Secondary metabolites in fungus-plant interactions

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Secondary metabolites in fungus-plant interactions

Fungi and plants are rich sources of thousands of secondary metabolites. The genetically coded possibilities for secondary metabolite production, the stimuli of the production, and the special phytotoxins basically determine the microscopic fungi-host plant interactions and the pathogenic lifestyle of fungi. The rewiew introduces plant secondary metabolites usually with antifungal effect as well as the importance of signaling molecules in induced systemic resistance and systemic acquired resistance processes. The review also concerns the mimicing of plant effector molecules like auxins, gibberellins and abscisic acid by fungal secondary metabolites that modulate plant growth or even can subvert the plant defense responses such as programmed cell death to gain nutrients for fungal growth and colonization. It also looks through the special secondary metabolite production and host selective toxins of some significant fungal pathogens and the plant response in form of phytoalexin production. New results coming from genome and transcriptional analyses in context of selected fungal pathogens and their hosts are also discussed

Genome Mining-based Studies on the Biosynthesis of Terpenoids and Alkyl Salicylaldehyde Derivatives in Aspergillus ustus

Natural products (NPs), defined here as those derived from secondary metabolism, are produced by bacteria, fungi and plants. They are not essential for growth, development, and reproduction of an organism, but equip their producers with specific advantages. As a result, many secondary metabolites exhibit bioactivities that can benefit human health as drugs or drug leads. In context of the discovery of NPs, fungi serve as a prolific source. With advances in sequencing technologies and bioinformatics analysis tools the great potential deposited in genomes can be disclosed. Genetic engineering methods and improvements in analytical technologies provide the toolbox for exploiting that hidden potential of fungal genomes. The discovery of biosynthetic genes is facilitated by their cluster architecture. Based on the gene coding for the respective backbone enzyme, the produced secondary metabolites can be classified, e.g. polyketides, non-ribosomal peptides, and terpenoids. Genome mining of Aspergillus ustus 3.3904 unveiled several uncharacterized biosynthetic gene clusters (BGCs) with genes coding for different backbone enzymes and several tailoring enzymes. Among them, two putative terpene cyclases and a presumably twelve-gene BGC that are subject in this thesis. In the first project, a sesquiterpene cyclase, GdlS, was identified and confirmed as germacradienol synthase by heterologous expression and biochemical characterization including testing substrate specificity, ion dependence and determination of the kinetic parameters. Germacradienol is the key intermediate in the biosynthesis of the ‘earthy odor’ geosmin. The biosynthetic mechanism of a bifunctional germacradienol/geosmin synthase has been extensively studied in bacteria over the past 25 years. Only two reports on the biosynthesis of germacradienol and geosmin in fungi were described. Phylogenetic analysis of both N-termini und C-termini of the germacradienol/geosmin synthase SCO6073 from Streptomyces coelicolor with homologues from other bacteria and fungi revealed unequivocally the existence of distinct clades for bacteria, ascomycetous fungi, and basidiomycetous fungi. In particular, the existence of putative bifunctional enzymes in bacteria that catalyze both the conversion of FPP to germacradienol and its fragmentation to geosmin, and the existence of homologues for fungi that most likely code for two distinct enzymes catalyzing the two reactions independently. This led to the suggestion of a different strategy for germacradienol and geosmin production in bacteria and fungi. The workflow for the second project was the same as for the first project. Heterologous expression and in vitro reaction with recombinant protein unveiled MfdS as malfilanol D synthase. The enzyme’s promiscuity was tested by using different substrates and metal ions as additives. The kinetic parameters were determined. Malfilanol D is a sesquiterpenoid of the less investigated bicyclo[5.4.0]undecane class. The biosynthesis of other compounds belonging to this class, e.g. β-himachalene, is proposed via a C-1 to C-11 ring closure after isomerization of FPP to NPP. As a result of this cyclization procedure, the geminal dimethyl group is directly attached to the 6/7 fused ring. Malfilanol D and its congeners, malfilanol A – C, differ in that the geminal dimethyl group is separated from the fused ring by one CH2 group. Therefore, a different cyclization strategy is assumed. In order to get deep insights into the mechanism of MfdS, feeding with 13C labeled precursors was performed. Subsequent isolation and interpretation of the 13C NMR data provided evidence for a C-1 to C-10 cyclization with successive alkyl and hydride migrations and C-1 to C-6 ring closure to form the core skeleton of malfilanols. These results confirm a novel cyclization mechanism for sesquiterpenoids with a bicyclo[5.4.0]undecane skeleton. This work was done in collaboration with Zheng-Xi Zhang. In the third project, a putative twelve-gene psa BGC was identified with genes coding for a highly-reducing polyketide synthase (HR-PKS), several short-chain dehydrogenases / reductases (SDRs), and a cupin-domain containing protein, among other modifying enzymes. Because the formation of alkyl salicylaldehydes and derivatives requires the involvement of a HR-PKS, two SDRs, and a cupin-domain containing protein that acts as aromatase, similar cluster products were proposed. Indeed, heterologous expression of these genes confirmed this hypothesis, but with one major exception. More precisely, heterologous expression of merely psaPS (HR-PKS), psaOX1 (SDR), and psaOX2 (SDR) led to the accumulation of four compounds, 6-propyl salicylaldehyde, 6-propyl salicyl alcohol, 6-propyl salicylic acid, and a dihydro-γ-pyrone derivative. Single gene deletion proved the necessity of those three genes for the formation of the aromatic compounds. The introduction of psaCP (cupin-domain containing protein) does not increase the production of aromatic products by a water elimination catalyzed aromatization, as reported in a very recent publication. PsaCP directs the biosynthetic pathway towards the main products rather than shunt products and most likely acts as a reductase. This hypothesis will be the subject of further biochemical investigation

