Akimitsu Tanaka

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.

Analysis of Expressed Sequence Tags from the Fungus Aspergillus oryzae Cultured Under Different Conditions

Abstract We performed random sequencing of cDNAs from nine biologically or industrially important cultures of the industrially valuable fungus Aspergillus oryzae to obtain expressed sequence tags (ESTs). Consequently, 21 446 raw ESTs were accumulated and subsequently assembled to 7589 non-redundant consensus sequences (contigs). Among all contigs, 5491 (72.4%) were derived from only a particular culture. These included 4735 (62.4%) singletons, i.e. lone ESTs overlapping with no others. These data showed that consideration of culture grown under various conditions as cDNA sources enabled efficient collection of ESTs. BLAST searches against the public databases showed that 2953 (38.9%) of the EST contigs showed significant similarities to deposited sequences with known functions, 793 (10.5%) were similar to hypothetical proteins, and the remaining 3843 (50.6%) showed no significant similarity to sequences in the databases. Culture-specific contigs were extracted on the basis of the EST frequency normalized by the total number for each culture condition. In addition, contig sequences were compared with sequence sets in eukaryotic orthologous groups (KOGs), and classified into the KOG functional categories.

Isolation of genes encoding novel transcription factors which interact with the Hap complex from Aspergillus species

The Saccharomyces cerevisiae CCAAT-binding factor is composed of four subunits Hap2p, Hap3p, Hap4p and Hap5p. Three subunits, Hap2/3/5p, are required for DNA-binding and Hap4p is involved in transcriptional activation. Although homologues of Hap2/3/5p (in the case of Aspergillus nidulans; HapB/C/E, respectively) were found in many eukaryotes, no Hap4p homologues have been found except for the other yeast, Kluyveromyces lactis. With the lexA-hap2, -hapB, -hapC, or -hapE fusion gene, we evaluated the ability of interaction between Aspergillus Hap subunits and S. cerevisiae Hap4p subunit in S. cerevisiae. Using the system with lexA-hapB, a gene encoding a novel transcriptional activator, which interacted with the Hap complex, was isolated from A. nidulans and designated hapX.

An Aspergillus oryzae CCAAT-binding protein, AoCP, is involved in the high-level expression of the Taka-amylase A gene.

Aspergillus oryzae contains a nuclear protein designated AoCP, which binds specifically to a CCAAT sequence in the promoter region of the A. oryzae Taka-amylase A gene. A gene encoding a homologue of Aspergillus nidulans HAPC, a subunit of the A. nidulans CCAAT binding complex, was isolated from A. oryzae and designated AohapC. AoHAPC comprises 215 amino acids and shows 84% identity to A. nidulans HAPC. Transformation of the A. nidulans hapC deletion strain with the AohapC gene restored the CCAAT binding activity, resulting in both enhancement of taa gene expression and complementation for the poor growth phenotype of this strain. Furthermore, introduction of the AohapC gene also restored the expression of the A. nidulans eglA gene, encoding an endo-β-1,4-glucanase, in the deletion strain. These results indicate functional interchangeability of the genes from two species.

AoHapB, AoHapC and AoHapE, subunits of the Aspergillus oryzae CCAAT-binding complex, are functionally interchangeable with the corresponding subunits in Aspergillus nidulans

Abstract. Two genes, AohapB and AohapE, encoding subunits of the Aspergillus oryzae CCAAT-binding complex were cloned and sequenced. The polypeptides encoded by AohapB and AohapE were expressed in Escherichia coli and used to reconstitute a DNA-binding complex with recombinant AoHapC. The DNA-binding activity was observed only in the presence of all three subunits, indicating that AoHapB, AoHapE and AoHapC are essential for CCAAT- binding. Furthermore, introduction of the AohapB, AohapC and AohapE genes into the A. nidulanshapBΔ, hapCΔ and hapEΔ strains, respectively, revealed that the A. oryzae Hap subunits are functionally interchangeable with the corresponding subunits in A. nidulans.

The Region in a Subunit of the Aspergillus CCAAT-Binding Protein Similar to the HAP4p-Recruiting Domain of Saccharomyces cerevisiae Hap5p Is Not Essential for Transcriptional Enhancement

The CCAAT-binding complex in Aspergillus species, known as the Hap complex, consists of at least three subunits, HapB, HapC, and HapE. Each Hap subunit contains an evolutionarily conserved core domain. In this study, a series of the truncated gene, which encodes the HapE subunit of Aspergillus oryzae, was constructed to survey the regions essential for the transcriptional enhancement of fungal genes. It was revealed that the non-conserved regions and the conserved region similar to the Hap4p recruiting domain of Saccharomyces cerevisiae were not necessary for Hap complex-mediated transcriptional enhancement.

A quantity control mechanism regulating levels of the HapE subunit of the Hap complex in Aspergillus nidulans: no accumulation of HapE in hapC deletion mutants.

The Aspergillus nidulans CCAAT‐binding complex (Hap complex) consists of at least three subunits, HapB, HapC and HapE. To investigate the quantity control mechanisms of the subunits during assembly of the Hap complex, reconstitution studies with the recombinant subunits and extracts prepared from the respective hap subunit deletion mutants were carried out. Furthermore, Western blot analysis of the Hap subunits and Northern blot analysis of the hap genes with the respective deletion mutants were also performed. From all the results together, it was suggested that the number of the HapC molecule could adjust that of the HapE molecule by forming stable heterodimers prior to assembly of the Hap complex.

Structural features of the glycogen branching enzyme encoding genes from aspergilli

A maltose binding protein, p78, was purified to homogeneity from Aspergillus nidulans by a single column chromatography step on cross-linked amylose. The partial amino acid sequence was highly homologous to the glycogen branching enzymes (GBEs) of human and yeast, and p78 did show branching enzyme activity. The genomic gene and its cDNA encoding GBE (p78) were isolated from the A. nidulans genomic and cDNA libraries. Furthermore, a cDNA encoding A. oryzae GBE was entirely sequenced. A. nidulans GBE shared overall and significant amino acid sequence identity with GBEs from A. oryzae (83.9%), Saccharomyces cerevisiae (61.1%) and human (63.0%), and with starch branching enzymes from green plants (55-56%).