• Journal of Microbiology and Biotechnology
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• 제34권5호
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• pp.1178-1187
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• 2024

Cordyceps militaris is a significant edible fungus that produces a variety of bioactive compounds. We have previously established a uridine/uracil auxotrophic mutant and a corresponding Agrobacterium tumefaciens-mediated transformation (ATMT) system for genetic characterization in C. militaris using pyrG as a screening marker. In this study, we constructed an ATMT system based on a dual pyrG and hisB auxotrophic mutant of C. militaris. Using the uridine/uracil auxotrophic mutant as the background and pyrG as a selection marker, the hisB gene encoding imidazole glycerophosphate dehydratase, required for histidine biosynthesis, was knocked out by homologous recombination to construct a histidine auxotrophic C. militaris mutant. Then, pyrG in the histidine auxotrophic mutant was deleted to construct a ΔpyrG ΔhisB dual auxotrophic mutant. Further, we established an ATMT transformation system based on the dual auxotrophic C. militaris by using GFP and DsRed as reporter genes. Finally, to demonstrate the application of this dual transformation system for studies of gene function, knock out and complementation of the photoreceptor gene CmWC-1 in the dual auxotrophic C. militaris were performed. The newly constructed ATMT system with histidine and uridine/uracil auxotrophic markers provides a promising tool for genetic modifications in the medicinal fungus C. militaris.

• The Plant Pathology Journal
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• 제25권3호
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• pp.205-212
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• 2009

Colletotrichum acutatum was the main cause of the recent outbreaks of anthracnose on pepper fruit in Korea. To facilitate molecular analysis of C. acutatum, we generated an arginine auxotrophic mutant of the C acutatum strain JC24 using a targeted gene replacement strategy. A 3.3-kb genomic region carrying an ortholog (designated CaARG2) of the fungal gene encoding N-acetylglutamate synthase, the first enzyme of arginine biosynthesis in fungi, was deleted from the fungal genome. The mutant exhibited normal growth only when arginine was exogenously supplied into the culture medium. Transformation of the arginine auxotrophic mutant with a plasmid DNA carrying an intact copy of CaARG2, which was smaller than the deleted region in the mutant, not only caused random vector insertions in the fungal genome, but also recovered both hyphal growth and pathogenicity of the mutant to the wild-type level. Using this new selection system, we have successfully developed a restriction enzyme-mediated integration procedure, which would provide an economically efficient random mutagenesis method in C. acutatum.

• Journal of Microbiology and Biotechnology
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• 제2권2호
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• pp.102-107
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• 1992

For the purpose of producing L-ornithine by microbial fermentation, mutant strains were developed from glutamate-producing bacteria by mutagenesis using N-methyl-N'-nitro-N-nitrosoguanidine (NTG) and UV irradiation. Brevibacterium ketoglutamicum BK1046, a L-citrulline auxotroph which is also resistant to arginine hydroxamate (Arghx), was isolated and selected as the best producer of L-ornithine. This strain was capable of producing L-ornithine at a concentration of 24 g/l after 69 hours of cultivation in the 21 jar fermentor. The optimum supplementary level of L-arginine, a substitute for L-citrulline, was found to be about 0.2 g/l in the shake-flask fermentation.

• Applied Biological Chemistry
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• 제11권
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• pp.137-141
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• 1969

In order to obtain amino acids and nucleic acid derivatives from adenine auxotrophic mutants of Bacillus subtilis and Escherichia coli, vitamin, nucleic acid analogue, streptomycin as well as ultraviolet light were adopted for the production of adenine auxotrophic mutants and the results showed efficient production of desired mutants. 1. Ultraviolet ray $(2530\;{\AA}\;2080\;erg/mm^2)$ irradiation to Bacillus subtilis and E. coli at a distance of 30 cm for 80-90 sec. and for 15-20 sec. respetively induced four and eight strains of auxotrophic mutants. 2. Treatment of aminopterine$(200\;{\mu}g/ml)$ inhibited the growth of Bacillus sultilis significantly but a subsequent irradiation of ultraviolet light at the above mentioned conditions induced six times as much mutants as compared to the irradiation alone. In case of E. coli a similar tendency was observed with treatment of streptomycin$(200\;{\mu}g/ml)$ with doubled induction rate of adenine auxotrophic mutants as compared to the irradiation alone.

