Targeted Tandem Duplication of a Large Chromosomal Segment in Aspergillus oryzae

We describe here the first successful construction of a targeted tandem duplication of a large chromosomal segment in Aspergillus oryzae. The targeted tandem chromosomal duplication was achieved by using strains that had a 5′-deleted pyrG upstream of the region targeted for tandem chromosomal duplication and a 3′-deleted pyrG downstream of the target region. Consequently, strains bearing a 210-kb targeted tandem chromosomal duplication near the centromeric region of chromosome 8 and strains bearing a targeted tandem chromosomal duplication of a 700-kb region of chromosome 2 were successfully constructed. The strains bearing the tandem chromosomal duplication were efficiently obtained from the regenerated protoplast of the parental strains. However, the generation of the chromosomal duplication did not depend on the introduction of double-stranded breaks (DSBs) by I-SceI. The chromosomal duplications of these strains were stably maintained after five generations of culture under nonselective conditions. The strains bearing the tandem chromosomal duplication in the 700-kb region of chromosome 2 showed highly increased protease activity in solid-state culture, indicating that the duplication of large chromosomal segments could be a useful new breeding technology and gene analysis method.     

Cell biology of the Koji mold Aspergillus oryzae

Koji mold, Aspergillus oryzae, has been used for the production of sake, miso, and soy sauce for more than one thousand years in Japan. Due to the importance, A. oryzae has been designated as the national micro-organism of Japan (Koku-kin). A. oryzae has been intensively studied in the past century, with most investigations focusing on breeding techniques and developing methods for Koji making for sake brewing. However, the understanding of fundamental biology of A. oryzae remains relatively limited compared with the yeast Saccharomyces cerevisiae. Therefore, we have focused on studying the cell biology including live cell imaging of organelles, protein vesicular trafficking, autophagy, and Woronin body functions using the available genomic information. In this review, I describe essential findings of cell biology of A. oryzae obtained in our study for a quarter of century. Understanding of the basic biology will be critical for not its biotechnological application, but also for an understanding of the fundamental biology of other filamentous fungi.     

Homologs of Aflatoxin Biosynthesis Genes and Sequence of aflR in Aspergillus oryzae and Aspergillus sojae

The presence, but not expression, of homologs of three structural genes and a regulatory gene necessary for aflatoxin biosynthesis in Aspergillus parasiticus and A. flavus was shown for A. oryzae and A. sojae. Homologs of the regulatory gene aflR were cloned and sequenced from A. oryzae and A. sojae.     

Mirin-Making from Koji Prepared with a Mutant Aspergillus oryzae Producing High Activity of Carboxypeptidase

It has been desired to improve the quality of mirin without decreasing the level of productivity. Accordingly, by ultraviolet irradiation to cause mutation, we screened for mutants with high acid carboxypeptidase (ACPase) activity from Aspergillus oryzae IFO 4079 (parental strain). We obtained a mutant with ACPase with the activity of 20,000 units · g⁻¹ of koji, about three times that of the parental strain. In mirin-making, koji prepared with this mutant had the same productivity, but the concentration of amino nitrogen in the mirin obtained with this mutant was 1.2–1.4 times that of the parental strain. Results of sensory tests showed that the mirin prepared with koji of the mutant had a good taste on the whole. The degree of clouding In mirin made with glutinous rice steamed at atmospheric pressure was much less with koji of this mutant than that of the parental strain.     

Purification and characterization of chitosanase and Exo-beta-D-glucosaminidase from a Koji mold, Aspergillus oryzae IAM2660

Chitosan-degrading activity was detected in the culture fluid of Aspergillus oryzae, A. sojae, and A. flavus among various fungal strains belonging to the genus Aspergillus. One of the strong producers, A. oryzae IAM2660 had a higher level of chitosanolytic activity when N-acetylglucosamine (GlcNAc) was used as a carbon source. Two chitosanolytic enzymes, 40 kDa and 135 kDa in molecular masses, were purified from the culture fluid of A. oryzae IAM2660. Viscosimetric assay and an analysis of reaction products by thin-layer chromatography clearly indicated the endo- and exo-type cleavage manner for the 40-kDa and 135-kDa enzymes, respectively. The 40-kDa enzyme, designated chitosanase, catalyzed a hydrolysis of glucosamine (GlcN) oligomers larger than pentamer, glycol chitosan, and chitosan with a low degree of acetylation (0-30%). The 135-kDa exo-beta-D-glucosaminidase,enzyme,named released a single GlcN residue from the GlcN oligomers and chitosan, but did not release GlcNAc residues from either GlcNAc oligomer or colloidal chitin.     

