Toshitaka Kumagai

Sequencing of Aspergillus nidulans and comparative analysis with A. fumigatus and A. oryzae

The aspergilli comprise a diverse group of filamentous fungi spanning over 200 million years of evolution. Here we report the genome sequence of the model organism Aspergillus nidulans, and a comparative study with Aspergillus fumigatus, a serious human pathogen, and Aspergillus oryzae, used in the production of sake, miso and soy sauce. Our analysis of genome structure provided a quantitative evaluation of forces driving long-term eukaryotic genome evolution. It also led to an experimentally validated model of mating-type locus evolution, suggesting the potential for sexual reproduction in A. fumigatus and A. oryzae. Our analysis of sequence conservation revealed over 5,000 non-coding regions actively conserved across all three species. Within these regions, we identified potential functional elements including a previously uncharacterized TPP riboswitch and motifs suggesting regulation in filamentous fungi by Puf family genes. We further obtained comparative and experimental evidence indicating widespread translational regulation by upstream open reading frames. These results enhance our understanding of these widely studied fungi as well as provide new insight into eukaryotic genome evolution and gene regulation.

Genomic sequence of the pathogenic and allergenic filamentous fungus Aspergillus fumigatus.

Aspergillus fumigatus is exceptional among microorganisms in being both a primary and opportunistic pathogen as well as a major allergen. Its conidia production is prolific, and so human respiratory tract exposure is almost constant. A. fumigatus is isolated from human habitats and vegetable compost heaps. In immunocompromised individuals, the incidence of invasive infection can be as high as 50% and the mortality rate is often about 50% (ref. 2). The interaction of A. fumigatus and other airborne fungi with the immune system is increasingly linked to severe asthma and sinusitis. Although the burden of invasive disease caused by A. fumigatus is substantial, the basic biology of the organism is mostly obscure. Here we show the complete 29.4-megabase genome sequence of the clinical isolate Af293, which consists of eight chromosomes containing 9,926 predicted genes. Microarray analysis revealed temperature-dependent expression of distinct sets of genes, as well as 700 A. fumigatus genes not present or significantly diverged in the closely related sexual species Neosartorya fischeri, many of which may have roles in the pathogenicity phenotype. The Af293 genome sequence provides an unparalleled resource for the future understanding of this remarkable fungus.

Genome sequencing and analysis of Aspergillus oryzae

The genome of Aspergillus oryzae, a fungus important for the production of traditional fermented foods and beverages in Japan, has been sequenced. The ability to secrete large amounts of proteins and the development of a transformation system have facilitated the use of A. oryzae in modern biotechnology. Although both A. oryzae and Aspergillus flavus belong to the section Flavi of the subgenus Circumdati of Aspergillus, A. oryzae, unlike A. flavus, does not produce aflatoxin, and its long history of use in the food industry has proved its safety. Here we show that the 37-megabase (Mb) genome of A. oryzae contains 12,074 genes and is expanded by 7–9 Mb in comparison with the genomes of Aspergillus nidulans and Aspergillus fumigatus. Comparison of the three aspergilli species revealed the presence of syntenic blocks and A. oryzae-specific blocks (lacking synteny with A. nidulans and A. fumigatus) in a mosaic manner throughout the genome of A. oryzae. The blocks of A. oryzae-specific sequence are enriched for genes involved in metabolism, particularly those for the synthesis of secondary metabolites. Specific expansion of genes for secretory hydrolytic enzymes, amino acid metabolism and amino acid/sugar uptake transporters supports the idea that A. oryzae is an ideal microorganism for fermentation.

Ustiloxins, fungal cyclic peptides, are ribosomally synthesized in Ustilaginoidea virens

MOTIVATION Ustiloxins A and B are toxic cyclic tetrapeptides, Tyr-Val/Ala-Ile-Gly (Y-V/A-I-G), that were originally identified from Ustilaginoidea virens, a pathogenic fungus affecting rice plants. Contrary to our report that ustiloxin B is ribosomally synthesized in Aspergillus flavus, a recent report suggested that ustiloxins are synthesized by a non-ribosomal peptide synthetase in U.virens. Thus, we analyzed the U.virens genome, to identify the responsible gene cluster. RESULTS The biosynthetic gene cluster was identified from the genome of U.virens based on homologies to the ribosomal peptide biosynthetic gene cluster for ustiloxin B identified from A.flavus. It contains a gene encoding precursor protein having five Tyr-Val-Ile-Gly and three Tyr-Ala-Ile-Gly motifs for ustiloxins A and B, respectively, strongly indicating that ustiloxins A and B from U.virens are ribosomally synthesized. AVAILABILITY AND IMPLEMENTATION Accession codes of the U.virens and A.flavus gene clusters in NCBI are BR001221 and BR001206, respectively. Supplementary data are available at Bioinformatics online.

Genome Sequence of Fungal Species No.11243, Which Produces the Antifungal Antibiotic FR901469.

