Identification and characterization of the asperthecin gene cluster of Aspergillus nidulans

The sequencing of Aspergillus genomes has revealed that the products of a large number of secondary metabolism pathways have not yet been identified. This is probably because many secondary metabolite gene clusters are not expressed under normal laboratory culture conditions. It is, therefore, important to discover conditions or regulatory factors that can induce the expression of these genes. We report that the deletion of sumO, the gene that encodes the small ubiquitin-like protein SUMO in A. nidulans, caused a dramatic increase in the production of the secondary metabolite asperthecin and a decrease in the synthesis of austinol/dehydroaustinol and sterigmatocystin. The overproduction of asperthecin in the sumO deletion mutant has allowed us, through a series of targeted deletions, to identify the genes required for asperthecin synthesis. The asperthecin biosynthesis genes are clustered and include genes encoding an iterative type I polyketide synthase, a hydrolase, and a monooxygenase. The identification of these genes allows us to propose a biosynthetic pathway for asperthecin.     

Phosphopantetheinyl transferase CfwA/NpgA is required for Aspergillus nidulans secondary metabolism and asexual development

Polyketide synthases (PKSs) and/or nonribosomal peptide synthetases (NRPSs) are central components of secondary metabolism in bacteria, plants, and fungi. In filamentous fungi, diverse PKSs and NRPSs participate in the biosynthesis of secondary metabolites such as pigments, antibiotics, siderophores, and mycotoxins. However, many secondary metabolites as well as the enzymes involved in their production are yet to be discovered. Both PKSs and NRPSs require activation by enzyme members of the 4'-phosphopantetheinyl transferase (PPTase) family. Here, we report the isolation and characterization of Aspergillus nidulans strains carrying conditional (cfwA2) and null (DeltacfwA) mutant alleles of the cfwA gene, encoding an essential PPTase. We identify the polyketides shamixanthone, emericellin, and dehydroaustinol as well as the sterols ergosterol, peroxiergosterol, and cerevisterol in extracts from A. nidulans large-scale cultures. The PPTase CfwA/NpgA was required for the production of these polyketide compounds but dispensable for ergosterol and cerevisterol and for fatty acid biosynthesis. The asexual sporulation defects of cfwA, DeltafluG, and DeltatmpA mutants were not rescued by the cfwA-dependent compounds identified here. However, a cfwA2 mutation enhanced the sporulation defects of both DeltatmpA and DeltafluG single mutants, suggesting that unidentified CfwA-dependent PKSs and/or NRPSs are involved in the production of hitherto-unknown compounds required for sporulation. Our results expand the number of known and predicted secondary metabolites requiring CfwA/NpgA for their biosynthesis and, together with the phylogenetic analysis of fungal PPTases, suggest that a single PPTase is responsible for the activation of all PKSs and NRPSs in A. nidulans.     

Aspergillus nidulans verA is required for production of the mycotoxin sterigmatocystin.

Aspergillus nidulans produces the carcinogenic mycotoxin sterigmatocystin (ST), the next-to-last precursor in the aflatoxin (AF) biosynthetic pathway found in the closely related fungi Aspergillus flavus and Aspergillus parasiticus. We identified and characterized an A. nidulans gene, verA, that is required for converting the AF precursor versicolorin A to ST. verA is closely related to several polyketide biosynthetic genes involved in polyketide production in Streptomyces spp. and exhibits extended sequence similarity to A. parasiticus ver-1, a gene proposed to encode an enzyme involved in converting versicolorin A to ST. By performing a sequence analysis of the region 3' to verA, we identified two additional open reading frames, designated ORF1 and ORF2. ORF2 is closely related to a number of cytochrome P-450 monooxygenases, while ORF1 shares identity with the gamma subunit of translation elongation factor 1. Given that several steps in the ST-AF pathway may require monooxygenase activity and that AF biosynthetic genes are clustered in A. flavus and A. parasiticus, we suggest that verA may be part of a cluster of genes required for ST biosynthesis. We disrupted the verA coding region by inserting the A. nidulans argB gene into the center of the coding region and transformed an A. nidulans argB2 mutant to arginine prototrophy. Seven transformants that produced DNA patterns indicative of a verA disruption event were grown under ST-inducing conditions, and all of the transformants produced versicolorin A but negligible amounts of ST (200-fold to almost 1,000-fold less than the wild type), confirming the hypothesis that verA encodes an enzyme necessary for converting versicolorin A to ST.     

