Reducing Polyketide Synthase Research Articles

Repurposing a Fully Reducing Polyketide Synthase toward 2-Methyl Guerbet-like Lipids.

In nature, thousands of diverse and bioactive polyketides are assembled by a family of multifunctional, "assembly line" enzyme complexes called polyketide synthases (PKS). Since the late 20th century, there have been several attempts to decode, rearrange, and "reprogram" the PKS assembly line to generate valuable materials such as biofuels and platform chemicals. Here, the first module from Mycobacterium tuberculosis (Mt) PKS12, an unorthodox, "modularly iterative" PKS, was modified and repurposed toward the formation of 2-methyl Guerbet lipids, which have wide applications in industry. We established a robust method for the recombinant expression and purification of this modified module (named [M1*]), and we demonstrated its ability to catalyze the formation of several 2-methyl Guerbet-like lipids (C13-C21). Furthermore, we studied and applied the promiscuous thioesterase activity of a neighboring β-ketoacyl synthase (KS) to release [M1*]-bound condensation products in a one-pot biosynthetic cascade. Finally, starting from lauric acid, we could generate our primary target compound (2-methyltetradecanoic acid) by coupling the Escherichia coli fatty acyl-CoA synthetase FadD to [M1*]. This work supports the biosynthetic utility of engineered PKS modules such as [M1*] and their ability to derive valuable Guerbet-like lipids from inexpensive fatty acids.

Production of Ochratoxin A and Citrinin and the Expression of Their Biosynthetic Genes from Penicillium verrucosum in Liquid Culture.

Penicillium verrucosum is a fungal pathogen capable of producing two mycotoxins of concern, ochratoxin A (OTA) and citrinin (CIT). The production profile of these two mycotoxins is not well understood but could help mitigate co-contamination in the food supply. As such, the production of OTA and CIT from P. verrucosum DAOMC 242724 was investigated under different growing conditions in liquid culture. We found that among the different liquid media chosen, liquid YES (yeast extract sucrose) medium induced the highest production of both OTA and CIT, when P. verrucosum DAOMC 242724 was cultured in stationary mode. Shake culture significantly reduced the amounts of OTA and CIT produced. Among all culture conditions tested, far greater amounts of CIT were produced compared to OTA. Consequently, upon transcriptomic data analysis, a statistically significant increase in the expression of CIT biosynthetic genes was easier to detect than the expression of OTA biosynthetic genes. Our study also revealed that the putative biosynthetic gene clusters of OTA and CIT in P. verrusocum DAOMC 242724 are likely distinct from each other. It appears that despite sharing a highly similar structure, the isocoumarin rings of OTA and CIT are each assembled by a specialized polyketide synthase enzyme. Our data identified a putative nonreducing polyketide synthase responsible for assembling the carbo-skeleton of CIT. In contrast, a highly reducing polyketide synthase appears to be involved in the biosynthesis of OTA.

α-Pyrone Derivatives from Calcarisporium arbuscula Discovered by Genome Mining.

A highly reducing polyketide synthase (HRPKS) gene cluster from the genome of Calcarisporium arbuscula was identified through genome mining. Heterologous expression of this cluster led to the production of four new α-pyrone compounds, calcapyrones A (1) and B (2), along with their biosynthetic intermediates calcapyrones C (3) and D (4). The structures of these compounds were elucidated on the basis of extensive spectroscopic experiments, and the absolute configurations of the 7,8-diol moieties in 1 and 2 were assigned using Snatzke's method. The biosynthetic pathway of 1 and 2 was established through in vivo and in vitro experiments.

Genome mining of multi-substituted alkylresorcinols from a hybrid highly reducing- and type III- polyketide pathway

Fungal alkylresorcinols are a class of polyketides, which are commonly synthesized by the hybridization of highly reducing polyketide synthase (hrPKS) with non-reducing polyketide synthase (nrPKS). In this study, we identified and demonstrated a new assembly model for synthesizing alkylresorcinol (scirpilin A, 1), which was accomplished by collaboration of a hrPKS (FscA) and a type III PKS (FscB). Furthermore, three post-tailoring enzymes (FscC, FscD, and FscE) act iteratively on 1 skeleton, including successive 14e− oxidation of inert carbons, di-halogenation, and di-methylation, to form highly oxidized and multi-substituted alkylresorcinols. Our work presents an unusual synthesis manner of alkylresorcinols, sheds light on the collaborative mechanism between hrPKS and type III PKS and provides three valuable enzymatic catalysts for the tailoring of alkylresorcinol family natural products in future.