The effect of temperature, initial moisture content, and level of inoculum of aflatoxin and ochratoxin production in corn

Mycotoxin contamination of grains can occur as a result of invasion by field fungi or by growth of storage fungi in improperly stored crops. Environmental factors and competing mycoflora can affect fungal growth and mycotoxin production. Since mycotoxins pose a potential health hazard to both man and animals, this study was con-ducted to evaluate combinations of temperature, initial moisture content (MC), and level of inoculum during storage of corn to determine optimal and limiting conditions for aflatoxin and ochratoxin production during fungal competition and growth in pure culture. Two fungi.were used in this study, Aspergillus parasiticus NRRL 3145 and A. sulphureus NRRL 4077. Fungal spores were added to sterilized corn at levels of 101, 103, 105, and 107 per 50g corn at 16%, 20%, 24%, and 28% MC. Samples were incubated at 20, 24, 28, and 32C for 3 weeks. Aflatoxins and ochratoxin A were then extracted from corn samples and analyzed by HPLC and TLC. More toxin was produced during fungal competition than when each fungus was grown alone. Maximum production of aflatoxin B1, ochratoxin A, and total toxin was at a lower temperature (24C) in mixed culture than in pure culture. Maximum production of aflatoxin G1 occurred at 24C in both pure and mixed culture. For aflatoxin B1, G1, ochratoxin A, and total toxin in mixed culture, 24C resulted in significantly more toxin production (P\u3c0.05) than 20, 28, and 32C. In pure culture, 20C resulted in significantly less aflatoxin B1, G22, ochratoxin A, and total toxin production (P\u3c0.05) than 24, 28, and 32C. Competition affected the temperature dependent relationship of aflatoxin B1 and G1. In both pure and mixed culture, 16% was the minimum MC for toxin production. For pure cultures, 16% MC resulted in significantly less aflatoxin Bi1, aflatoxin G1, ochratoxin A, and total toxin pro duction (P\u3c0.05) than 20, 24, and 28% MC. Optimum conditions for toxin production were more limited during fungal competition. Inoculum was not a significant source of variation. Only for aflatoxin G1 and ochratoxin A in pure culture did the 101 inoculum level result in significantly less toxin production (P\u3c0.05) than the other three inoculum levels. No significant differences between inoculum levels were found in mixed culture. In summary, the presence of A. parasiticus enhanced ochratoxin A production by A. sulphureus and the presence of A. sulphureus enhanced aflatoxin production by A. parasiticus. Toxin production increased probably as a means of survival. Based on the results of this experiment, recommendations for corn storage to reduce or prevent toxin formation by A. parasiticus and A. sulphureus are: a moisture content \u3c 16%; a temperature \u3c 20%; and an inoculum level \u3c 101 spores/50g since in some cases, 10 spores per 50 grams of corn resulted in toxin production

