Osamu Hatamoto

Cloning and sequencing of the gene encoding tannase and a structural study of the tannase subunit from Aspergillus oryzae.

We cloned the Aspergillus oryzae tannase gene using three oligodeoxyribonucleotide (oligo) probes synthesized according to the tannase N-terminal and an internal amino acid (aa) sequence. The nucleotide (nt) sequence of the tannase gene was determined and compared with that of a tannase DNA complementary to RNA (cDNA) by means of reverse transcriptase PCR. The results indicated that there was no intron in the tannase gene and that it coded for 588 aa with a molecular weight of about 64,000. The tannase low-producing strain A. oryzae AO1 was transformed with the plasmid pT1 which contained the tannase gene, and tannase activities of the transformants increased in proportion to the number of copies. Tannase consisted of two kinds of subunits, linked by a disulfide bond(s) with molecular weights of about 30,000 and 33,000, respectively. We purified these subunits and determined their N-terminal aa sequences. The large and small subunits of tannase were encoded by the first and second halves, respectively. Judging from the above results, the tannase gene product is translated as a single polypeptide that is cleaved by post-translational modification into two tannase subunits linked by a disulfide bond(s). We concluded that native tannase consisted of four pairs of the two subunits, forming a hetero-octamer with a molecular weight of about 300,000.

Efficient gene disruption in the koji -mold Aspergillus sojae using a novel variation of the positive-negative method

When no phenotypic screen is available, gene disruption in the koji -mold Aspergillus sojae is a time-consuming process, owing to the low frequency of homologous recombination. To achieve efficient gene disruption in the koji -mold, we developed a novel positive-negative selection method to enrich for homologous recombinants. The pyrG gene from A. sojae was used as a positive selection marker for transformants, and the oliC31 gene of A. nidulans, which codes for a mutant form of subunit 9 of the F1FO-ATPase, was employed as a negative selection marker to facilitate elimination of non-homologous recombinants among the transformants. The positive-negative selection markers, in combination with a pyrG deletion strain as a host, enabled enrichment for homologous recombinants, and disruption of the genes niaD, areA and tannase was successfully demonstrated. In order to examine whether the positive-negative selection technique is effective for targeting any locus, even in the absence of information on gene function or phenotype, we attempted to disrupt the aflR gene of A. sojae, which codes for a putative transcription factor for the aflatoxin biosynthetic pathway, using the method. Despite the fact that this gene is not transcribed in A. sojae, aflR disruptants were efficiently obtained, suggesting that the method is indeed capable of targeting any locus, without additional ectopic integration, and is thus applicable for functional genomics studies in filamentous fungi, including A. sojae.

A simple method for enrichment of uninucleate conidia of Aspergillus oryzae.

Aspergillus oryzae produces multinucleate conidia, which makes the obtaining of homokaryons labor-intensive. Analysis of conidia by flow cytometry clarified the relationship that conidia of lower nuclear number were smaller in size. Based on this, we have developed a simple way to enrich uninucleate conidia with a membrane filter. Our results also suggest that the method is useful for elimination of heterokaryons.