Nonhomologous end-joining deficiency allows large chromosomal deletions to be produced by replacement-type recombination in Aspergillus oryzae

Gene targeting is a technique of introducing a genetic trait at a predetermined site within a genome; it is also used to eliminate undesirable chromosomal regions from the relevant genome. Thus far, replacement-type recombination between two homologous regions separated by a large nonhomologous sequence has been hardly achieved probably due to the low frequency of homologous recombination in filamentous fungi. In this study, we report the successful and highly efficient deletion by replacement-type recombination of up to 470-kb regions of chromosome 8 and 200-kb region in chromosome 3, which includes a homologue of aflatoxin gene cluster, by nonhomologous end-joining deficient strains of Aspergillus oryzae. Our study results indicate that the deficiency of nonhomologous end-joining increases the distance of nonhomologous regions in replacement-type recombination, i.e., the possible deletion range in generation of large chromosomal deletion by one cycle of replacement-type recombination is increased in nonhomologous end-joining deficient strains.     

Analysis of the functions of recombination-related genes in the generation of large chromosomal deletions by loop-out recombination in Aspergillus oryzae

Loop-out-type recombination is a type of intrachromosomal recombination followed by the excision of a chromosomal region. The detailed mechanism underlying this recombination and the genes involved in loop-out recombination remain unknown. In the present study, we investigated the functions of ku70, ligD, rad52, rad54, and rdh54 in the construction of large chromosomal deletions via loop-out recombination and the effect of the position of the targeted chromosomal region on the efficiency of loop-out recombination in Aspergillus oryzae. The efficiency of generation of large chromosomal deletions in the near-telomeric region of chromosome 3, including the aflatoxin gene cluster, was compared with that in the near-centromeric region of chromosome 8, including the tannase gene. In the Δku70 and Δku70-rdh54 strains, only precise loop-out recombination occurred in the near-telomeric region. In contrast, in the ΔligD, Δku70-rad52, and Δku70-rad54 strains, unintended chromosomal deletions by illegitimate loop-out recombination occurred in the near-telomeric region. In addition, large chromosomal deletions via loop-out recombination were efficiently achieved in the near-telomeric region, but barely achieved in the near-centromeric region, in the Δku70 strain. Induction of DNA double-strand breaks by I-SceI endonuclease facilitated large chromosomal deletions in the near-centromeric region. These results indicate that ligD, rad52, and rad54 play a role in the generation of large chromosomal deletions via precise loop-out-type recombination in the near-telomeric region and that loop-out recombination between distant sites is restricted in the near-centromeric region by chromosomal structure.     

Biosynthesis of L-DOPA by Aspergillus oryzae

The present investigation deals with the biosynthesis of L-DOPA by parental (GCB-6) and mutant (UV-7) strains of Aspergillus oryzae. There was a marked difference between the mycelial morphology and pellet type of parental and UV-irradiated mutant culture. The mutant strain of A. oryzae UV-6 exhibited pellet-like mycelial morphology and improved tyrosinase activity. Mould mycelium was used for biochemical conversion of L-tyrosine to L-DOPA because tyrosinase is an intracellular enzyme. The mutant was found to yield 3.72 fold higher production of L-DOPA than the parental strain. The mutant strain is stable and D-glc-resistant. The comparison of kinetic parameters was also done which showed the greater ability of the mutant to yield L-DOPA (i.e., Yp/x 40.00+/-0.01 d mg/mg with parent and 182.86+/-0.02a mg/mg in case of mutant). When cultures grown for various incubation periods, were monitored for Qp, Qs and q(p), there was significant enhancement (p < 0.0025-0.005) in these variables by the mutant strain of A. oryzae UV-7 over GCB-6 on all the rates. L-DOPA (3,4-dihydroxy phenyl L-alanine) is a drug of choice in the treatment of Parkinson's disease and myocardium following neurogenic injury.     

Characterization of deletions in common wheat induced by an Aegilops cylindrica chromosome: detection of multiple chromosome rearrangements

An Aegilops cylindrica chromosome induces terminal deletions of chromosomes in wheat as identified by C-banding. We are constructing high-density physical maps of wheat chromosomes and have detected additional chromosome rearrangements. Among 63 lines with chromosomal subarm deletions in group 7 chromosomes, 7 lines (11.1%) were shown to harbor additional chromosome rearrangements. Two other lines were also omitted from the physical mapping because of the nature of the breakpoint calculations. The presence or absence of chromosome-specific restriction fragment length polymorphism (RFLP) or random amplified polymorphic DNA (RAPD) markers indicated that additional interstitial deletions are present in 3 lines (4.8%) with deletions in the short chromosome arms and in 4 lines (6.3%) with deletions in the long chromosome arms. We also used chromosome pairing analysis of F₁ plants of deletion lines with double ditelosomic lines of Chinese Spring wheat to detect small terminal deletions. The deletion of the most distal 1% of chromosome arm 7AL was associated with a pairing reduction of 60%.     