In Vivo Antioxidant Activity of Okara Koji,a Fermented Okara,by Aspergillus oryzae

The antioxidants in Okara Koji (OK), an okara (OC) fermented by Aspergillus oryzae, γ-tocopherol, δ-tocopherol, genistin, daizein, genistein, and 3-hydroxyanthranilic acid were identified by HPLC. OK’s extract with 80% methanol strongly inhibited linoleate peroxidation, much more than other OK’s extracts with hexane or hot water. The methanol extract accelerated 12-hydroxyeicosatetraenoic acid formation in membrane lipids at 10^?3 concentration, but inhibited the formation at higher concentrations than 10^?3 ex vivo. To confirm the total effect of all components of OK on lipid peroxidation in vivo, rats fed food deficient in vitamin E were put on diets containing OK or OC with oxidized oil. In rats fed the OK diet, no effect of oxidized oil feeding on the body weight gain, of the TBA value in plasma, or of glutathione peroxidase activities of plasma and liver was observed. But in rats fed the OC diet, the effect of oxidized oil feeding was apparently observed on all of those values. These results suggested that OK would scavenge lipid peroxides in vivo.     

Enhanced gene targeting frequency in ku70 and ku80 disruption mutants of Aspergillus sojae and Aspergillus oryzae

In the koji molds Aspergillus sojae and Aspergillus oryzae, exogenous DNA is integrated in the genome, in most cases irrespective of the sequence homology, suggesting that DNA integration occurs predominantly through a nonhomologous end joining pathway where two ku genes, namely, ku70 and ku80, play a key role. To determine the effect of ku gene disruption on the gene targeting frequency, we constructed ku70-, ku80-, and ku70–ku80-disrupted strains of A. sojae and A. oryzae. The gene targeting frequency of the tannase gene in ku70 and ku80 strains of both Aspergillus species was markedly enhanced as compared with that of the parental strains. The gene targeting frequency of the aflR and ku80 genes was also enhanced in an A. sojae ku70 background. Therefore, the koji mold strains with ku-disrupted genes will be excellent tools as hosts for efficient gene targeting.     

Identification and analysis of Ku70 and Ku80 homologs in the koji molds Aspergillus sojae and Aspergillus oryzae

Ku genes play a key role in the non-homologous end-joining pathway. We have identified Ku70 and Ku80 homologs in the koji molds Aspergillus sojae and Aspergillus oryzae, and have constructed the disruption mutants of Ku70, Ku80, and Ku70-80 to characterize the phenotypic change in these mutants. Neither Ku70- nor Ku80-disrupted strains show hypersensitivity to the DNA damaging agents methylmethane sulfonate (MMS) and phleomycin. Moreover, undesirable phenotypes, such as poor growth or repressed conidiospore formation, were not observed in the Ku-disrupted A. sojae and A. oryzae.     

Use of glutaminase for soy sauce made by Koji or a preparation of proteases from Aspergillus oryzae

Although a small amount of glutamic acid was released in the hydrolysis of protein or soy sauce made by a preparation of proteases containing little glutaminase, a large amount of glutamic acid was formed in such hydrolyzate or soy sauce made by the addition of mycelia of black Aspergilli or glutaminase from Cryptococcus albidus. The former effect was caused mainly by glutaminase produced by black Aspergilli. The former crude enzyme showed an optimum pH of 5.0, broad pH stability and salt tolerance. The addition of glutaminase from C. albidus ATCC 20293 in soy sauce manufacture using a preparation of proteases resulted in a 42% increase in glutamic acid per total nitrogen content.     

Isolation and Analysis of Molds from Soy Sauce Koji in Thailand

Five different isolates of Aspergillus and one of Mucor were compared with a Japanese commercial strain of Aspergillus oryzae for proteolytic activity on wheat bran substrate. One isolate of Aspergillus with superior protease production, identified as Aspergillus flavus var. columnaris, showed no detectable aflatoxin production on glutinous rice or soybean substrate. Preliminary tests using this fungus as a koji mold in a traditionally operated factory resulted in a soy sauce superior in quality to that usually produced.     

Metabolism of Phenylalanine in Aspergillus sojae

It was found that a new compound of phenylalanine metabolites (2-hydroxy-3-phenylpropenoic acid) and phenylacetic acid were formed in the cultured Czapek medium containing phenylalanine by Aspergillus sojae. 2-Hydroxy-3-phenylpropenoic acid (HPPA) was formed from phenylalanine (d- and l-form) via phenyllactic acid (d- and l-form), and degraded to benzoic acid, p-hydroxybenzoic acid, protocatechuic acid, and catechol in this order.

