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.     

Neutral Proteinases I and II of Aspergillus sojae

Epimerization of (?)-isodihydrocarvone (I) to (?)-dihydrocarvone (II) by Pseudomonas fragi IFO 3458 was studied. I was easily epimerized to II by the growing cells, the resting cells or the cell-free extracts.

An epimerase catalyzing the conversion of I to II was partially purified from the bacterial extracts about 56-fold with heat-treatment, ammonium sulfate precipitation and DEAE-Sephadex A-50 column chromatography. By the action of this epimerase, the ratio of I to II becomes about 25: 75 (K=3). It appeared that the epimerase is very stable to heat; the activity of epimerization remains 66 and 36% after treatment at 97°C for 60 and 120 min, respectively.     

Generation of Large Chromosomal Deletions in Koji Molds Aspergillus oryzae and Aspergillus sojae via a Loop-Out Recombination

We established a technique for efficiently generating large chromosomal deletions in the koji molds Aspergillus oryzae and A. sojae by using a ku70-deficient strain and a bidirectional marker. The approach allowed deletion of 200-kb and 100-kb sections of A. oryzae and A. sojae, respectively. The deleted regions contained putative aflatoxin biosynthetic gene clusters. The large genomic deletions generated by a loop-out deletion method (resolution-type recombination) enabled us to construct multiple deletions in the koji molds by marker recycling. No additional sequence remained in the resultant deletion strains, a feature of considerable value for breeding of food-grade microorganisms. Frequencies of chromosomal deletions tended to decrease in proportion to the length of the deletion range. Deletion efficiency was also affected by the location of the deleted region. Further, comparative genome hybridization analysis showed that no unintended deletion or chromosomal rearrangement occurred in the deletion strain. Strains with large deletions that were previously extremely laborious to construct in the wild-type ku70⁺ strain due to the low frequency of homologous recombination were efficiently obtained from Δku70 strains in this study. The technique described here may be broadly applicable for the genomic engineering and molecular breeding of filamentous fungi.     

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.     

On the mechanism of mitotic recombination in Aspergillus nidulans. I. Intragenic recombination and DNA replication

Summary Adenine or pABA starvation induce mitotic recombination within the ad9 and paba1 cistrons respectively. Adenine concentrations in the plating medium as low as 1×10^-8 M increase recombination frequency; the concentration optimal in respect to induced recombination frequency is 5×10^-7 M.Recombination within the paba1 cistron is stimulated by low pABA concentrations, or caseine hydrolysate, or methionine.Aminopterin applied for one or two hours before conidia of pABA-requiring diploid are plated on proper selective media, induces recombination within the pro1, ad9 and paba1 cistrons. Conclusion is drawn that it is adenine or thymine starvation which induce mitotic recombination.The implications of this and other similar evidence are discussed.     

Positive correlation between recombination rates and levels of genetic variation in natural populations of sea beet (Beta vulgaris subsp. maritima).

The relation between the level of genetic variation and the rate of recombination per physical unit was investigated in sea beet (Beta vulgaris subsp. maritima). The rate of recombination per physical unit was estimated indirectly through marker density in an RFLP linkage map of sugar beet. From this map, we also selected RFLP markers covering two of the nine chromosomes in Beta. The markers were used to estimate the level of genetic variation in three populations of sea beet, two from Italy and one from England. Two estimates of genetic variation were employed, one based on the number of alleles in the sample and the other on heterozygosity. A statistically significant positive correlation was found between recombination rate and genetic variation. Several theoretical explanations for this are discussed, background selection being one. A correlation similar to this has been observed previously in Drosophila, one that was higher than what we obtained for Beta. This is consistent with various biological differences between the two species.     

Natural genetic variation in plant photosynthesis

Natural genetic variation in plant photosynthesis is a largely unexplored and as a result an underused genetic resource for crop improvement. Numerous studies show genetic variation in photosynthetic traits in both crop and wild species, and there is an increasingly detailed knowledge base concerning the interaction of photosynthetic phenotypes with their environment. The genetic factors that cause this variation remain largely unknown. Investigations into natural genetic variation in photosynthesis will provide insights into the genetic regulation of this complex trait. Such insights can be used to understand evolutionary processes that affect primary production, allow greater understanding of the genetic regulation of photosynthesis and ultimately increase the productivity of our crops.     