Engineering large-scale chromosomal deletions by CRISPR-Cas9

Large-scale chromosomal deletions are a prevalent and defining feature of cancer. A high degree of tumor-type and subtype specific recurrencies suggest a selective oncogenic advantage. However, due to their large size it has been difficult to pinpoint the oncogenic drivers that confer this advantage. Suitable functional genomics approaches to study the oncogenic driving capacity of large-scale deletions are limited. Here, we present an effective technique to engineer large-scale deletions by CRISPR-Cas9 and create isogenic cell line models. We simultaneously induce double-strand breaks (DSBs) at two ends of a chromosomal arm and select the cells that have lost the intermittent region. Using this technique, we induced large-scale deletions on chromosome 11q (65 Mb) and chromosome 6q (53 Mb) in neuroblastoma cell lines. A high frequency of successful deletions (up to 30% of selected clones) and increased colony forming capacity in the 11q deleted lines suggest an oncogenic advantage of these deletions. Such isogenic models enable further research on the role of large-scale deletions in tumor development and growth, and their possible therapeutic potential.     

Microbial degradation of monocrotophos by Aspergillus oryzae

Organophosphorus pesticides are widely used in India for protection of agricultural yields. However, these pesticides pose various threats to organisms, including humans, and hamper soil microbial activity; thus, they are a cause for concern. As a measure of bioremediation, soil fungi capable of degrading monocrotophos (MCP) were isolated from various geographical and ecological sites. Twenty-five strains were isolated by an enrichment method using MCP as a carbon and phosphorus source. On the basis of MCP tolerance capacity exhibited in gradient agar plate assay the isolate M-4, identified as Aspergillus oryzae ARIFCC 1054, was selected for further studies. The ability of the isolate to mineralize MCP was investigated under different culture conditions. The isolate was found to possess phosphatase activity. The course of the degradation process was studied using HPTLC and FTIR analyses. The results suggest that this organism could be used for bioaugmentation of soil contaminated with MCP and for treatment of aqueous wastes.     

米曲霉发酵纯化无患子皂苷的工艺研究

采用微生物发酵法对无患子皂苷水提取液进行纯化.比较了采用自然发酵、接种酵母菌发酵和接种米曲霉发酵纯化无患子皂苷的效果.结果表明,提取液不灭菌,接种米曲霉发酵纯化效果较为明显,优化后的发酵条件为:温度30℃、接种龄12 h、接种量为3％、摇床转速150 r/min,发酵7d后,皂苷含量稍有下降,但皂苷纯度可从48.71％提高到82.47％.米曲霉发酵法明显优于水提醇沉法、絮凝法和正丁醇萃取法.     

Invasion of paranasal sinuses by Aspergillus oryzae

A 24-year-old male patient receiving chemotherapy for acute promyelocytic leukemia developed fever, right periorbital swelling and mild right proptosis. A head scan showed opacification of the right maxillary and ethmoid sinuses with adjacent soft tissue swelling. Biopsy of the nasal mucosa demonstrated the typical septate hyphae of Aspergillus species which was later shown on culture to be Aspergillus oryzae. A. oryzae has only rarely been reported in human disease and there is confusion as to its precise identification and role. We would like to confirm the pathogenicity of A. oryzae with this uncommon presentation of aspergillosis and also emphasize the need to take adequate and multiple cultures in suspected cases so that the possibility of species identification will be maximized.     

Alpha-galactosidase production by Aspergillus oryzae in solid-state fermentation

Comparisons were made for alpha-galactosidase production using red gram plant waste (RGPW) with wheat bran (WB) and other locally available substrates using the fungus Aspergillus oryzae under solid-state fermentation (SSF). RGPW proved to be potential substrate for alpha-galactosidase production as it gave higher enzyme titers (3.4 U/g) compared to WB (2.7 U/g) and other substrates tested. Mixing WB with RGPW (1:1, w/w) resulted enhanced alpha-galactosidase yield. The volume of moistening agent in the ratio of 1:2 (w/v), pH 5.5 and 1 ml (1 x 10(6) spores) of inoculum volume and four days incubation were optimum for alpha-galactosidase production. Increase in substrate concentration (RGPW+WB) did not decrease enzyme yield in trays.     

