An Unconventional Melanin Biosynthetic Pathway in Ustilago maydis

ABSTRACTUstilago maydis is a phytopathogenic fungus responsible for corn smut disease. Although it is a very well established model organism for the study of plant-microbe interactions, its biosynthetic potential has not been totally explored. By analyzing U. maydis genome, we identified a biosynthetic gene cluster whose activation led to the production of a black melanin pigment. Single deletion mutants of the cluster genes revealed that five encoded enzymes are required for the accumulation of the black pigment, including three polyketide synthases (pks3, pks4 and pks5), a cytochrome P450 monooxygenase (cyp4) and a protein with similarity to versicolorin B-synthase (vbs1). Moreover, metabolic profiles of the mutants defective for pks3 and pks4 indicated that the products of these genes catalyze together the first step in the melanin biosynthetic pathway since none of the mutants accumulated any melanin or intermediate products. Mutants deleted for pks5 produced orsellinic acid (OA) and triacetic acid lactone (TAL), suggesting that both products are produced by Pks3 and Pks4. It might thus demonstrate that Pks5 plays a role in a reaction downstream of that catalyzed by Pks3 and Pks4. OA and TAL were also found in extracts of a cyp4 deletion mutant along with several heterodimers of TAL and Pks5-derived orsellinic aldehyde compounds. According to their phenotypes and the intermediate products isolated from these strains, Cyp4 and Vbs1 seem to be involved in reactions downstream of Pks5. Our findings suggest that U. maydis synthesizes a new melanin based on coumarin and pyran-2-one intermediates, while most fungal melanins are derived from 1,8-dihydroxynaphthalene (DHN) or L-3,4-dihydroxyphenylalanine (L-DOPA). Along with these observations, this work also provides an insight into the mechanisms of polyketide synthases in this filamentous fungus.IMPORTANCEUstilago maydis represents one of the major threats for maize plants since it is responsible for corn smut disease, which generates considerable economical losses around the world. Therefore, contributing to a better understanding of the biochemistry of defense mechanisms used by U. maydis to protect itself against harsh environments, as the synthesis of melanin, could provide improved biological tools for tackling the problem and protect the crops. In addition, the fact that this fungus synthesizes melanin in a very unique way, requiring more than one polyketide synthase for producing this secondary metabolite, gives a different perspective on the complexity of these multimodular enzymes and their evolution in the fungal kingdom.

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An Unconventional Melanin Biosynthetic Pathway in Ustilago maydis

Applied and Environmental Microbiology ◽

10.1128/aem.01510-20 ◽

2020 ◽

Author(s):

Esmeralda Z. Reyes-Fernández ◽

Yi-Ming Shi ◽

Peter Grün ◽

Helge B. Bode ◽

Michael Bölker

Keyword(s):