Aflatoxin in corn new perspectives

Preharvest contamination of com (Zea mays L.) with aflatoxin, a metabolite produced by the fungus Aspergillus flavus Link: Fr., is a recurrent problem in the southeastern United States, but occasional serious outbreaks also occur in the Midwest Com Belt (21). Aflatoxins are recognized as potent hepatotoxins and carcinogens, causing mortality or reducing the productivity of farm animals (89). Aflatoxin-contaminated foodstuffs also have been associated with increased incidence of liver cancer in humans (39). In com-producing regions, the economic impact from yield loss is not very large, but A. flavus contaminates the grain with aflatoxin. Fungal toxins reduce the value of grain as an animal feed and devalue it as an export commodity (74). Any strategy that reduces the extent of aflatoxin contamination of com will result in a safer and more valuable food supply for humans and animals. Plant pathologists and com breeders have thus far been unable to identify com genotypes with substantial resistance to aflatoxin contamination. On ears in the field, A. flavus grows saprophytically on the remains of kernels damaged by insects or birds. These damaged kernels can become contaminated with substantial quantities of aflatoxin (i.e., to as much as 300,000-600,000 ppb). Aflatoxin also accumulates (to as much as 4,000 ppb) in many of the adjacent intact kernels. It does not take a large quantity of these aflatoxin-contaminated kernels to contaminate bulk grain with \u3e20 ppb aflatoxin. At present, the only reliable method for preventing aflatoxin from entering the food chain has been the detection and segregation of aflatoxin-contaminated produce

Enzymatic conversion of sterigmatocystin to aflatoxin B1.

Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1984.The age of Aspergillus parasiticus (1-11-105Wh1) mycelium was found to have an influence on the level of enzymes, responsible for the conversion of sterigmatocystin to aflatoxin B[1] and O-methylsterigmatocystin, present. These enzymes were active over a wide range of temperature and pH. Production of a cell free system by lyophiliization yielded the highest aflatoxin B[1] synthesising activity. Three other methods of preparing the cell free system capable of synthesising aflatoxin B[1] were also studied, ie,: french press, protoplast, and grinding, but with limited success. The lyophilized preparation had narrower temperature and pH optima for the conversion than whole mycelia. Initial purification of the aflatoxin B[1] synthesising enzyme was achieved by separating the crude cell free extract by gel filtration. The enzyme activity was located in a membrane fraction. The involvement of endoplasmic reticulum was indirectly concluded by the use of marker enzyme and chelating agents. This membrane fraction was ultracentrifuged and the released extrinsic proteins were separated by gel filtration. A fraction containing two proteins which were capable of converting sterigmatocystin to aflatoxin B[1] was isolated and characterised by isoelectric focusing and gel electrophoresis. The temperature and pH optima together with the cofactor requirements were studied. The Michaelis-Menten constant (Km) and the stoichiometry for the conversion of sterigmatocystin to aflatoxin B[1] was determined

Genomic Analysis of Secondary Metabolism in U. maydis

Summary Ustilago maydis is a well established model organism for the study of plant-microbe interactions although its biosynthetic potential has not been totally explored. Therefore, in this work we focused our attention on identifying potential secondary metabolite (SM) gene clusters by mining U. maydis genome. The combination of different strategies as manual annotation and bioinformatic approaches allowed us the detection of 4 potential SM gene clusters (A-D). The further selection of cluster A as a subject of this study, was based on its chromosomal location and the analysis of gene expression profiles among members of each cluster. Such analysis was possible due to the construction of an excel table in which all available U. maydis gene expression data from Gene Expression Omnibus were compiled and normalized. Overexpression of the transcription factor Mtf1 in cluster A resulted in the activation of at least 12 genes including three polyketide synthases (pks3, pks4 and pks5), a cytochrome P450 (cyp4) and a versicolorin B synthase (vbs1), among others. Prolonged induction of cluster A triggered the production of a black-greenish pigment mainly composed of 1,3,6,8-tetrahydroxynaphthalene (T4HN), therefore cluster A was named as the melanin-like cluster. This result showed that U. maydis synthesizes melanin using an unusual pathway, since most fungal melanins are derived from DHN, whose precursor is T4HN. Mutants defective for pks3, pks4, pks5 and cyp4 did not accumulate melanin, indicating a crucial role of these genes at the first stages of its biosynthesis. Deletion of cyp4 produced orsellinic acid (OA) and two of its derivatives. Interestingly, a feeding experiment with OA rescued the melanization defect of pks3 and pks4 deletion mutants. Moreover, the simultaneous expression of the pks3 and pks4 genes produced OA, suggesting that both genes are involved in OA biosynthesis, which is then used as a substrate for further chemical conversion into T4HN, a reaction presumably catalyzed by Cyp4 and/or Pks5. Overexpression of pks1, a polyketide synthase gene in U. maydis previously reported to play a role in melanization together with pks2 and lac1, could rescue the phenotype in the strain MB215 pks3 Pcrg::mtf1, suggesting that Pks3 and Pks1 have complementary functions. Maize seedlings infected with single deletion mutants of the melanin-like cluster genes showed no effect on spore coloration and had only a minor effect on virulence, supporting the previous finding that pks1 and pks2 are the major contributors of melanization during sporulation. On the other hand, SG200 pks3, SG200 pks4 and SG200 pks5 showed no significant differences compared to the wild type (SG200) when exposed to hydrogen peroxide, indicating that melanin-like cluster genes may be involved in other kind of stress responses, hence further experiments need to be performed to understand the conditions under which the melanin-like cluster is activated