The ability of ionizing radiations of different LET to induce chromosomal deletions in Aspergillus nidulans.

Conidia, derived from a strain of Aspergillus nidulans known to carry a specific chromosomal duplication, were irradiated. The duplicated segment had genetic markers, which, when eliminated from the genome, allowed the easy detection of deletion mutants. Survival curves derived following 15 MeV electron and gamma-ray irradiation were characterised by the presence of an appreciable shoulder, whilst 50 kvp X-rays gave a much smaller shoulder. Irradiation with beta-particles and alpha-particles gave rise to exponential survival curves. The RBE values for these radiations, based on the D37 value were for gamma-rays, 1.0, 15 MeV electrons 1.0, 50 kvp X-rays 1.9, beta-particles 2.1 and alpha-particles 3.4. With the exception of gamma-rays the radiations described were compared with respect to their ability to induce chromosomal deletions. When the number of deletants amongst survivors was plotted against dose, a linear relationship was found for electrons, X-rays and beta-particles. The response recorded for alpha-particles was essentially linear but with a biphasic component. The RBE values for the radiations, based on a value of unity for 15 MeV electrons were as follows: X-rays 1.3, beta-particles 0.8, alpha-particles above 7.5 krad 2.3 and below 7.5 krad 3.5. When these same data were re-plotted with number of deletants amongst survivors against log survival, electrons appeared the most efficient radiation at producing deletants amongst survivors, with an "m value" of 283 X 10(-5). Tritiated water was least efficient, the corresponding value being 182 X 10(-5). The number of deletants per 10(4) conidia plated, when plotted against dose yielded a curve which increased to a peak and then decreased linearly for all radiations. The peaks for electrons, X-rays and alpha-particles each had a value of about 14 deletants per 10(4) conidia plated and the peaks roughly corresponded with the point at which the survival curve became exponential and was clearly indicative of the accumulation of sub-lethal damage. However, for beta-particles the peak had a value of 7 deletants per 10(4) conidia plated. A non-DNA target has been implicated for cellular death following beta-particle irradiation.     

Repetitive attack by Aspergillus oryzae alpha amylase

The action of Aspergillus oryzae alpha amylase on reducing-end, and uniformly radiolabeled maltotriose through maltodecaose has been studied. The enzyme is found to hydrolyze more than a single glycosidic bond during some enzyme-substrate encounters. The extent of this repetitive attack is quantitated.     

Bioremediation of dyes in textile effluents by Aspergillus oryzae

In this study Aspergillus oryzae was utilized to remove azo dyes from aqueous solution. Physically induced in its paramorphogenic form to produce standardized mycelial pellets, the non-autoclaved and autoclaved hyphae biomass was applied to biosorb the reactive dyes Procion Red HE7B (PR-HE7B) and Procion Violet H3R (PV-H3R) at different pH values (2.50, 4.50, and 6.50). The best pH for biosorption was 2.50, though the autoclaved demonstrated a higher biosorption capacity than the non-autoclaved pellets. The toxicity level was determined using the Trimmed Spearman–Karber method with Daphnia similis in all bioassays. The calculated toxicity of PV-H3R (LC₁₀₀ 62.50 μg mL⁻¹) was higher than to PR-HE7B (LC₁₀₀ 300.00 μg mL⁻¹), and its results brought out that the decrease of toxicity levels to zero might be accomplished by adding small quantities of pelletized A. oryzae to the solutions.     

Trehalase in conidia of Aspergillus oryzae

Horikoshi, Koki (The Institute of Physical and Chemical Research, Bunkyo-ku, Tokyo, Japan), and Yonosuke Ikeda. Trehalase in conidia of Aspergillus oryzae. J. Bacteriol. 91:1883-1887. 1966.-Trehalases (soluble trehalase and coat-bound trehalase) were found in the conidia of Aspergillus oryzae, and the total activity of the trehalases increased during the germination process. The soluble trehalase was purified by diethylaminoethyl-cellulose column chromatography; its optimal pH, Michaelis constant, and heat stability were studied. In vitro, the trehalases were competitively inhibited by d-mannitol, which was also contained in the conidia. Since the trehalose content in the conidia decreased at an early stage of germination, it was assumed that trehalase might begin to hydrolyze trehalose after the inhibitory effect of d-mannitol decreased.