Gene Cluster ◽

Polyketide Synthase ◽

Model Organism ◽

Ustilago Maydis ◽

Biosynthetic Gene Cluster ◽

Polyketide Synthases ◽

Phytopathogenic Fungus ◽

Deletion Mutants ◽

P450 Monooxygenase ◽

Corn Smut

Ustilago maydis is a phytopathogenic fungus responsible for corn smut disease. Although it is a very well established model organism for the study of plant-microbe interactions, its potential to produce specialized metabolites, which might contribute to this interaction, has not been studied in detail. By analyzing the U. maydis genome, we identified a biosynthetic gene cluster whose activation led to the production of a black melanin pigment. Single deletion mutants of the cluster genes revealed that five encoded enzymes are required for the accumulation of the black pigment, including three polyketide synthases (pks3, pks4 and pks5), a cytochrome P450 monooxygenase (cyp4) and a protein with similarity to versicolorin B synthase (vbs1). Metabolic profiles of deletion mutants in this gene cluster suggested that Pks3 and Pks4 act in concert as heterodimer to generate orsellinic acid (OA) which is reduced to the corresponding aldehyde by Pks5. The OA-aldehyde can then react with triacetic acid lactone (TAL) also derived from Pks3/Pks4 heterodimers to form larger molecules including novel coumarin derivatives. Our findings suggest that U. maydis synthesizes a novel type of melanin based on coumarin and pyran-2-one intermediates, while most fungal melanins are derived from 1,8-dihydroxynaphthalene (DHN) or L-3,4-dihydroxyphenylalanine (L-DOPA). Along with these observations, this work also provides an insight into the mechanisms of polyketide synthases in this filamentous fungus. IMPORTANCE The fungus Ustilago maydis represents one of the major threats for maize plants since it is responsible for corn smut disease, which generates considerable economical losses around the world. Therefore, contributing to a better understanding of the biochemistry of defense mechanisms used by U. maydis to protect itself against harsh environments, as the synthesis of melanin, could provide improved biological tools for tackling the problem and protect the crops. In addition, the fact that this fungus synthesizes melanin in an unconventional way, requiring more than one polyketide synthase for producing melanin precursors, gives a different perspective on the complexity of these multimodular enzymes and their evolution in the fungal kingdom.

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Pheromone-Induced G2 Arrest in the Phytopathogenic Fungus Ustilago maydis

Eukaryotic Cell ◽

10.1128/ec.2.3.494-500.2003 ◽

2003 ◽

Vol 2 (3) ◽

pp. 494-500 ◽

Cited By ~ 63

Author(s):

Tatiana García-Muse ◽

Gero Steinberg ◽

José Pérez-Martín

Keyword(s):

Cell Cycle ◽

Ustilago Maydis ◽

Phytopathogenic Fungus ◽

Microtubule Organization ◽

Mating Response ◽

Cycle Arrest ◽

Synthetic Pheromone ◽

Genes Encoding ◽

The Relationship ◽

Corn Smut

ABSTRACT In the corn smut fungus Ustilago maydis, pathogenic development is initiated when two compatible haploid cells fuse and form the infectious dikaryon. Mating is dependent on pheromone recognition by compatible cells. In this report, we set out to evaluate the relationship between the cell cycle and the pheromone response in U. maydis. To achieve this, we designed a haploid pheromone-responsive strain that is able to faithfully reproduce the native mating response in nutrient-rich medium. Addition of synthetic pheromone to the responsive strain induces the formation of mating structures, and this response is abolished by mutations in genes encoding components of the pheromone signal transduction cascade. After recognition of pheromone, U. maydis cells arrest the cell cycle in a postreplicative stage. Visualization of the nucleus and microtubule organization indicates that the arrest takes place at the G2 phase. Chemical-induced cell cycle arrest and release in the presence of pheromone further support this conclusion.

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Huitlacoche (corn smut), caused by the phytopathogenic fungus Ustilago maydis, as a functional food

Revista Iberoamericana de Micología ◽

10.1016/j.riam.2011.01.001 ◽

2011 ◽

Vol 28 (2) ◽

pp. 69-73 ◽

Cited By ~ 21

Author(s):

Margarita Juárez-Montiel ◽

Sandra Ruiloba de León ◽

Griselda Chávez-Camarillo ◽

César Hernández-Rodríguez ◽

Lourdes Villa-Tanaca

Keyword(s):

Functional Food ◽

Ustilago Maydis ◽

Phytopathogenic Fungus ◽

Corn Smut

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The plmS2-Encoded Cytochrome P450 Monooxygenase Mediates Hydroxylation of Phoslactomycin B in Streptomyces sp. Strain HK803

Journal of Bacteriology ◽

10.1128/jb.187.23.7970-7976.2005 ◽

2005 ◽

Vol 187 (23) ◽

pp. 7970-7976 ◽

Cited By ~ 8

Author(s):

Mohini S. Ghatge ◽

Kevin A. Reynolds

Keyword(s):