Biosynthesis of rumbrins and inspiration for discovery of HIV inhibitors

Investigation on how nature produces natural compounds with chemical and biological diversity at the genetic level offers inspiration for the discovery of new natural products and even their biological targets. The polyketide rumbrin (1) is a lipid peroxide production and calcium accumulation inhibitor, which contains a chlorinated pyrrole moiety that is a rare chemical feature in fungal natural products. Here, we identify the biosynthetic gene cluster (BGC) rum of 1 and its isomer 12E-rumbrin (2) from Auxarthron umbrinum DSM3193, and elucidate their biosynthetic pathway based on heterologous expression, chemical complementation, and isotopic labeling. We show that rumbrins are assembled by a highly reducing polyketide synthase (HRPKS) that uniquely incorporates a proline-derived pyrrolyl-CoA starer unit, and followed by methylation and chlorination. Sequent precursor-directed biosynthesis was able to yield a group of rumbrin analogues. Remarkably, inspired by the presence of a human immunodeficiency virus (HIV)-Nef-associated gene in the rum cluster, we predicted and pharmacologically demonstrated rumbrins as potent inhibitors of HIV at the nanomolar level. This work enriches the recognition of unconventional starter units of fungal PKSs and provides a new strategy for genome mining-guided drug discovery.

Putative Biosynthesis of Talarodioxadione & Talarooxime from Talaromyces stipitatus.

Polyesters containing 2,4-dihydroxy-6-(2-hydroxypropyl)benzoate and 3-hydroxybutyrate moieties have been isolated from many fungal species. Talaromyces stipitatus was previously reported to produce a similar polyester, talapolyester G. The complete genome sequence and the development of bioinformatics tools have enabled the discovery of the biosynthetic potential of this microorganism. Here, a putative biosynthetic gene cluster (BGC) of the polyesters encoding a highly reducing polyketide synthase (HR-PKS) and nonreducing polyketide synthase (NR-PKS), a cytochrome P450 and a regulator, was identified. Although talapolyester G does not require an oxidative step for its biosynthesis, further investigation into the secondary metabolite production of T. stipitatus resulted in isolating two new metabolites called talarodioxadione and talarooxime, in addition to three known compounds, namely 6-hydroxymellein, 15G256α and transtorine that have never been reported from this organism. Interestingly, the biosynthesis of the cyclic polyester 15G256α requires hydroxylation of an inactive methyl group and thus could be a product of the identified gene cluster. The two compounds, talarooxime and transtorine, are probably the catabolic metabolites of tryptophan through the kynurenine pathway. Tryptophan metabolism exists in almost all organisms and has been of interest to many researchers. The biosynthesis of the new oxime is proposed to involve two subsequent N-hydroxylation of 2-aminoacetophenone.

Biosynthesis of Xylariolide D in Penicillium crustosum Implies a Chain Branching Reaction Catalyzed by a Highly Reducing Polyketide Synthase.

Fungi are important sources for the discovery of natural products. During the last decades, technological progress and the increasing number of sequenced genomes facilitated the exploration of new secondary metabolites. Among those, polyketides represent a structurally diverse group with manifold biological activities. In this study, we successfully used genome mining and genetic manipulation for functional proof of a polyketide biosynthetic gene cluster from the filamentous fungus Penicillium crustosum. Gene activation in the native host and heterologous expression in Aspergillus nidulans led to the identification of the xil cluster, being responsible for the formation of the 6-methyl-2-pyrone derivative xylariolide D. Feeding with 13C-labeled precursors supported the hypothesis of chain branching during the backbone formation catalyzed by a highly reducing fungal polyketide synthase. A cytochrome P450-catalyzed hydroxylation converts the PKS product to the final metabolite. This proved that just two enzymes are required for the biosynthesis of xylariolide D.

C-Methylation controls the biosynthetic programming of alternapyrone.

Alternapyrone is a highly methylated polyene α-pyrone biosynthesised by a highly reducing polyketide synthase. Mutations of the catalytic dyad residues, H1578A/Q and E1604A, of the C-methyltransferase domain resulted in either significantly reduced or no production of alternapyrone, indicating the importance of C-methylation for alternapyrone biosynthesis.

Biochemistry‐Guided Prediction of the Absolute Configuration of Fungal Reduced Polyketides

AbstractHighly 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.

Biochemistry-Guided Prediction of the Absolute Configuration of Fungal Reduced Polyketides.

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.