GAC1, a gene encoding a putative GTPase-activating protein, regulates bikaverin biosynthesis in Fusarium verticillioides

Fusarium verticillioides (teleomorph Gibberella moniliformis) is an ascomycete known to produce a variety of secondary metabolites, including fumonisins, fusaric acid and bikaverin. These metabolites are synthesized when the fungus is under stress, notably nutrient limitations. To date we have limited understanding of the complex regulatory process associated with fungal secondary metabolism. In this study we generated a collection of F. verticillioides mutants by using REMI (restriction enzyme mediated integration) mutagenesis and in the process identified a strain, R647, that carries a mutation in a gene designated GAC1. Mutation in the GACI locus, which encodes a putative GTPase activating protein, resulted in the increased production of bikaverin, suggesting that GAC1 is negatively associated with bikaverin biosynthesis. Complementation of R647 with the wildtype GAC1 gene restored the bikaverin production level to that of the wild-type progenitor, demonstrating that gac1 mutation was directly responsible for the overproduction of bikaverin. We also demonstrated that AREA, encoding global nitrogen regulator, and PKS4, encoding polyketide synthase, are downstream genes that respectively are regulated positively and negatively by GAC1. Our results suggest that GAC1 plays an important role in signal transduction regulating bikaverin production in F. verticillioides.     

A putative high affinity hexose transporter, hxtA, of Aspergillus nidulans is induced in vegetative hyphae upon starvation and in ascogenous hyphae during cleistothecium formation

Fungi employ different carbohydrate uptake systems to adapt to certain environmental conditions and to different carbon source concentrations. The hydrolysis of polymeric carbohydrates and the subsequent uptake of monomeric forms may also play a role in development. Aspergillus nidulans accumulates cell wall components during vegetative growth and degrades them during sexual development. We have identified the hxtA (high affinity hexose transporter) gene in a differential library, which was enriched for sexual-specific genes. The hxtA gene is disrupted by 6 introns and predicted to encode a 531 amino acid protein with high similarity to major facilitator superfamily members including the high affinity hexose transporter Gtt1 from Trichoderma harzianum. A. nidulans HxtA contains the 12 predicted transmembrane domains characteristic for this family. Deletion of hxtA did not impair growth of A. nidulans on a variety of carbon sources nor did it inhibit sexual development suggesting redundant sugar uptake systems. We found at least 17 putative hexose transporters in the genome of A. nidulans. Despite the high similarity of HxtA to fungal high affinity glucose transporters, the hxtA gene did not restore growth on glucose of a Saccharomyces cerevisiae mutant, in which all hexose transporters were deleted. Northern blot analysis revealed that the A. nidulans hxtA gene was repressed under high glucose conditions and expressed in vegetative hyphae upon carbon starvation and during sexual development. We found hxtA(p)::sgfp expression in developing cleistothecia specifically in ascogenous hyphae and propose that HxtA is a high affinity glucose transporter involved in sugar metabolism during sexual development.     

