• 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

Amino 산(酸) 핵산관련물질등(核酸關連物質等)을 발효생산(醱酵生産)하는데 응용(應用)키 위(爲)하여 Adenine auxotrophic mutants를 분리(分離)하려고 Gram positive이며 Spore forming bacteria 인 Bacillus subtilis 및 Gram negative bacteria 인 Escherichia coli 를 시험균주(試驗菌株)로 하였다. 이들 균주(菌株)에 U.V-light 처리(處理)와 그리고 Vitamin 및 핵산(核酸) Analogue와 Aminopterine, Streptomycin 처리(處理)로서 효율(效率)좋게 Adenine auxotrophic mutants를 분리(分離)하였다. 1. U.V. -light$(2530\;{\AA}\;2080\;erg/mm^2)$, distance 30 cm) $80{\sim}90sec$. 조사(照射)가 Bacillus subtilis의 Auxotrophic mutants 4주(株) 그리고 E. coli에 $15{\sim}20sec$. 조사(照射)는 Auxotrophic mutants 8주(株)를 분리(分離)하는데 최적조건(最適條件)임을 알았다. 2. Bacillus subtilis 에 대(對)하여 위의 최적조건(最適條件)으로 조사(照射)하는데 Aminopterine$(200\;{\mu}g/ml)$. 처리구(處理區)가 생육(生育)을 현저(顯著)히 조지(阻止)시켰고 무처리구(無處理區)보다 더욱 효과적(效果的) 으로 Adenine 요구변이주(要求變異株)를 약(約) 6배정도(培程度) 더 분리(分離)케 하였다. E. coli에 대(對)하여도 위의 최적조건(最適條件)으로 조사(照射)하는데 Streptomycin$(200\;{\mu}g/ml)$ 처리구(處理區)는 생육(生育)을 현저(顯著)히 조지(阻止)시켰고 무처리구(無處理區)보다 Adenine요구변이주(要求變異株)를 2배(倍)나 효과적(效果的)으로 분리(分離)할 수 있었다.

• 한국균학회지
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• 제14권2호
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• pp.185-188
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• 1986

노랑 느타리버섯의 유전 연구 및 다른 느타리종과의 융합을 위해서 노랑 느타리의 담자포자에 자외선을 조사하여 영양요구성 균주를 선발하였다. 노랑 느타리 포자에 UV를 조사한 결과 UV조사시간이 $40{\sim}70$초이고 생존율 $0.4{\sim}1.4%$ 일때 많은 mutants를 얻을 수 있었고, 선발된 14 auxotrophic mutants의 유전 marker는 amino acid-requiring strain 3균주, nucleic acid base-requiring strain 1 균주, vitamin-requiring strain 3 균주, amino acid/nucleic acid base-requiring strain 2 균주, amino acid/vitamin-requiring strain 5 균주로 되었다.

• 미생물학회지
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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.

• 한국미생물학회:학술대회논문집
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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

• 미생물학회지
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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.

• 한국균학회지
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• 제25권1호통권80호
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• pp.26-29
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• 1997

여름느타리버섯의 담자포자에 자외선을 조사하여 5'-fluoro-orotic acid(FOA) 선발배지에서 pyrimidine 영양요구성 균주를 직접 선발하였다. 24개의 5'-FOA 저항성 균주중 13개의 pyrimidine 영양요구성 균주를 선발할 수 있었으며, 이들의 mating type group과 성장속도를 측정하여 형질전환을 위한 최적의 DNA수용체를 선발할 수 있었다.

• 미생물학회지
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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 $1.6{\times}10^{-2}\;and\;4.1{\times}10^{-2}$.