• 한국균학회지
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• 제21권3호
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• pp.230-234
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• 1993

R. nigricans Ehrenberg의 돌연변이주를 분리하기 위하여 화학적인 돌연변이원인 N-Methyl-N'-Nitro-N-Nitrosoguanidine(MNNG)과 Ethyl Methane Sulphonate(EMS)의 최적 처리 조건을 조사하였을 때, MNNG농도는 $125{\mu}g/ml$, 처리시간이 60분일 때 돌연변이율이 가장 높은 생존율 0.1-1.0%를 나타내었다. MNNG를 이용하여 영양요구성 돌연변이주 Leucine auxotroph를 분리하였으며, 포자낭병의 길이가 축소된 것, 포자낭병의 모양이 나선형으로 변한 것, 그리고 포자낭과 포자낭포자의 크기가 감소된 것 등 세 가지 형태적 변이주를 분리하였다.

• Journal of Microbiology and Biotechnology
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• 제13권6호
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• pp.949-954
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• 2003

The argG gene of Corynebacterium glutamicum encoding argininosuccinate synthetase (EC6345) was cloned and sequenced. The gene was cloned by heterologous complementation of an Escherichia coli arginine auxotrophic mutant (argG/sup -/). The cloned DNA fragment also complements E. coli argD, argF, and argH mutants, suggesting a clustered organization of the genes in the chromosome. The coding region of the argG gene is 1,206 nucleotides long with a deduced molecular weight of about 44 kDa, comparable with the predicted size of the expressed protein on the SDS-PAGE. Computer analysis revealed that the amino acid sequence of the argG gene product had a high similarity to that of Mycobacterium tuberculosis and Streptomyces clavuligerus. Two conserved sequence motifs within the ArgG appear to be ATP-binding sites which correspond to 2 of the 3 conserved regions found in sequences of all known argininosuccinate synthetases.

• Journal of Microbiology and Biotechnology
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• 제1권3호
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• pp.151-156
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• 1991

In order to construct a yeast strain having high ethanol tolerance together with good lactose fermentation ability, the protoplast fusion using Saccharomyces cerevisiae STV 89 and Kluyveromyces fragilis CBS 397 was carried out. Auxotrophic mutants of K. fragilis were obtained as a selection marker by treatment of ethylmethane sulfonate. The best mutant for protoplast fusion was selected based on the capabilities of ${\beta}-galactosidase$ production and lactose fermentation. The protoplast fusion using polyethylene glycol and calcium chloride solution led to the fusion frequence of $3{\times}10^{-6}$ and a number of fusants were obtained. Among these fusants, a fusant F-3-19 showed the best results in terms of ethanol tolerance, ${\beta}-galactosidase$ activity and lactose fermentation. The performance of lactose fermentation and ethanol tolerance by this fusant were better than those of K. fragilis. Study on the ethanol tolerance having relation to fatty acid composition and intracellular ethanol concentration revealed that the fusant F-3-19 had a higher unsaturated fatty acids content and accumulated less amount of intracellular ethanol compared with a parent of K. fragilis.

• 한국균학회지
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• 제17권1호
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• pp.27-30
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• 1989

팽나무버섯 ${\beta}-isopropylmalate$ dehydrogenase의 유전자가 PBR322에 크로닝된 백터를 이용하여 사철느타리버섯 Leucine 영양요구성 균주를 형질전환 시켰다. 형질전환율은 저조하였으나 형질전환된 균총은 버섯 최소배지에서 다소 느리지만 생육할 수 있었다. 형질전환은 Southern hybridization과 형질 전환된 사철 느타리버섯 염색체 DNA를 다시 대장균에 형질전환시켜 확인하였다.

