Yoji Hata

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 7–9u2009Mb 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.

Regulation of the glucoamylase-encoding gene (glaB), expressed in solid-state culture (Koji) of Aspergillus oryzae

Abstract Aspergillus oryzae has two glucoamylase-encoding genes, glaA and glaB, the patterns of expression of which are different. Expression of the glaB gene is marked in solid-state culture (koji), but low in submerged culture. To elucidate the induction mechanism of the glaB promoter in solid-state culture (koji), we employed a fusion gene system using the glaA or glaB promoter and the Escherichia coli uidA gene encoding β-glucuronidase (GUS). The expression of glaB-GUS was induced by starch or maltooligosaccharides in a similar manner to that glaA-GUS, but other physical factors were found to be required for the maximal expression of the glaB gene in solid-state culture (koji). The time-course of glaB-GUS expression in solid-state culture (rice-koji making) suggested that its expression is induced by low water activity (Aw) of the medium and high temperature. When mycelia grown on a membrane under standard conditions were transferred to low-Aw and high-temperature conditions (membrane-transfer culture, MTC), glaB expression was markedly induced, but that of glaA was not. Additionally, glaB-GUS production was induced in MTC using a membrane with smaller pore size, suggesting that a physical barrier against hyphal extension could regulate glaB expression. Under conditions found to induce glaB expression, namely, starch, low-Aw, high-temperature and physical barriers, approximately 6400 U/mg-protein was obtained, equivalent to that in solid-state culture (koji). In conclusion, glucoamylase production under these induction conditions achieved in MTC reached 274 U/ml-broth, which was equivalent to the level observed in solid-state culture (koji). Northern blot analysis indicated that glaB expression was induced at the level of transcription 4 h after the transfer to the inducible conditions described above.

Comparison of two glucoamylases produced by Aspergillus oryzae in solid-state culture (koji) and in submerged culture

Abstract Two extracellular glucoamylases (EC 3.2.1.3) of Aspergillus oryzae were purified from solid-state culture (S-GA) and from submerged culture (L-GA). The two glucoamylases have different molecular masses, 65 kDa for L-GA; 63–99 kDa for S-GA, and different isoelectric points, 4.2 for L-GA; 3.9 for S-GA. Almost all of the enzymatic characteristics of the two glucoamylases were similar, except for thermal stability, initial reaction velocity on pullulan and K m value with soluble starch. Although L-GA could digest raw starch, S-GA demonstrated little activity with raw starch. Peptide mapping and amino acid composition showed that L-GA must be encoded by the glaA gene previously cloned as the glucoamylase-encoding gene from A. oryzae , but S-GA had a different primary structure than the deduced glaA product. Introduction of multiple copies of the glaA gene to A. oryzae caused on elevation of glucoamylase productivity of transformant in submerged culture but not in solid-state culture. These results suggested that the two forms of glucoamylases arise from different genes rather than result from proteolytic processing after polypeptide synthesis of a single protein.

Nucleotide sequence of an alternative glucoamylase-encoding gene (glaB) expressed in solid-state culture of Aspergillus oryzae.

The DNA (glaB) and a cDNA-encoding glucoamylase produced in solid-state culture of Aspergillus oryzae were cloned using oligodeoxyribonucleotide probes derived from internal amino acid sequences of the enzyme. Comparison of the nucleotide sequences of a genomic DNA fragment with its cDNA showed the glaB gene carried three exons interrupted by two introns and had an open reading frame encoding 493 aa residues. The 5-flanking region had a TATA box at nt -87 from the start codon and two putative CAAT sequences at nt -276 and -288. The glaB gene shared 57% homology at the aa level with the glaA gene which was cloned previously from A. oryzae. Interestingly, the glucoamylase encoded by the glaB gene had no C-terminal domain such as that proposed to have starch binding activity in Aspergillus glucoamylases. Introduction of cDNA of the glaB gene to Saccharomyces cerevisiae caused the secretion of active glucoamylase to culture medium and introduction of the glaB gene to A. oryzae increased glucoamylase productivity in solid-state culture. Northern blot analysis showed the glaB gene was expressed in solid-state culture, but not in submerged culture.