ABSTRACT Fungal species No.11243 was originally isolated from a decayed leaf sample collected in Kyoto, Japan. It produces FR901469, a 1,3-beta-glucan synthase inhibitor. The genome sequence of No.11243 was determined and annotated to obtain useful information for improving productivity of the effective antifungal agent FR901469.

Identification of a putative FR901469 biosynthesis gene cluster in fungal sp. No. 11243 and enhancement of the productivity by overexpressing the transcription factor gene frbF

FR901469 is an antifungal antibiotic produced by fungal sp. No. 11243. Here, we searched for FR901469 biosynthesis genes in the genome of No. 11243. Based on the molecular structure of FR901469 and endogenous functional motifs predicted in each genomic NRPS gene, a putative FR901469 biosynthesis gene cluster harboring the most plausible NRPS gene was identified. A transcription factor gene, designated frbF, was found in the cluster. To improve FR901469 productivity, we constructed a strain in which frbF was overexpressed and named it TFH2-2. FR901469 productivity of TFH2-2 was 3.4 times higher than that of the wild-type strain. Transcriptome analysis revealed that most of the genes in the putative FR901469 biosynthesis gene cluster were upregulated in TFH2-2. It also showed that the expression of genes related to ergosterol biosynthesis, β-1,3-glucan catabolism, and chitin synthesis was inclined to exhibit significant differences in TFH2-2.

Further enhancement of FR901469 productivity by co-overexpression of cpcA, a cross-pathway control gene, and frbF in fungal sp. No. 11243

FR901469 is a secondary metabolite with antifungal activity, produced by fungal sp. No. 11243. In our previous study, we constructed the frbF overexpression mutant (TFH2-2) from the wild-type strain. FR901469 productivity of TFH2-2 was 3.4 times higher than that of the wild-type strain. To further enhance FR901469 productivity in TFH2-2, we attempted to find genes from the genome that limited the productivity as bottlenecks in this study. Based on both correlation analysis of gene expression level against FR901469 productivity and genome annotation information, the cross-pathway control gene A (cpcA) was most predicted as the bottleneck. The cpcA and frbF co-overexpression mutant named TFCH3 was then constructed from TFH2-2. As a result, FR901469 productivity of TFCH3 was enhanced at 1.8 times higher than that of TFH2-2. Transcriptome analysis revealed that many genes involved in amino acid biosynthesis and encoding tRNA ligases were significantly upregulated in TFCH3, which implied increase of amino acids as the substrates of FR901469 would be a reason of further productivity enhancement.

Knockout of the SREBP system increases production of the polyketide FR901512 in filamentous fungal sp. No. 14919 and lovastatin in Aspergillus terreus ATCC20542

In the production of useful microbial secondary metabolites, the breeding of strains is generally performed by random mutagenesis. However, because random mutagenesis introduces many mutations into genomic DNA, the causative mutations leading to increased productivity are mostly unknown. Therefore, although gene targeting is more efficient for breeding than random mutagenesis, it is difficult to apply. In this study, a wild-type strain and randomly mutagenized strains of fungal sp. No. 14919, a filamentous fungus producing the HMG-CoA reductase inhibitor polyketide FR901512, were subjected to point mutation analysis based on whole genome sequencing. Among the mutated genes found, mutation of the sterol regulatory element-binding protein (SREBP) cleavage-activating protein (SCAP) had a positive effect on increasing FR901512 productivity. By complementing the SCAP gene in the SCAP-mutated strain, productivity was decreased to the level of the SCAP-intact strain. Conversely, when either the SCAP or SREBP gene was deleted, the productivity was significantly increased. By genomic transcriptional analysis, the expression levels of three enzymes in the ergosterol biosynthesis pathway were shown to be decreased by SCAP mutation. These findings led to the hypothesis that raw materials of polyketides, such as acetyl-CoA and malonyl-CoA, became more available for FR901512 biosynthesis due to depression in sterol biosynthesis caused by knockout of the SREBP system. This mechanism was confirmed in Aspergillus terreus producing the polyketide lovastatin, which is structurally similar to FR901512. Thus, knockout of the SREBP system should be considered significant for increasing the productivities of polyketides, such as HMG-CoA reductase inhibitors, by filamentous fungi.

Genome Sequence of the Fungal Strain 14919 Producing 3-Hydroxy-3-Methylglutaryl–Coenzyme A Reductase Inhibitor FR901512

ABSTRACT Fungal strain 14919 was originally isolated from a soil sample collected at Mt. Kiyosumi, Chiba Prefecture, Japan. It produces FR901512, a potent and strong 3-hydroxy-3-methylglutaryl–coenzyme A (HMG-CoA) reductase inhibitor. The genome sequence of fungal strain 14919 was determined and annotated to improve the productivity of FR901512.

Genome Sequence of Ustilaginoidea virens IPU010, a Rice Pathogenic Fungus Causing False Smut

ABSTRACT Ustilaginoidea virens is a rice pathogenic fungus that causes false smut disease, a disease that seriously damages the yield and quality of the grain. Analysis of the U. virens IPU010 33.6-Mb genome sequence will aid in the understanding of the pathogenicity of the strain, particularly in regard to effector proteins and secondary metabolic genes.