Cytochrome P450 ◽

Biosynthetic Pathway ◽

Sequence Similarity ◽

Biosynthetic Gene Cluster ◽

Conjugative Plasmid ◽

Cytochrome P450 Monooxygenases ◽

Phosphate Ester ◽

High Sequence Similarity ◽

P450 Monooxygenase ◽

Complementation Experiment

ABSTRACT Streptomyces sp. strain HK803 produces six analogues of phoslactomycin (Plm A through Plm F). With the exception of Plm B, these analogues contain a C-18 hydroxyl substituent esterified with a range of short-alkyl-chain carboxylic acids. Deletion of the plmS 2 open reading frame (ORF), showing high sequence similarity to bacterial cytochrome P450 monooxygenases (CYPs), from the Plm biosynthetic gene cluster has previously resulted in an NP1 mutant producing only Plm B (N. Palaniappan, B. S. Kim, Y. Sekiyama, H. Osada, and K. A. Reynolds, J. Biol. Chem. 278:35552-35557, 2003). Herein, we report that a complementation experiment with an NP1 derivative (NP2), using a recombinant conjugative plasmid carrying the plmS 2 ORF downstream of the ermE* constitutive promoter (pMSG1), restored production of Plm A and Plm C through Plm F. The 1.2-kbp plmS 2 ORF was also expressed efficiently as an N-terminal polyhistidine-tagged protein in Streptomyces coelicolor. The recombinant PlmS2 converted Plm B to C-18-hydroxy Plm B (Plm G). PlmS2 was highly specific for Plm B and unable to process a series of derivatives in which either the lactone ring was hydrolyzed or the C-9 phosphate ester was converted to C-9/C-11 phosphorinane. This biochemical analysis and complementation experiment are consistent with a proposed Plm biosynthetic pathway in which the penultimate step is hydroxylation of the cyclohexanecarboxylic acid-derived side chain of Plm B by PlmS2 (the resulting Plm G is then esterified to provide Plm A and Plm C through Plm F). Kinetic parameters for Plm B hydroxylation by PlmS2 (Km of 45.3 ± 9.0 μM and k cat of 0.27 ± 0.04 s−1) are consistent with this step being a rate-limiting step in the biosynthetic pathway. The penultimate pathway intermediate Plm G has less antifungal activity than Plm A through Plm F and is not observed in fermentations of either the wild-type strain or NP2/pMSG1.

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Organization of the biosynthetic gene cluster for the macrolide concanamycin A in Streptomyces neyagawaensis ATCC 27449

Microbiology ◽

10.1099/mic.0.28194-0 ◽

2005 ◽

Vol 151 (10) ◽

pp. 3161-3169 ◽

Cited By ~ 59

Author(s):

Stephen F. Haydock ◽

Anthony N. Appleyard ◽

Tatiana Mironenko ◽

John Lester ◽

Natasha Scott ◽

...

Keyword(s):

Gene Cluster ◽

Biosynthetic Pathway ◽

Polyketide Synthase ◽

Building Blocks ◽

Biosynthetic Gene Cluster ◽

Sequence Motif ◽

Biosynthetic Gene ◽

Concanamycin A ◽

Modular Polyketide Synthase

The macrolide antibiotic concanamycin A has been identified as an exceptionally potent inhibitor of the vacuolar (V-type) ATPase. Such compounds have been mooted as the basis of a potential drug treatment for osteoporosis, since the V-ATPase is involved in the osteoclast-mediated bone resorption that underlies this common condition. To enable combinatorial engineering of altered concanamycins, the biosynthetic gene cluster governing the biosynthesis of concanamycin A has been cloned from Streptomyces neyagawaensis and shown to span a region of over 100 kbp of contiguous DNA. An efficient transformation system has been developed for S. neyagawaensis and used to demonstrate the role of the cloned locus in the formation of concanamycin A. Sequence analysis of the 28 ORFs in the region has revealed key features of the biosynthetic pathway, in particular the biosynthetic origin of portions of the backbone, which arise from the unusual polyketide building blocks ethylmalonyl-CoA and methoxymalonyl-ACP, and the origin of the pendant deoxysugar moiety 4′-O-carbamoyl-2′-deoxyrhamnose, as well as the presence of a modular polyketide synthase (PKS) encoded by six giant ORFs. Examination of the methoxymalonyl-specific acyltransferase (AT) domains has led to recognition of an amino acid sequence motif which can be used to distinguish methylmalonyl-CoA- from methoxymalonyl-ACP-specific AT domains in natural PKSs.