Polyketides produced by the entomopathogenic fungus Metarhizium anisopliae induce Candida albicans growth

Metarhizium anisopliae is an important entomopathogenic species and model for arthropod-fungus interaction studies. This fungus harbors a diverse arsenal of unexplored secondary metabolite biosynthetic gene clusters, which are suggested to perform diverse roles during host interaction and soil subsistence as a saprophytic species. Here we explored an unusual carnitine acyltransferase domain-containing highly reducing polyketide synthase found in the genome of M. anisopliae. Employing heterologous expression in Aspergillus nidulans, two new polyketides were obtained, named BAA and BAB, as well as one known polyketide [(2Z,4E,6E)-octa-2,4,6-trienedioic acid]. Intra-hemocoel injection of the most abundant compound (BAA) in the model-arthropod Galleria mellonella larvae did not induce mortality or noticeable alterations, suggesting that this compound may not harbor insecticidal activity. Also, the potential role of such molecules in polymicrobial interactions was evaluated. Determination of minimum inhibitory concentration assays using distinct fungal species revealed that BAA and BAB did not alter Cryptococcus neoformans growth, while BAA exhibited weak antifungal activity against Saccharomyces cerevisiae. Unexpectedly, these compounds increased Candida albicans growth compared to control conditions. Furthermore, BAA can mitigate the fungicidal effects of fluconazole over C. albicans. Although the exact role of these compounds on the M. anisopliae life cycle is elusive, the described results add up to the complexity of secondary metabolites produced by Metarhizium spp. Moreover, up to our knowledge, these are the first polyketides isolated from filamentous fungi that can boost the growth of another fungal species.

Vital roles of Pks11, a highly reducing polyketide synthase, in fungal conidiation, antioxidant activity, conidial cell wall integrity, and UV tolerance of Beauveria bassiana

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.

Biosynthesis of 6‐Hydroxymellein Requires a Collaborating Polyketide Synthase‐like Enzyme

AbstractThe polyketide synthase (PKS)‐like protein TerB, consisting of inactive dehydratase, inactive C‐methyltransferase, and functional ketoreductase domains collaborates with the iterative non reducing PKS TerA to produce 6‐hydroxymellein, a key pathway intermediate during the biosynthesis of various fungal natural products. The catalytically inactive dehydratase domain of TerB appears to mediate productive interactions with TerA, demonstrating a new mode of trans‐interaction between iterative PKS components.

Biosynthesis of 6-Hydroxymellein Requires a Collaborating Polyketide Synthase-like Enzyme.

The polyketide synthase (PKS)‐like protein TerB, consisting of inactive dehydratase, inactive C‐methyltransferase, and functional ketoreductase domains collaborates with the iterative non reducing PKS TerA to produce 6‐hydroxymellein, a key pathway intermediate during the biosynthesis of various fungal natural products. The catalytically inactive dehydratase domain of TerB appears to mediate productive interactions with TerA, demonstrating a new mode of trans‐interaction between iterative PKS components.

Self‐Resistance in the Biosynthesis of Fungal Macrolides Involving Cycles of Extracellular Oxidative Activation and Intracellular Reductive Inactivation

AbstractSelf‐resistance genes are employed by many microbial producers of bioactive natural products to avoid self‐harm. Herein, we describe a unique strategy for self‐resistance toward a macrolide antibiotic, A26771B (1), identified by elucidating its biosynthetic pathway in the fungus Penicillium egyptiacum. A highly reducing polyketide synthase and a trans‐acting thioesterase generate the macrolide backbone, and a cytochrome P450 and an acyltransferase, respectively catalyze hydroxylation and succinylation to form the prodrug berkeleylactone E (2). Then, extracellular oxidative activation by a secreted flavin‐dependent oxidase forms 1, while intracellular reductive inactivation by a short‐chain reductase reforms 2, forming a redox cycle. Our work illustrates a unique redox‐mediated resistance mechanism for fungal antibiotics and contributes to the understanding of antibiotic biosynthesis and resistance.

Self-Resistance in the Biosynthesis of Fungal Macrolides Involving Cycles of Extracellular Oxidative Activation and Intracellular Reductive Inactivation.

Self-resistance genes are employed by many microbial producers of bioactive natural products to avoid self-harm. Herein, we describe a unique strategy for self-resistance toward a macrolide antibiotic, A26771B (1), identified by elucidating its biosynthetic pathway in the fungus Penicillium egyptiacum. A highly reducing polyketide synthase and a trans-acting thioesterase generate the macrolide backbone, and a cytochrome P450 and an acyltransferase, respectively catalyze hydroxylation and succinylation to form the prodrug berkeleylactone E (2). Then, extracellular oxidative activation by a secreted flavin-dependent oxidase forms 1, while intracellular reductive inactivation by a short-chain reductase reforms 2, forming a redox cycle. Our work illustrates a unique redox-mediated resistance mechanism for fungal antibiotics and contributes to the understanding of antibiotic biosynthesis and resistance.

Biosynthesis of Mycotoxin Fusaric Acid and Application of a PLP-Dependent Enzyme for Chemoenzymatic Synthesis of Substituted l-Pipecolic Acids.