Purification and Properties of Acid Carboxypeptidase I from Aspergillus oryzae

To elucidate the constitution of peptidases from Aspergillus oryzae, systematic separation of the enzymes was carried out by batchwise treatment with Amberlite IRC-50 and precipitation with rivanol. Proteases were separated to two fractions. They were Amberlite IRC-50 adsorbed and the non-adsorbed fractions and the latter fraction was further separated to two fractions, rivanol precipitable and non-precipitable fractions.

Acid carboxypeptidase I was purified from the rivanol non-precipitable fraction by column chromatography on DEAE-cellulose, DEAE-Sephadex A-50 and SE-cellulose. The purified enzyme was not homogeneous on disc electrophoresis, although symmetric peaks were obtained for enzyme protein and activity in Sephadex gel filtration. The optimum pH is at pH 4.0 for carbobenzoxy-l-alanyl-l-glutamic acid. The enzyme activity was inhibited by SH reagents, but not inhibited by metal chelating agents. The molecular weight of the enzyme was estimated to be about 120,000 by gel filtration.     

Development of enzyme technology for Aspergillus oryzae,A. sojae,and A. luchuensis,the national microorganisms of Japan

This paper describes the modern enzymology in Japanese bioindustries. The invention of Takadiastase by Jokiti Takamine in 1894 has revolutionized the world of industrial enzyme production by fermentation. In 1949, a new γ-amylase (glucan 1,4-α-glucosidase, EC 3.2.1.3) from A. luchuensis (formerly designated as A. awamori), was found by Kitahara. RNase T₁ (guanyloribonuclease, EC 3.1.27.3) was discovered by Sato and Egami. Ando discovered Aspergillus nuclease S₁ (single-stranded nucleate endonuclease, EC 3.1.30.1). Aspergillopepsin I (EC 3.4.23.18) from A. tubingensis (formerly designated as A. saitoi) activates trypsinogen to trypsin. Shintani et al. demonstrated Asp76 of aspergillopepsin I as the binding site for the basic substrate, trypsinogen. The new oligosaccharide moieties Man₁₀GlcNAc₂ and Man₁₁GlcNAc₂ were identified with α-1,2-mannosidase (EC 3.2.1.113) from A. tubingensis. A yeast mutant compatible of producing Man₅GlcNAc₂ human compatible sugar chains on glycoproteins was constructed. The acid activation of protyrosinase from A. oryzae at pH 3.0 was resolved. The hyper-protein production system of glucoamylase was established in a submerged culture.     

Detection of genetic variation with radioactive ligands. IV. X-linked, polymorphic genetic variation of thyroxin-binding globulin (TBG).

A genetically determined, polymorphic electrophoretic variant of thyroxin-binding alpha-globulin (TBG) is found in sera from populations of African and Oceania origin, although not in Caucasians nor Orientals. The TBG polymorphism is inherited in X-linked fashion, based on data from American blacks, and thus provides an X-chromosome marker with a relatively high gene frequency in this ethnic group (frequency of the slow allele, TBGs, is 11%). This slow variant should prove valuable in expanding the map of the X chromosome and in linkage studies. An additional family exhibiting X-linked TBG deficiency is also described.     

Purification,characterization, and genetic analysis of a leucine aminopeptidase from Aspergillus sojae