• The Korean Journal of Mycology
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• 제14권2호
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• pp.185-188
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• 1986

After treatment of basidiospores of P. cornucopiae with ultraviolet light, 84 putative mutants from 4671 isolates were obtained. The highest proportion of auxotrophic mutants was obtained from the isolates irradiated to give $0.4{\sim}1.0%$ survival. Fourteen auxotrophs were selected for protoplast fusion and each genetic marker was identified.

• Korean Journal of Microbiology
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• 제20권1호
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• pp.1-10
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• 1982

From Aspergillus nidulans, six suppressor mutants, MSI-MS6, were isolated, and their characteristics were analysed. These were the suppressor mutants for acriflavin resistant and nicotinamide auxotrophic mutant phenotypes. MS1, MS2, MS5 and MS6, were linked to the chromosome IV, I, II respectively, and MS2 was linked to one of the rest chromosomes, MS3 and MS6 mutants had both suppressors for acriflavin resistant marker and for nicotinamide auxotrophic marker. In order to know the stability and efficiency of the suppressors, their reversion frequencies, that is, frequencies of losing the suppressibility, were analysed. Only MS3 and MS5 were reversed with high frequency. The four mutants didn't lose their suppressibilities, and this meant that the suppressors of these four were very stable and highly effcient. The suppressor specificities of these mutants were tested for other mutant's phenotype marker. One of the six suppressors, MS1, had the suppressor specificity for acriflavin resistant marker of 163 strain.

• Proceedings of the Microbiological Society of Korea Conference
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• 한국미생물학회 2005년도 International Meeting of the Microbiological Society of Korea
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• pp.225.4-225
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• 2005

• Korean Journal of Microbiology
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• 제29권2호
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• pp.117-122
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• 1991

In order to improve the removal ability of phosphate, Spheroplast fusions were performed among auxotrophic mutants of Aeromonas hydrophila isolated from waste water, named A13 and A14, Aci37 auxotrophic mutant of Acinetobactercalcoaceticus, and auxotrophic E. coli HR262/pCE27 carring pit gene. Eight fusants obtained from this experiment showed different biochemical characteristics. When the rate of phosphate uptake among fusants (F1-F8) was investigated in Phosphate Uptake Medium (PUM), F8 strain showed the highest rate for phosphate removal, 7 times as much as control after two hours incubation. The role of cations ($Mg^{++}$ ,$Ca^{++}$ , $K^{+}$ in phosphate uptade by F8 was also investigated in PUM without each salt. $K^{+}$ seemed to be crucial. Being compared with phosphate untake rate in PUM, that in PUM without $K^{+}$ was reduced 1.5 times. Therefore, by applying F8 strain and $K^{+}$ in practical environmental system, the increased efficiency in phosphate removal can be derived.

• The Korean Journal of Mycology
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• 제25권1호통권80호
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• pp.26-29
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• 1997

Pyrimidine auxotrophic basidiospores of Pleurotus sajor-caju were selected using positive selection method. Wild type basidiospores could not grow on minimal medium containing the pyrimidine analog 5'-fluoro-orotic acid (5'-FOA) whereas pyrimidine auxotrophs grew normally. After treatment of basidiospores with ultraviolet light, a total of 13 pyrimidine auxotrophic basidiospores were isolated among 24 5'-FOA resistant mutants. These mutants require the pyrimidine such as uracil, cytosine, thymine. Mating type group and growth rate of their mutants were determined.

• Korean Journal of Microbiology
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• 제22권2호
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• pp.103-110
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• 1984

The conditions for the protoplast fusion of auxotrophic mutants of Trichoderma koningii were determined. A preparation of commercial enzyme Driselase was used successfully to isolate protoplasts from the 18 hr old mycelium of T. koningii. The yields of protoplasts production were ranged from $0.3{\times}10^8$ to $2.5{\times}10^8$ protoplasts per mg of damp mycelium of various auxotrophic mutant strains. The regeneration frequencies from $9.3{\times}10^{-3}\;to\;2.0{\times}10^{-1}$ were obtained when the protoplasts from auxotrophic mutants were plated on the malt extract medium containing 0.6M $MgSO_4$, and 2% agar, and the optimal concentration of PEG for protoplst fusion was 30%. Exposure of protoplasts to PEG for 10 min was found to be sufficient to induce high frequency heterokaryon formation. Optimal pH of fusion mixture was determined as 5.5, and 1 mM of calcium chloride in fusion mixture was found to be sufficient to enhance protoplast fusion frequency. Under optimal condition, the fusion frequency of the cross between protoplasts from various auxotrophic mutants were $1.6{\times}10^{-2}\;and\;4.1{\times}10^{-2}$.