Functional elements of the promoter region of the Aspergillus oryzae glaA gene encoding glucoamylase

SummaryAnalysis was made of the promoter region of the Aspergillus oryzae glaA gene encoding glucoamylase. Northern blots using a glucoamylase cDNA as a probe indicated that the amount of mRNA corresponding to the glaA gene increased when expression was induced by starch or maltose. The promoter region of the glaA gene was fused to the Escherichia coli uidA gene, encoding β-glucuronidase (GUS), and the resultant plasmid was introduced into A. oryzae. Expression of GUS protein in the A. oryzae transformants was induced by maltose, indicating that the glaA-GUS gene was regulated at the level of transcription in the presence of maltose. The nucleotide sequence 1.1 kb upstream of the glaA coding region was determined. A comparison of the nucleotide sequence of the A. oryzae glaA promoter with those of A. oryzae amyB, encoding α-amylase, and A. niger glaA showed two regions with similar sequences. Deletion and site-specific mutation analysis of these homologous regions indicated that both are essential for direct high-level expression when grown on maltose.

Identification of functional elements that regulate the glucoamylase-encoding gene (glaB) expressed in solid-state culture of Aspergillus oryzae

Aspergillus oryzae has two glucoamylase-encoding genes, glaA and glaB, whose expressions are distinguished by the type of culture used. The glaB gene is markedly expressed in solid-state culture but is little expressed in submerged culture. In solid-state culture, glaB expression at the transcriptional level is enhanced by low-Aw (water activity), high-temperature, and physical barriers to hyphal extension, as well as by starch. To determine the cis-acting factors in the glaB promoter, deletion analysis of the promoter was done with GUS (β-glucuronidase) as the reporter. Deletion of the 27u2009bp from −350 to −324 (Region A) in 1.1u2009kb of the glaB promoter completely abolished starch, low-Aw, and high-temperature induction. Substitution of the 12-bp GC-rich motif from −335 to −324 (GC-box) resulted in significant loss of starch and low-Aw inductivities. These findings suggest that the GC-box is a cis-element essential for the high-level expression of glaB in solid-state culture.

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.

Post-Translational His-Cys Cross-Linkage Formation in Tyrosinase Induced by Copper(II)−Peroxo Species

Autocatalytic formation of His-Cys cross-linkage in the enzyme active site of tyrosinase from Aspergillus oryzae has been demonstrated to proceed by the treatment of apoenzyme with Cu(II) under aerobic conditions, where a (μ-η(2):η(2)-peroxo)dicopper(II) species has been suggested to be involved as a key reactive intermediate.

Crawler, a novel Tc1/mariner-type transposable element in Aspergillus oryzae transposes under stress conditions

A novel active transposable element, designated Crawler, has been isolated from an industrial strain (OSI1013) of Aspergillus oryzae as an insertion sequence within the niaD gene encoding nitrate reductase. It is 1290bp in length with imperfect terminal inverted repeats of 28bp and is flanked by 2bp (TA) target site duplications. It contains an open reading frame with no introns that encodes a putative transposase (AotA) of 357 amino acid residues, which is highly homologous to the transposase existing in impala, a member of Tc1/mariner superfamily class II DNA transposon from Fusarium oxysporum. Southern blot analysis revealed that the OSI1013 strain has multiple copies (at least 16) of the element in the genome. Transcription of Crawler occurred under standard growth conditions, and was up-regulated in the presence of CuSO(4) or by heat shock at 42 degrees C. Moreover, transposition events of Crawler induced by various stress treatments were observed by transposon trapping, in which crnA and niaD genes were used as targets for insertion of the element. The excision analysis of Crawler inserted within promoter regions of the crnA gene revealed that CuSO(4) stress and heat shock treatment for conidia were most effective on its excision/transposition, and that acidic environment, oxidative stress, and UV irradiation also slightly induced transposition. To our knowledge, this is the first study reporting the observation of active transpositions of a resident class II transposon under various stress conditions in filamentous fungi.

Multifunctions of MelB, a Fungal Tyrosinase from Aspergillus oryzae

The pro form of melB tyrosinase from the melB gene of Aspergillus oryzae was over‐produced from E. coli and formed a homodimer that exhibited the spectral features of met‐tyrosinase. In the presence of NH2OH (reductant), the proenzyme bound dioxygen to give a stable (μ‐η2:η2‐peroxo)dicopper(II) species (oxy form), thus indicating that the pro form tyrosinase can function as an oxygen carrier or storage protein like hemocyanin. The pro form tyrosinase itself showed no catalytic activity toward external substrates, but proteolytic digestion with trypsin activated it to induce tyrosinase activity. Mass spectroscopy analyses, mutagenesis experiments, and colorimetry assays have demonstrated that the tryptic digestion induced cleavage of the C‐terminal domain (Glu458–Ala616), although the dimeric structure of the enzyme was retained. The structural changes induced by proteolytic digestion might open the entrance to the enzyme active site for substrate incorporation.