a type I PKS gene cluster. Biochemical analysis confirmed that the catalytic activity of Nat-hyg5 generates 3-HBA from chorismate. Finally, we achieved selective production of unantimycins B and C by engineering a chassis host. On the basis of these findings, we propose that unantimycin biosynthesis is directed by the neoantimycin-producing NRPS-PKS complex and initiated with the starter unit of 3-HBA. The elucidation of the biosynthetic unantimycin pathway reported here paves the way to improve the yield of these compounds for evaluation in oncotherapeutic applications.

Tuberculosis, a major global health concern, and its drug development toward the disease are too devastating to meet the clinical demands. The present work emphasizes a detailed QSAR study using QSARINS which developed descriptors favoring an excellent model equation. The best model equation generated has four variables namely AlogP, ATSc4, mindssC, and MDEC23 with statistical values

Polyketide synthases (PKSs) use simple extender units to synthesize complex natural products. A fundamental question is how different extender units are site‐specifically incorporated into the growing polyketide. Here we established phoslactomycin (Pn) PKS, which incorporates malonyl‐ and ethylmalonyl‐CoA, as an in vitro model to study substrate specificity. We combined up to six Pn PKS modules with different termination sites for the controlled release of tetra‐, penta‐ and hexaketides, and challenged these systems with up to seven different extender units in competitive assays to test for the specificity of Pn modules. While malonyl‐CoA modules of Pn PKS exclusively accept their natural substrate, the ethylmalonyl‐CoA module PnC tolerates different α‐substituted derivatives, but discriminates against malonyl‐CoA. We show that the ratio of extender transacylation to hydrolysis controls incorporation in PnC, thus explaining site‐specific selectivity and promiscuity in the natural context of Pn PKS., Going to any length: Polyketide synthases produce compounds of high structural and functional diversity. We have created phoslactomycin polyketide synthase‐derived in vitro systems and studied their substrate selectivity. We were able to produce various phoslactomycin derivatives with altered side‐chain lengths and characterized the transacylation and hydrolysis activity of the involved acyltransferases for all substrates tested.

Growth from spores activated a biosynthetic gene cluster in Actinomadura sp. RB29, resulting in the identification of two novel groups of halogenated polyketide natural products, named maduralactomycins and actinospirols. The unique tetracyclic and spirocyclic structures were assigned based on a combination of NMR analysis, chemoinformatic calculations, X-ray crystallography, and 13C labeling studies. On the basis of HRMS2 data, genome mining, and gene expression studies, we propose an underlying noncanonical angucycline biosynthesis and extensive post-polyketide synthase (PKS) oxidative modifications.

Miharamycins are peptidyl nucleoside antibiotics with a unique branched C9 pyranosyl amino acid core and a rare 2-aminopurine moiety. Inactivation of 19 genes in the biosynthetic gene cluster and identification of several unexpected intermediates suggest an alternative biosynthetic pathway, which is further supported by feeding experiments and in vitro characterization of an unusual adenylation domain recognizing a complex nucleoside derivative as the substrate. These results thereby provide an unprecedented biosynthetic route of high-carbon sugar catalyzed by atypical hybrid nonribosomal peptide synthetase-polyketide synthase.

Background Acridone alkaloids are heterocyclic compounds that exhibit a broad-range of pharmaceutical and chemotherapeutic activities, including anticancer, antiviral, anti-inflammatory, antimalarial, and antimicrobial effects. Certain plant species such as Citrus microcarpa, Ruta graveolens, and Toddaliopsis bremekampii synthesize acridone alkaloids from anthranilate and malonyl-CoA. Results We synthesized two acridones in Escherichia coli. Acridone synthase (ACS) and anthraniloyl-CoA ligase genes were transformed into E. coli, and the synthesis of acridone was examined. To increase the levels of endogenous anthranilate, we tested several constructs expressing proteins involved in the shikimate pathway and selected the best construct. To boost the supply of malonyl-CoA, genes coding for acetyl-coenzyme A carboxylase (ACC) from Photorhabdus luminescens were overexpressed in E. coli. For the synthesis of 1,3-dihydroxy-10-methylacridone, we utilized an N-methyltransferase gene (NMT) to supply N-methylanthranilate and a new N-methylanthraniloyl-CoA ligase. After selecting the best combination of genes, approximately 17.3 mg/L of 1,3-dihydroxy-9(10H)-acridone (DHA) and 26.0 mg/L of 1,3-dihydroxy-10-methylacridone (NMA) were synthesized. Conclusions Two bioactive acridone derivatives were synthesized by expressing type III plant polyketide synthases and other genes in E. coli, which increased the supplement of substrates. This study showed that is possible to synthesize diverse polyketides in E. coli using plant polyketide synthases.