Fusaric acid (FA) is a well-known mycotoxin that plays an important role in plant pathology. The biosynthetic gene cluster for FA has been identified, but the biosynthetic pathway remains unclarified. Here, we elucidated the biosynthesis of FA, which features a two-enzyme catalytic cascade, a pyridoxal 5'-phosphate (PLP)-dependent enzyme (Fub7), and a flavin mononucleotide (FMN)-dependent oxidase (Fub9) in synthesizing the picolinic acid scaffold. FA biosynthesis also involves an off-line collaboration between a highly reducing polyketide synthase (HRPKS, Fub1) and a nonribosomal peptide synthetase (NRPS)-like carboxylic acid reductase (Fub8) in making an aliphatic α,β-unsaturated aldehyde. By harnessing the stereoselective C-C bond-forming activity of Fub7, we established a chemoenzymatic route for stereoconvergent synthesis of a series of 5-alkyl-, 5,5-dialkyl-, and 5,5,6-trialkyl-l-pipecolic acids of high diastereomeric ratio.

The Laetiporus polyketide synthase LpaA produces a series of antifungal polyenes

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.

Genome-based mining of new antimicrobial meroterpenoids from the phytopathogenic fungus Bipolaris sorokiniana strain 11134.

Polyketide-terpenoid hybrid compounds are one of the largest families of meroterpenoids, with great potential for drug development for resistant pathogens. Genome sequence analysis of secondary metabolite gene clusters of a phytopathogenic fungus, Bipolaris sorokiniana 11134, revealed a type I polyketide gene cluster, consisting of highly reducing polyketide synthase, non-reducing polyketide synthase, and adjacent prenyltransferase. MS- and UV-guided isolations led to the isolation of ten meroterpenoids, including two new compounds: 19-dehydroxyl-3-epi-arthripenoid A (1) and 12-keto-cochlioquinone A (2). The structures of 1-10 were elucidated by the analysis of NMR and high-resolution electrospray ionization mass spectroscopy data. Compounds 5-8 and 10 showed moderate activity against common Staphylococcus aureus and methicillin-resistant S. aureus, with minimum inhibitory concentration (MIC) values of 12.5-100μg/mL. Compound 5 also exhibited activity against four clinical resistant S. aureus strains and synergistic antifungal activity against Candida albicans with MIC values of 12.5-25μg/mL. The biosynthetic gene cluster of the isolated compounds and their putative biosynthetic pathway are also proposed. KEY POINTS: • Ten meroterpenoids were identified from B. sorokiniana, including two new compounds. • Cochlioquinone B (5) showed activity against MRSA and synergistic activity against C. albicans. • The biosynthetic gene cluster and biosynthetic pathway of meroterpenoids are proposed. • Genome mining provided a new direction to uncover the diversity of meroterpenoids.

Genetic evidences for the core biosynthesis pathway, regulation, transport and secretion of liamocins in yeast-like fungal cells.

So far, it has been still unknown how liamocins are biosynthesized, regulated, transported and secreted. In this study, a highly reducing polyketide synthase (HR-PKS), a mannitol-1-phosphate dehydrogenase (MPDH), a mannitol dehydrogenase (MtDH), an arabitol dehydrogenase (ArDH) and an esterase (Est1) were found to be closely related to core biosynthesis of extracellular liamocins in Aureobasidium melanogenum 6-1-2. The HR-PKS was responsible for biosynthesis of 3,5-dihydroxydecanoic acid. The MPDH and MtDH were implicated in mannitol biosynthesis and the ArDH was involved in arabitol biosynthesis. The Est1 catalyzed ester bond formation of them. A phosphopantetheine transferase (PPTase) activated the HR-PKS and a transcriptional activator Ga11 activated expression of the PKS1 gene. Therefore, deletion of the PKS1 gene, all the three genes encoding MPDH, MtDH and ArDH, the EST1, the gene responsible for PPTase and the gene for Ga11 made all the disruptants (Δpks13, Δpta13, Δest1, Δp12 and Δg11) totally lose the ability to produce any liamocins. A GLTP gene encoding a glycolipid transporter and a MDR1 gene encoding an ABC transporter took part in transport and secretion of the produced liamocins into medium. Removal of the GLTP gene and the MDR1 gene resulted in a Δgltp1 mutant and a Δmdr16 mutant, respectively, that lost the partial ability to secrete liamocins, but which cells were swollen and intracellular lipid accumulation was greatly enhanced. Hydrolysis of liamocins released 3,5-dihydroxydecanoic acid, mannitol, arabitol and acetic acid. We proposed a core biosynthesis pathway, regulation, transport and secretion of liamocins in A. melanogenum.