Biochemical characteristization of propionyl-coenzyme a carboxylase complex of <Emphasis Type="Italic">Streptomyces toxytricini</Emphasis>

Acyl-CoA carboxylases (ACC) are involved in important primary or secondary metabolic pathways such as fatty acid and/or polyketides synthesis. In the 62 kb fragment of pccB gene locus of Streptomyces toxytricini producing a pancreatic inhibitor lipstatin, 3 distinct subunit genes of presumable propionyl-CoA carboxylase (PCCase) complex, assumed to be one of ACC responsible for the secondary metabolism, were identified along with gene for a biotin protein ligase (Bpl). The subunits of PCCase complex were a subunit (AccA3), P subunit (PccB), and auxiliary ɛ subunit (PccE). In order to disclose the involvement of the PCCase complex in secondary metabolism, some biochemical characteristics of each subunit as well as their complex were examined. In the test of substrate specificity of the PCCase complex, it was confirmed that this complex showed much higher conversion of propionyl-CoA rather than acetyl-CoA. It implies the enzyme complex could play a main role in the production of methylmalonyl-CoA from propionyl-CoA, which is a precursor of secondary polyketide biosynthesis.     

Understanding substrate specificity of polyketide synthase modules by generating hybrid multimodular synthases

Modular polyketide biosynthesis can be harnessed to generate rationally designed complex natural products through bioengineering. A detailed understanding of the features that govern transfer and processing of polyketide biosynthetic intermediates is crucial to successfully engineer new polyketide pathways. Previous studies have shown that substrate stereochemistry and protein-protein interactions between polyketide synthase modules are both important factors in this process. Here we investigated the substrate tolerance of different polyketide modules and assessed the relative importance of inter-module chain transfer versus chain elongation activity of some of these modules. By constructing a variety of hybrid modular polyketide synthase systems and assaying their ability to generate polyketide products, it was determined that the substrate tolerance of each individual ketosynthase domain is an important parameter for the successful recombination of polyketide synthase modules. Surprisingly, however, failure by a module to process a candidate substrate was not due to its inability to bind to it. Rather, it appeared to result from a blockage in carbon-carbon bond formation, suggesting that proper orientation of the initially formed acyl thioester in the ketosynthase active site was important for the enzyme-catalyzed decarboxylative condensation reaction.     

Methylcitrate synthase from Aspergillus fumigatus. Propionyl-CoA affects polyketide synthesis, growth and morphology of conidia

Methylcitrate synthase is a key enzyme of the methylcitrate cycle and required for fungal propionate degradation. Propionate not only serves as a carbon source, but also acts as a food preservative (E280-283) and possesses a negative effect on polyketide synthesis. To investigate propionate metabolism from the opportunistic human pathogenic fungus Aspergillus fumigatus, methylcitrate synthase was purified to homogeneity and characterized. The purified enzyme displayed both, citrate and methylcitrate synthase activity and showed similar characteristics to the corresponding enzyme from Aspergillus nidulans. The coding region of the A. fumigatus enzyme was identified and a deletion strain was constructed for phenotypic analysis. The deletion resulted in an inability to grow on propionate as the sole carbon source. A strong reduction of growth rate and spore colour formation on media containing both, glucose and propionate was observed, which was coincident with an accumulation of propionyl-CoA. Similarly, the use of valine, isoleucine and methionine as nitrogen sources, which yield propionyl-CoA upon degradation, inhibited growth and polyketide production. These effects are due to a direct inhibition of the pyruvate dehydrogenase complex and blockage of polyketide synthesis by propionyl-CoA. The surface of conidia was studied by electron scanning microscopy and revealed a correlation between spore colour and ornamentation of the conidial surface. In addition, a methylcitrate synthase deletion led to an attenuation of virulence, when tested in an insect infection model and attenuation was even more pronounced, when whitish conidia from glucose/propionate medium were applied. Therefore, an impact of methylcitrate synthase in the infection process is discussed.     

On the mechanism of action of the antifungal agent propionate.