The architecture of polyketide synthases

Since the discovery of penicillin over a century ago, secondary metabolites from all kingdoms of life have proven to be of high medical value. One class of proteins prevalent in the production of secondary metabolites are polyketide synthases (PKSs). Their polyketide products are complex organic compounds based on carbon chains assembled from carboxylic acid precursors. Many polyketides are produced by their hosts with the primary purpose of gaining an advantage in their ecological niche. To contribute to such an advantage, a significant proportion of polyketides are active against pro- and eukaryotic microorganisms. Type I PKSs are giant multienzyme proteins employing an assembly line logic for the synthesis of the most complex polyketides. They are composed of one or more functional and structural modules, each capable of carrying out one step of precursor elongation during the formation of an extended polyketide product. In this thesis, I address two fundamental and open questions in the biosynthesis of polyketides: First, what is the unique architecture underlying the assembly line logic of multimodular PKS assembly lines; and second, how is atomic accuracy achieved in cyclization and aromatic ring formation in the final step of PKS action. The first aim is addressed in chapter two, which provides for the first time detailed structural insights into the organization of type I PKS multimodules. This is achieved by cryo-electron microscopic analysis of filamentous and non-filamentous forms of K3DAK4, a bimodular trans-acyltransferase (AT) PKS fragment from Brevibacillus brevis. Overall reconstructions are provided at an intermediate resolution of 7 Å, with detailed insights into individual domains at sub-3Å resolution from cryo-electron microscopy and X-ray crystallography. The bimodule core displays a vertical stacking of its two modules along the central dimer axis of all three enzymatic domains involved. Additionally, K3DAK4 oligomerizes into filaments horizontally via small scaffolding domains in a trans-AT PKS-specific manner. In chapter three the second aim is tackled, as I visualize an intermediate of the enigmatic targeted cyclization and aromatic ring formation in the product template domain (PT) of the aflatoxin-producing PksA at 2.7 Å resolution using X-ray crystallography. To this end a substrate-analogue mimicking the transient intermediate after the first of two cyclization steps facilitated by the enzyme is covalently crosslinked to the active site. The positioning of the ligand relative to previously known ligands representing the pre-and post-cyclization states indicate an outward movement of the substrate throughout the process and a substantial effect of progressing cyclization on the meticulous positioning of the intermediates. The work provides detailed insights into core aspects of PKS biology from the atomistic picture of guided product modification to the giant overall assembly line architecture. In chapter four, both of these levels are put into context with current advances in the analysis of modular structure and dynamics of PKSs, such as recent structural models of cis-AT PKS modules and iterative PKSs. Furthermore, it addresses currently open questions, such as the interaction of trans-AT PKS with their cognate trans-acting enzymes. Altogether, the current progress in mechanistic understanding of PKS systems makes systematic and structure-guided efforts to unleash the full potential of PKS bioengineering ever more achievable

Biosynthesis of alkaloids from Penicillium palitans and malfilanol D from Aspergillus ustus