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

10.26434/chemrxiv.12897095 ◽

2020 ◽

Author(s):

Yang Hai ◽

Mengbin Chen ◽

Arthur Huang ◽

Yi Tang

Keyword(s):

Biosynthetic Pathway ◽

Polyketide Synthase ◽

Picolinic Acid ◽

Fusaric Acid ◽

Biosynthetic Gene Cluster ◽

Chemoenzymatic Synthesis ◽

Flavin Mononucleotide ◽

Biosynthetic Gene ◽

Bond Forming ◽

Peptide Synthetase

<div><p>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 alpha,beta-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. </p></div>

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

10.26434/chemrxiv.12897095.v1 ◽

2020 ◽

Author(s):

Yang Hai ◽

Mengbin Chen ◽

Arthur Huang ◽

Yi Tang

Keyword(s):

Biosynthetic Pathway ◽

Polyketide Synthase ◽

Picolinic Acid ◽

Fusaric Acid ◽

Biosynthetic Gene Cluster ◽

Chemoenzymatic Synthesis ◽

Flavin Mononucleotide ◽

Biosynthetic Gene ◽

Bond Forming ◽

Peptide Synthetase

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Aromatic polyketide biosynthesis: fidelity, evolution and engineering

10.1101/581074 ◽

2019 ◽

Author(s):

Zhiwei Qin ◽

Rebecca Devine ◽

Matthew I. Hutchings ◽

Barrie Wilkinson

Keyword(s):

Biosynthetic Pathway ◽

Polyketide Synthase ◽

Single Gene ◽

Biosynthetic Gene Cluster ◽

Type Ii ◽

Trace Level ◽

Aromatic Polyketides ◽

Pyridine Moiety ◽

Catalytic Role ◽

New Compounds

AbstractWe report the formicapyridines which are structurally and biosynthetically related to the pentacyclic fasamycin and formicamycin aromatic polyketides but comprise a rare pyridine moiety. These new compounds are trace level metabolites formed by derailment of the major biosynthetic pathway. Inspired by evolutionary logic we show that rational mutation of a single gene in the biosynthetic gene cluster leads to a significant increase both in total formicapyridine production and their enrichment relative to the fasamycins/formicamycins. Our observations broaden the polyketide biosynthetic landscape and identify a non-catalytic role for ABM superfamily proteins in type II polyketide synthase assemblages for maintaining biosynthetic pathway fidelity.

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A Consensus Ochratoxin A Biosynthetic Pathway: Insights from the Genome Sequence ofAspergillus ochraceusand a Comparative Genomic Analysis

Applied and Environmental Microbiology ◽

10.1128/aem.01009-18 ◽

2018 ◽

Vol 84 (19) ◽

Cited By ~ 16

Author(s):

Yan Wang ◽

Liuqing Wang ◽

Fan Wu ◽

Fei Liu ◽

Qi Wang ◽

...

Keyword(s):