Propionate is used to protect bread and animal feed from moulds. The mode of action of this short-chain fatty acid was studied using Aspergillus nidulans as a model organism. The filamentous fungus is able to grow slowly on propionate, which is oxidized to acetyl-CoA via propionyl-CoA, methylcitrate and pyruvate. Propionate inhibits growth of A. nidulans on glucose but not on acetate; the latter was shown to inhibit propionate oxidation. When grown on glucose a methylcitrate synthase deletion mutant is much more sensitive towards the presence of propionate in the medium as compared to the wild-type and accumulates 10-fold higher levels of propionyl-CoA, which inhibits CoA-dependent enzymes such as pyruvate dehydrogenase, succinyl-CoA synthetase and ATP citrate lyase. The most important inhibition is that of pyruvate dehydrogenase, as this affects glucose and propionate metabolism directly. In contrast, the blocked succinyl-CoA synthetase can be circumvented by a succinyl-CoA:acetate/propionate CoA-transferase, whereas ATP citrate lyase is required only for biosynthetic purposes. In addition, data are presented that correlate inhibition of fungal polyketide synthesis by propionyl-CoA with the accumulation of this CoA-derivative. A possible toxicity of propionyl-CoA for humans in diseases such as propionic acidaemia and methylmalonic aciduria is also discussed.     

Molecular characterization of a blue-copper laccase, TILA, of Aspergillus nidulans

Laccases are blue-copper enzymes, which oxidize phenolic substrates and thereby reduce molecular oxygen. They are widespread within fungi and are involved in lignin degradation or secondary metabolism such as pigment biosynthesis. Many fungi contain several laccases, not all of whose functions are known. In Aspergillus nidulans one, yA, is expressed during asexual development and converts a yellow precursor to the green pigment. We identified a second laccase gene, which encodes a 66.3-kDa protein 37.6% identical to laccase I of A. nidulans. The protein harbors an N-terminal secretion signal, and three characteristic copper-binding centers. The enzyme localizes at the growing hyphal tip. The gene was therefore named tilA (=tip laccase). Deletion or overexpression of the gene had no discernible phenotype under laboratory conditions.     

宏基因组文库技术获得聚酮化合物

聚酮化合物是一类重要的具有生物活性的次级代谢物。由于运用传统方法从自然界中直接筛选新型天然聚酮化合物的重现率很高, 近年来出现了很多开发新型聚酮化合物的新方法, 文章主要介绍通过环境宏基因组文库获得新聚酮类化合物的方法。     

Genomics reveals traces of fungal phenylpropanoid-flavonoid metabolic pathway in the f ilamentous fungus Aspergillus oryzae

Fungal secondary metabolites constitute a wide variety of compounds which either play a vital role in agricultural, pharmaceutical and industrial contexts, or have devastating effects on agriculture, animal and human affairs by virtue of their toxigenicity. Owing to their beneficial and deleterious characteristics, these complex compounds and the genes responsible for their synthesis have been the subjects of extensive investigation by microbiologists and pharmacologists. A majority of the fungal secondary metabolic genes are classified as type I polyketide synthases (PKS) which are often clustered with other secondary metabolism related genes. In this review we discuss on the significance of our recent discovery of chalcone synthase (CHS) genes belonging to the type III PKS superfamily in an industrially important fungus, Aspergillus oryzae. CHS genes are known to play a vital role in the biosynthesis of flavonoids in plants. A comparative genome analyses revealed the unique character of A. oryzae with four CHS-like genes (csyA, csyB, csyC and csyD) amongst other Aspergilli (Aspergillus nidulans and Aspergillus fumigatus) which contained none of the CHS-like genes. Some other fungi such as Neurospora crassa, Fusarium graminearum, Magnaporthe grisea, Podospora anserina and Phanerochaete chrysosporium also contained putative type III PKSs, with a phylogenic distinction from bacteria and plants. The enzymatically active nature of these newly discovered homologues is expected owing to the conservation in the catalytic residues across the different species of plants and fungi, and also by the fact that a majority of these genes (csyA, csyB and csyD) were expressed in A. oryzae. While this finding brings filamentous fungi closer to plants and bacteria which until recently were the only ones considered to possess the type III PKSs, the presence of putative genes encoding other principal enzymes involved in the phenylpropanoid and flavonoid biosynthesis (viz., phenylalanine ammonia-lyase, cinnamic acid hydroxylase and p-coumarate CoA ligase) in the A. oryzae genome undoubtedly prove the extent of its metabolic diversity. Since many of these genes have not been identified earlier, knowledge on their corresponding products or activities remain undeciphered. In future, it is anticipated that these enzymes may be reasonable targets for metabolic engineering in fungi to produce agriculturally and nutritionally important metabolites.     