• KSBB Journal
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• 제6권1호
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• pp.63-68
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• 1991

본 연구에서는 L-phcn ylalaninc을 생산하는 조절기작을 상실한 영양요구성 변이주인 Corynebacterium glulamicum ATCC 21674를 이용하여 플라스크내에서의 회분식 배양시의 특성을 조사하였다. 이 균주는 회분반효시 2.1-3.4 g/I 의 phcnllalanine파 2.9-4.4 g/I 의 tyrosine을 생산하였고, 당농도가 높을 경우 생산성이 저하됨을 알 수 있었다. 또한 온도의 변화는 이들 아미노산 생산에 큰 영향을 미침이 관찰되었다. $30^{\circ}C$에사 배양하는 경우, $37^{\circ}C$에서 배양하는 것보다 훨씬 많은 아미노산이 생산되었다. 배양도즙 pH는 급격한 변화를 보였다, 이 균주는 tyrosine이 없는 최소배지에서도 자라는 것이 확인되었고, tyrosine를 과량 생성까시 함으로 보아 영양 요구 성질을 상실한 revertant로서 조전기작 상실성을 ­유지한 것으로 판단된다.

• 미생물학회지
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• 제29권1호
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• pp.8-15
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• 1991

Interspecific hybrids between the ASpergillus spp., A. awamori, A. usamii and A. oryzae, were obtained by nuclear transfer technique. Nuclei isolated from an auxotrophic mutant strain were transferred into the protoplasts of a recipient strain of different species. The frequency of interspecific hybrid formation by nuclear transfer was $2*10^{-5}$ $-7*10^{-4}$ In contrast, no interspecific hybrid was isolated by protoplast fusion. Among the hybrids tested, 10 strains showed increased activity of some or all components of cellulases, xylanases and amylase up to more than two times. Isozyme pattern of the hybrids were analyzed by polyacrylamide gel electrophoresis and isoelectric focusing followed by activity staining, which showed that some of the hybrids have isozyme patterns unidentical to either of the two parents. By measuring the DNA contents and the sizes ofthe conidia, the karyotypes of the hybrids were estimated to be aneuploid near to haploid, diploid or triploid. It was concluded that the unclear transfer technique is much more efficient in the formation of interspecific hybrids than protoplast fusion and is very useful for the improvement of Aspergillus strains.

• 미생물학회지
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• 제25권1호
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• pp.46-51
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• 1987

In order to increase the production of tryptophan by maximizing expression of recombinant trp plasmid, Klebsiella pneumoniae KC 105(pheA tyrA trpE trpR tyrR) was genetically modified. KC 107, inosine monophospate(IMP) auxotroph from KC 105 and KC 108, histidine(His) auxotroph from KC 107 were also derived respectively to increase phosphoribosylpyrophosphate(PRPP) production which is required for tryptophan biosynthesis. From KC 107 phosphoribosylpyrophosphate consumption which is required for tryptophan biosynthesis. From KC 107 and KC 108, KC 109 and KC 110, both arginine auxotrophs were derived respectively. To investigate the expression of recombinant trp plasmid in the selected K. pneumoniae mutants, the auxotrophic mutants were transformed with recombinant trp plasmids pSC 101-$trpE^{FBR}$, pSC 101-trpL(.DELTA.att) $trpE^{FBR}$ (pSC 101-trp-AF). Amount of tryptophan produced and activities of tryptophan synthase of $trpR^{-}$ mutant (KC 100) and $tyrR^{-}$ mutnat(KC 105) containing recombinant plasmid pSC 101-trp operon were increased by 30-40% as compared with KC 99(pheA tyrA trpE) containing recombinant plasmid pSC 101-trp operon. Activities of tryptophan synthase and production of tryptophan of KC 108 ($His^{-}$) and KC 109($Arg^{-}$) containing recombinant plasmid pSC 101-trp operon were increase by two-fold as compared with KC 107 containing pSC 101-trp operon.

• 미생물학회지
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• 제16권3호
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• pp.111-121
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• 1978

About 40 temperature-sensitive mutants have been isolated as a preliminary step to study the spore germination, the cell cycle, and the control of macromolecular synthesis in Aspergillus nidulans. To obtain temperature-sensitive mutants rapidly and effectively, the selective enrichment method using antifungal antibiotic nystatin was developed. Based on the data which had applied to the concentration of auxotrophic mutants by the earlier investigators, the optimal concentration and the time of treatment at the nonpermissive temeprature were determined as 50 to 100 units per ml and 4.5 hr., respectively. Out of 41 ts mutants assigned to the strain symbol PK, thirteen that seemed to be arrested at the earlystage of spore germination were subjected to the further cytological and genetic analysis. Elght of these mutants are able to form germ tube and five not. Staining with acid fuchsin for the 5PK strains shows that one irreversible mutant, PK6 strain able to form germ tube, accumulate mitotic spindle, being arrested in mitosis. Another PK15 and PK23 strain have more than one intact nucleolus without germ tube formation at the restrictive temperature. the temperature-senstive mutation in PK12 strain, the onlystrain which is able occurred in certain gene specific for the germination of spore. All of the ts markers are recessive and complement each other in heterokaryon between two different ts markers at the restrictive temperature.