Ochratoxin A ◽

Biosynthetic Pathway ◽

Polyketide Synthase ◽

Genomic Analysis ◽

Amide Bond ◽

Bzip Transcription Factor ◽

Economic Losses ◽

Comparative Genomic ◽

P450 Monooxygenase ◽

Content Type

ABSTRACTOchratoxin A (OTA) is a toxic secondary metabolite produced byAspergillusandPenicilliumspecies that widely contaminates food and feed. We sequenced and assembled the complete ∼37-Mb genome ofAspergillusochraceusfc-1, a well-known producer of OTA. Key genes of the OTA biosynthetic pathway were identified by comparative genomic analyses with five other sequenced OTA-producing fungi:A. carbonarius,A. niger,A. steynii,A. westerdijkiae, andPenicillium nordicum. OTA production was completely inhibited in the deletion mutants (ΔotaA, ΔotaB, ΔotaC, ΔotaD, and ΔotaR1), and OTA biosynthesis was restored by feeding a postblock substrate to the corresponding mutant. The OTA biosynthetic pathway was unblocked in the ΔotaDmutant by the addition of heterologously expressed halogenase. OTA biosynthesis begins with a polyketide synthase (PKS), OtaA, utilizing acetyl coenzyme A (acetyl-CoA) and malonyl-CoA to synthesize 7-methylmellein, which is oxidized to OTβ by cytochrome P450 monooxygenase (OtaC). OTβ andl-β-phenylalanine are combined by a nonribosomal peptide synthetase (NRPS), OtaB, to form an amide bond to synthesize OTB. Finally, OTB is chlorinated by a halogenase (OtaD) to OTA. TheotaABCDgenes were expressed at low levels in the ΔotaR1mutant. A second regulator,otaR2, which is adjacent to the biosynthetic gene, could modulate only the expression ofotaA,otaB, andotaD. Thus, we have identified a consensus OTA biosynthetic pathway that can be used to prevent and control OTA synthesis and will help us understand the variation and production of the intermediate components in the biosynthetic pathway.IMPORTANCEOchratoxin A (OTA) is a significant mycotoxin that contaminates cereal products, coffee, grapes, wine, cheese, and meat. OTA is nephrotoxic, carcinogenic, teratogenic, and immunotoxic. OTA contamination is a serious threat to food safety, endangers human health, and can cause huge economic losses. At present, >20 species of the generaAspergillusandPenicilliumare known to produce OTA. Here we demonstrate that a consensus OTA biosynthetic pathway exists in all OTA-producing fungi and is encoded by a gene cluster containing four highly conserved biosynthetic genes and a bZIP transcription factor.

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Characterization of the P450 Monooxygenase NysL, Responsible for C-10 Hydroxylation during Biosynthesis of the Polyene Macrolide Antibiotic Nystatin in Streptomyces noursei

Applied and Environmental Microbiology ◽

10.1128/aem.72.4.2514-2519.2006 ◽

2006 ◽

Vol 72 (4) ◽

pp. 2514-2519 ◽

Cited By ~ 33

Author(s):

Olga Volokhan ◽

Håvard Sletta ◽

Trond E. Ellingsen ◽

Sergey B. Zotchev

Keyword(s):

Polyketide Synthase ◽

Kinetic Studies ◽

Biosynthetic Gene Cluster ◽

Gene Replacement ◽

Gene Encoding ◽

P450 Monooxygenase ◽

Polyene Macrolide Antibiotic ◽

Streptomyces Noursei

ABSTRACT The nysL gene, encoding a putative P450 monooxygenase, was identified in the nystatin biosynthetic gene cluster of Streptomyces noursei. Although it has been proposed that NysL is responsible for hydroxylation of the nystatin precursor, experimental evidence for this activity was lacking. The nysL gene was inactivated in S. noursei by gene replacement, and the resulting mutant was shown to produce 10-deoxynystatin. Purification and an in vitro activity assay for 10-deoxynystatin demonstrated its antifungal activity being equal to that of nystatin. The NysL protein was expressed heterologously in Escherichia coli as a His-tagged protein and used in an enzyme assay with 10-deoxynystatin as a substrate. The results obtained clearly demonstrated that NysL is a hydroxylase responsible for the post-polyketide synthase modification of 10-deoxynystatin at position C-10. Kinetic studies with the purified recombinant enzyme allowed determination of Km and k cat and revealed no inhibition of recombinant NysL by either the substrate or the product. These studies open the possibility for in vitro evolution of NysL aimed at changing its specificity, thereby providing new opportunities for engineered biosynthesis of novel nystatin analogues hydroxylated at alternative positions of the macrolactone ring.

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