Natural products (NPs), strict limitation to secondary metabolites (SMs), have been noted to exhibit high structural diversity and complexity including examples such as alkaloids, phenylpropanoids, polyketides, and terpenoids with their unique pharmacological or biological activities. Many potent antibiotics were successively discovered from ascomycetous fungi Penicillium, Cephalosporium, Aspergillus, etc. Driven by that, more and more discoveries of natural products were conducted, leading to the dereplication. The drawback is obvious. Traditional isolation from natural resources is unable to meet the demands of medicinal treatment because of technical barriers, low yield, and damage to natural organisms. The lead compounds with complex structures, e.g., functional groups with chiral centers and unstable molecules with rearrangement, are difficult to be synthesized due to lengthy and jumbled steps and low yield. With the help of genetic tools, cell factories become one of the approaches to address such challenges by de novo design of biosynthetic pathways. To elucidate the biosynthetic mechanism, advanced bioinformatics, biological technologies, and biochemical tools have been utilized to investigate the coding genes, which are usually located together as a biosynthetic gene cluster (BGC). BGC normally contains backbone enzyme(s) responsible for NPs’ scaffold, and tailoring enzymes responsible for skeleton’s post-modification. Enzyme-mediated reactions extremely increase the diversity of NP’s structures. Besides, nonenzymatic reactions also occur occasionally in the formation of NPs. In this thesis, the biosynthesis of alkaloids from Penicillium palitans was elucidated in cooperation with Zhanghai Li, and the formation mechanism of a sesquiterpenoid from Aspergillus ustus by isotopic labelling was carried out in cooperation with Marlies Peter. To investigate the alkaloid biosynthesis in P. palitans (projects 3.1 and 3.2), the SMs were uncovered first. Cultivation of P. palitans in mCDH medium at 25 °C for 15 days and LC-MS analysis revealed the presence of at least eight metabolites. Among them, alkaloids cyclopenol (1), viridicatol (6), and cyclopiazonic acid (7, CPA) as dominant products, and speradine F (8) as a minor product were identified from the wild type. Then, related genome mining was performed via bioinformatics analysis. NRPS- or PKS-NRPS-derived three alkaloids containing amino acids were further investigated due to the enzymatic potentials in their corresponding BGCs for the formation of the meta-hydroxylation at a monosubstituted benzene ring and highly oxygenated tetrahydrofuran. Genome mining, gene deletion, and heterologous expression led to the identification of two separate clusters, the four-gene vdo cluster responsible for cyclopenin (2) skeleton formation and the five-gene spe cluster for the CPA skeleton formation. Feeding experiments proved that the cytochrome P450 enzyme VdoD catalyzes the key step in the conversion of cyclopenin (2) to cyclopenol (1), demonstrating a less studied enzymatic meta-hydroxylation at a monoalkylated benzene ring. Heterologous expression (HE) established that spe cluster is responsible for the formation of CPA and its derivatives (7 – 12), and the highly oxygenated speradine F (8) was synthesized by multiple nonenzymatic hydroxylations. The study completes the biosynthesis of viridicatol and speradine F and illustrates two hydroxylation reactions in biosynthesis. For the identification of catalytic mechanism of the sesquiterpenoid malfilanol D (project 3.3), we carried out genome analysis at the very beginning, then proved the function by HE, characterization biochemically with recombinant protein, and isotopic labelling experiments. Sequence analysis of the genome of A. ustus led to the identification of an uncharacterized putative terpene cyclase, termed mfdS hereafter. HE in the model organism Aspergillus nidulans LO8030 was used to prove the gene function. An additional metabolite was detected by LC-MS and then isolated from the culture of the transformant. Interpretation of the NMR data, including 1H NMR, 13C NMR, 1H-1H COSY, HSQC, HMBC, and 1H-1H NOESY revealed that the conspicuous peak is a new compound malfilanol D (13). To prove that MfdS is solely responsible for the formation of malfilanol D, enzyme assay of farnesyl diphosphate (FPP, C15) with the recombinant protein was conducted. Additionally, based on sodium 13C-acetate labelling experiments, a novel sesquiterpene cyclase from A. ustus was demonstrated in the biosynthesis of the less investigated bicyclo[5.4.0]undecane sesquiterpenes. 1,2-alkyl shift of allylic cation leads to ring expansion and successive 1,2-hydride transfer, C-1 to C-6 ring closure, and capture of water, provides the final product malfilanol D. The three cases, investigating products derived from NRPS, PKS-NRPS, and TC from ascomycetous fungus Penicillium palitans and Aspergillus ustus, illustrated that fungi are a promising pool to exploit bioactive SMs and interesting enzymes by elucidation of their complete biosynthesis