On the other hand, phenylacetic acid was formed from phenylpyruvic acid, and converted to homogentisic acid via o-hydroxyphenylacetic acid. From these results, a metabolic pathway of phenylalanine in Asp. sojae was proposed.     

Metabolism of l-Tyrosine in Aspergillus sojae

Circadian rhythmicity was investigated in isolated small intestine and mucosal epithelial cells from rats on restricted-feeding regimen (food available from 17:00 to 23:00 every day). In the isolated intestine, daily rhythms synchronized to meal-timing were found in the activity patterns of l-leucine, l-lysine and d-glucose transport, and mucosal γ-glutamyltransferase and sucrase, and in the rates of lactate formation from glucose; the nadirs occurred at 12:00 and the peaks at 23:00. These same patterns were also noted with the mucosal epithelial cells prepared at distinct times of day from rats on meal-feeding regimen. The fasted rat intestine responded to refeeding with prompt increase in transport activity, i.e., out of phase with the original rhythm. Intraperitoneal administration of cyloheximide suppressed the daily rise in leucine transport activity, indicating that the transport rhythm was entrained by or closely associated with the rhythmic fluctuation in protein synthesis in the epithelial cells, which in turns is cued by the feeding schedule. The kinetic parameters estimated for leucine transport, the apparent affinity constant for transport and the maximal transport rate, were significantly higher at high activity periods. It is suggested that the rhythmic increase in transport activity is not only associated with membrane hyperpolarization but may be mediated by the emergence of a high capacity transport system.     

Characteristics of Koji prepared from the transformant of Aspergillus oryzae with the glucoamylase gene of Aspergillus shirousamii,and its utilization for sake-brewing

Test sake fermentation was carried out using an Aspergillus oryzae transformant (TF2–5) which had the glucoamylase gene from Aspergillus shirousamii RIB2504. The fermentation progressed rapidly due to high glucoamylase activity, and the steamed rice rapidly dissolved in the moromi-mash. Consequently, the total alcohol yield increased. In addition, the obtained sake had a moderate sweetness and a rich fruity flavor.     

Conversion of Deletions during Recombination in Pneumococcal Transformation

Genetic analysis of 16 deletions obtained in the amiA locus of pneumococcus is described. When present on donor DNA, all deletions increased drastically the frequency of wild-type recombinants in two-point crosses. This effect was maximal for deletions longer than 200 bases. It was reduced for heterologies shorter than 76 bases and did not exist for very short deletions. In three-point crosses in which the deletion was localized between two point mutations, we demonstrated that this excess of wild-type recombinants was the result of a genetic conversion. This conversion extended over several scores of bases outside the deletion. Conversion takes place during the heteroduplex stage of recombination. Therefore, in pneumococcal transformation, long heterologies participated in this heteroduplex configuration. As this conversion did not require an active DNA polymerase A gene it is proposed that the mechanism of conversion is not a DNA repair synthesis but involves breakage and ligation between DNA molecules. Conversion of deletions did not require the Hex system of correction of mismatched bases. It differs also from localized conversion. It appears that it is a process that evolved to correct errors of replication which lead to long heterologies and which are not eliminated by other systems.     

Hereditary variation and genetic recombination in Koji-molds (Aspergillus oryzae and Asp. sojae). I. Natural variation

Natural variation in monospore lines of Koji-molds (Asp. oryzae and Asp. sojae), isolated from commercial Koji material or soil and from laboratory stock cultures, has been observed. We can divide the 58 strains of Koji-molds investigated into two groups; one group consists of inconstant strains which are very liable to produce natural variants, and the other consists of strains which remain constant through successive single spore culture. The inconstant strains develop colonies bearing various proportions of conidia and aerial mycelium (X-type). They generally form large conidia (Asp. oryzae var. magnasporus) but sometimes medium sized conidia (Asp. oryzae s. str.), which produce large conidia occasionally. The colonies of the constant strains show abundant conidial formation and smooth surfaces (C-type). The conidia are mostly small (Asp. oryzae var. microsporus) but sometimes medium in size (Asp. oryzae s. str.). The colony types of the variants are as follows: C (Conidial type, whole colony covered with conidia), M (mycelial type), R (restricted in growth rate), St (sterile type, little sporulation on all media tested), Nit (requiring reduced nitrate, very faint growth on Czapek's agar), and LS (semi lethal, growth cease immediately after germination). Pedigree cultures of the 8 inconstant strains have been made, but no definite segregation ratios for each variant type have been recognized through successive generations. The LS and N types commonly occur spontaneously from the M-type.