Extracellular leucine aminopeptidase (LAP) from Aspergillus sojae was purified to protein homogeneity by sequential fast protein liquid chromatography steps. LAP had an apparent molecular mass of 37 kDa, of which approximately 3% was contributed by N-glycosylated carbohydrate. The purified enzyme was most active at pH 9 and 70 degrees C for 30 min. The enzyme preferentially hydrolyzed leucine p-nitroanilide followed by Phe, Lys, and Arg derivatives. The LAP activity was strongly inhibited by metal-chelating agents, and was largely restored by divalent cations like Zn(2+) and Co(2+). The lap gene and its corresponding cDNA fragment of the A. sojae were cloned using degenerated primers derived from internal amino acid sequences of the purified enzyme. lap is interrupted by three introns and is transcribed in a 1.3-kb mRNA that encodes a 377-amino-acid protein with a calculated molecular mass of 41.061 kDa. The mature LAP is preceded by a leader peptide of 77 amino acids, predicted to include an 18-amino-acid signal peptide and an extra sequence of 59 amino acids. Two putative N-glycosylation sites are identified in Asn-87 and Asn-288. Southern blot analysis suggested that lap is a single-copy gene in the A. sojae genome. The deduced amino acid sequence of A. sojae LAP shares only 11-33.1% identity with those of LAPs from 18 organisms.     

Yellow Pigments of Aspergillus niger and Asp. awamori

From mycelia of Asp. niger and Asp. awamori aurasperones A, B and C along with related two yellow pigments have been isolated.

Aurasperone A, C₃₂H₂₆O₁₀, is obtained in yellow prisms; m.p. 207°C; [α]_d —136°; gives the diacetate and the dimethyl ether and is assumed to be a dimeric 2-methyl-5- hydroxy-6,8-dimethoxy-4H-naphtho [2,3-b] pyran-4-one (IV). Aurasperone B, [α]_D +46.3°, is the main yellow metabolite, m.p. 186°C, and affords aurasperone A on hydrochloric acid-treatment. It has molecular formula C₃₂H₃₀O₁₂ and is supposed to have the structure (V). The other yellow pigments have been found to be also congeners of aurasperone A.     

Pathotypic variation of Xanthomonas oryzae pv. oryzae in Bangladesh

Bacterial Blight (BB) caused by Xanthomonas oryzae pv. oryzae (Xoo), a destructive disease of rice. Altogether, 96 isolates of Xoo were collected from 19 rice growing districts of Bangladesh in irrigated and rainfed seasons during 2014 to assess pathotypic variation. Pathotypic analyses on a set of 12 Near Isogenic Lines (NILs) of rice containing resistance genes viz. Xa1, Xa2, Xa3, Xa4, Xa5, Xa7, Xa8, Xa10, Xa11, Xa13, Xa14 and Xa21 and two check varieties IR24 and TN1 by leaf clip-inoculation technique. A total of 24 pathotypes were identified based on their virulence patterns on NILs tested. Among these, pathotypes VII, XII, and XIV considered as major, containing maximum number of isolates, (9.38% each) frequently distributed in North to Mid-Eastern districts of Bangladesh. Most virulent pathotype I recorded from Habiganj and Brahmanbaria. This pathotypic variation explained the pathogenic relatedness of X. oryzae pv. oryzae populations from diverse geographic areas in Bangladesh.     

Kinetics of alpha-amylase secretion in Aspergillus oryzae.

Pulse and pulse-chase experiments have been performed to study L-[(35)S] methionine incorporation and protein secretion kinetics in Aspergillus oryzae. Pulse experiments confirmed the mechanism of methionine uptake reported previously for Penicillium chrysogenum (Benko et al., 1967). Pulse-chase experiments were carried out to investigate the alpha-amylase secretion kinetics in A. oryzae. No unglycosylated alpha-amylase was detected neither intracellularly nor extracellularly demonstrating that glycosylation was not the rate controlling step in the secretory pathway. The pulse chase experiments indicated that there are two pools of intracellular alpha-amylase: a fast secreted and a slow secreted. The secretion of those two pools were described with a kinetic model, which was fitted to the pulse chase experiments.     

Improvement in amyloglucosidase production following genetic recombination of Aspergillus niger strains

Summary Recombination has been demonstrated in amyloglucosidase-producing strains of Aspergillus niger. A high-yielding strain has been crossed, parasexually, with a low-yielding strain from the same genealogy. Recombinants have been produced, having the efficient broth filtration characteristics of the low-yielding strain. One such segregant was superior, for the industrial production of amyloglucosidase, to the best parent strain previously used for that purpose.