Fundamental contribution of beta-oxidation to polyketide mycotoxin production in planta

Seed contamination with polyketide mycotoxins, including aflatoxin (AF) and sterigmatocystin (ST) produced by Aspergillus spp., is an agricultural, economic, and medical issue worldwide. Acetyl-CoA, the fundamental building block of all known fungal polyketides, is generated by a large number of biochemical pathways, including beta-oxidation of fatty acids and glycolysis of sugars. We present several lines of evidence to support a major role for seed fatty acids in formation of AF and ST in A. flavus, A. parasiticus, and A. nidulans. Aspergillus strains exhibiting canonical signs of oleic acid-induced peroxisome proliferation, including increased catalase activity, beta-oxidation gene expression, and peroxisomal clustering, also exhibited a marked increase in toxin gene expression and biosynthesis. Furthermore, microscopic observations showed that the ST and AF precursor norsolorinic acid accumulated in peroxisomes of all three Aspergilli. While a peroxisomal beta-oxidation mutation eliminated oleic acid-induced increases in ST in A. nidulans, a mitochondrial beta-oxidation mutation played a larger role in eliminating ST formation on oatmeal medium and on live corn kernels, implicating a fundamental role for both peroxisomal and mitochondrial beta-oxidation in toxin production.     

Versatile enzyme expression and characterization system for Aspergillus nidulans, with the Penicillium brevicompactum polyketide synthase gene from the mycophenolic acid gene cluster as a test case

Assigning functions to newly discovered genes constitutes one of the major challenges en route to fully exploiting the data becoming available from the genome sequencing initiatives. Heterologous expression in an appropriate host is central in functional genomics studies. In this context, filamentous fungi offer many advantages over bacterial and yeast systems. To facilitate the use of filamentous fungi in functional genomics, we present a versatile cloning system that allows a gene of interest to be expressed from a defined genomic location of Aspergillus nidulans. By a single USER cloning step, genes are easily inserted into a combined targeting-expression cassette ready for rapid integration and analysis. The system comprises a vector set that allows genes to be expressed either from the constitutive PgpdA promoter or from the inducible PalcA promoter. Moreover, by using the vector set, protein variants can easily be made and expressed from the same locus, which is mandatory for proper comparative analyses. Lastly, all individual elements of the vectors can easily be substituted for other similar elements, ensuring the flexibility of the system. We have demonstrated the potential of the system by transferring the 7,745-bp large mpaC gene from Penicillium brevicompactum to A. nidulans. In parallel, we produced defined mutant derivatives of mpaC, and the combined analysis of A. nidulans strains expressing mpaC or mutated mpaC genes unequivocally demonstrated that mpaC indeed encodes a polyketide synthase that produces the first intermediate in the production of the medically important immunosuppressant mycophenolic acid.     

Streptomyces coelicolor DNA homologous with acyltransferase domains of type I polyketide synthase gene complex

An acyltransferase-homologous DNA fragment was amplified in a PCR reaction on a cosmid DNA template from the genomic DNA library of the soil bacterium Streptomyces coelicolor A3(2). The putative amino acid sequence of the fragment resembles acyl-CoA:ACP acyltransferase domains from several bacterial enzymatic complexes of polyketide synthase. There is a high similarity with acyltransferase domains from so-called type I polyketide synthases. Such synthases catalyze production of the aglycone portion of macrolides and polyethers that are important as antibiotics or immunosuppressants. The amplified fragment is considered to be a part of a larger gene complex.