• 미생물학회지
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• 제27권4호
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• pp.422-429
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• 1989

Auxotrophic mutant were isolated from wild types by the treatment with NTG as a mutagen, and the conditions of protoplast formation for them were established. The protoplasts of killer yeast Saccharomyces cerevisiae K52 were formed to the level of above 70% when cells grown for 20 hr in PM medium were treated with 200 unit/ml Lyticase 50,000 at $30^{\circ}C$ for 60 min after pretreatment of 50 mM 2-mercaptoethanol in 10mM potassium phosphate buffer (pH 7.5) containing EDTA and 0.6 M sorbitol for 15 min. Also, the protoplast of the recipient S. cerevisiae S 29 were formed to the level of above 85% as it was cultured to the log phase of 24 hr in PM medium under the same conditions. The fusion frequency between the protoplast of killer yeast S. cerevisiae K 52 and the protoplast of recipient S. cerevisiae S 29 was reached to $8.2\times 10^{-6}$ when the hypertonic regeneration medium embeded with the fused protoplasts after mixing the parental protoplasts to 10$^{8}$ cells/ml in SP buffer containing 20 mM $CaCl_{2}$ and 30% PEG 6,000 for 15 min at $30^{\circ}C$ were incubated.

• 한국식품영양과학회지
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• 제16권4호
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• pp.251-261
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• 1987

본(本) 실험(實驗)은 세균(細菌)에 의한 L-threonine의 효율적인 생성(生成)을 검토할 목적(目的)으로 Brevibacterium flavum ATCC 14067을 사용하여 L-threonine 생성능(生成能)이 우수한 균수(菌株)를 선발(選拔)하기 위해 변이원인 N-methyl-N'-nitro-N-nitrosoguanidine (NTG)로 처리하여 돌연변이주(突然變異株)를 유도(誘導)한 후(後), 다시 methionine 영양요구주, lysine 영양요구주, isoleucine 영양요구주, lysine 영양요구주, isoleucine 영양요구주, methionine 및 isoleucine 영양요구주를 선발(選拔)하였다. 또한 선발(選拔)된 영양요구성 변이주들 중에서 L-threonine 생성능(生成能)이 원균(原菌)에 비(比)해 $3{\sim}4$배(倍)정도 우수한 B-13균주(菌株)$(met^-)$를 선발(選拔)하여 L-threonine 생성력(生成力), 배지(培地) 조성(組成) 및 배양(培養)에 따른 몇가지 요인(要因)들에 대하여 실험한 결과 다음과 같은 결과(結果)를 얻었다. 1. L-threonine생성량은 원균주가 1.4mg/ml에 비해 methionine 영양요구성 변이주인 B-13은 4.86mg/ml로서 약 3.5배(倍)의 높은 생성량을 나타내었다. 2. B-13에 의한 L-threonine 생성(生成)에 적당한 배지조성(培地組成)은 glucose 10%, ammonium sulfate 2%, potassium phosphate monobasic 0.2%, magnesium sulfate 0.05%, biotin $200{\mu}g/l$ thiamine $300{\mu}g/l$이였으며 nicotinic acid 0.05% 첨가시 더욱 증가 되었다. 3. B-13에 있어서 유기영양원에 대한 효과는 yeast extract와 Peptone이 양호하였으며 영양요구물질인 metionine은 $100{\mu}g/ml$가 적당하였으며 aspartic acid와 homoserine 첨가시 L-threonine 생성이 증가되었으며 lysine 첨가시에는 감소하는 경향이 나타났다. 4. B-13에 의한 L-threonine 생성에 가장 적절한 pH는 $7.0{\sim}8.0$이였으며 배양일수(培養日數)는 4일(日)이 적당하였다.