• Journal of Life Science
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• v.2 no.1
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• pp.26-34
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• 1992

유전자 재조합 균주의 발효에 있어서 플라스미드의 안정성과 발효공정의 최적화에 대해 개략적으로 서술하였다. 클론된 DNA의 발현은 플라스미드의 안정성을 크게 저해하며, 저하된 플라스미드의 안정성은 재조합 균주의 생산성을 많이 떨어뜨린다. 최적 발효 조건은 각각의 숙주-벡터 시스템, 사용한 배지, 생성물 등에 따라 크게 변한다. 동일한 숙주-벡터 시스템의 경우도, 사용하는 배지에 따라 온도, 희석률 또는 성장속도에 의존하는 정도가 달라지고 또 최적값도 다 변한다. 또한 발효조건의 최적화가 균체 내 플라스미드의 자기복제,mRNA로의 전사, 단백질로의 translation, 더 나아가 미생물 전체의 생리와 밀접하게 관련이 있다.

• Korean Journal of Microbiology
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• v.46 no.4
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• pp.383-388
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• 2010

Alkaline protease-overproducing strains of Vibrio metschnikovii were developed by using the molecular evolution from the classical mutants V. metschnikovii L12-23, N4-8, and KS1. Each vapK (Vibrio alkaline protease K) was obtained from the genomic DNAs of mutants by PCR to carry out the DNA shuffling. The modified vapK-1 obtained by DNA shuffling was used again as a template for the error-prone PCR to make the vapK-2. Both genes were cloned in the plasmid pKF3 to construct the recombinant plasmids which have one or two copies of the modified genes. The recombinant plasmids were back-transformed to V. metschnikovii KS1 to construct recombinant V. metschnikovii that expresses the alkaline protease. About 3.9-fold more protease activity was measured in the strain which has the plasmid containing two copies of vapK-2 when compared to strain KS1. When compared to wild type V. metschnikovii RH530, 43-fold more activity was achieved. Comparison of amino acids among vapK, vapK-1, and vapK-2 revealed that the active sites was highly conserved and not changed. However, many amino acids except the active sites were changed. These results suggested that the changes in amino acids might play an important role in the increase of protease activity by allowing the easy access of substrate to active sites of the protease. The fermentation of alkaline protease from the V. metschnikovii KS1 harboring the plasmid that contains two copies of vapK-1 showed the possibility of this strain to be used as industrial producer.

• Journal of Soil and Groundwater Environment
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• v.11 no.5
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• pp.43-50
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• 2006

Bioreporter bacteria, such as recombinant bioluminescent bacteria, have been used for the detection of specific compounds in complex environmental media. In this study, optimum conditions for the preparation and application of deep-freezed and Iyophilized recombinant bioluminescent strain KG1206 were investigated for the future application on contaminated environmental sites. Genetically engineered microorganism, Pseudomonas putida mt-2 KG1206, contains TOL plasmid and the plasmid inserted $P_{m}$, promoter on the upper part of lux gone in vector pUCD615, and m-toluate and benzoate are considered direct inducers for bioluminescence. Optimum conditions determined for the preparation and application of the deep-freezed and lyophilized strain were followings: cryoprotective agent (24% sucrose), lyophilization time (12 hrs), strain concentration ($OD_{600}=0.6$), reconstitution for freezed strain (quick reconstitution at $35^{\circ}C$), reconstitution for lyophilized strain ($3{\sim}6$ hrs exposure on LB medium), carrying conditions (keep at $20^{\circ}C$ after reconstitution). These results demonstrate the feasibility of deep-freezed or lyophilized state of genetically engineered bioluminescent strain for environmental usage.

• Journal of Korean Society of Environmental Engineers
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• v.31 no.2
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• pp.147-152
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• 2009

In this study, the applicability of alginate-immobilized Pseudomonas putida mt-2 KG1206 on the environments, contaminated with toluene analogs was conducted. Genetically engineered strain KG1206 produces light by direct (m-toluate, benzoate) and indirect (toluene, xylenes) inducers. The protocol for the alginate-immobilization was determined in terms of the cell to alginate ratio, solution, proper number of alginate beads, and other conditions. Maximum bioluminescences of five chemicals by immobilized strain were generally observed in following orders: m-toluate > p-xylene > toluene > o-xylene > m-xylene. In relationship between bioluminescence activity and inducer reduction, initial m-toluate (5 mM) in solution was removed approximately 48% of initial at 5 h exposure, showing continuous decrease of inducer chemical in solution. These results of study with alginate-immobilized beads would be useful, especially, for biomonitoring of contaminated environments with specific compounds, such as petroleum hydrocarbon compounds including toluene analogs.

• Microbiology and Biotechnology Letters
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• v.26 no.5
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• pp.400-405
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• 1998

The ddh gene encoding meso-DAP-dehydrogenase (DDH) involved in the dehydrogenase pathway is essential for high-level lysine production in Brevibacterium lactofermentum. To investigate the effect of the ddh gene amplification on lysine production by B. lactofementum, we constructed two E. coli -B. lactofermentum shuttle vector, pEB1 and pEB2. The recombinant plasmids, pRK1 and pRK2, carrying the ddh gene were introduced into B. lactofermentum by electroporation. The specific activity of DDH by amplification of the ddh gene was increased 7-fold, and also L-Lysine production of B. lactofermentum strains harboring recombinant plasmids were 18∼20% higher than that of the control.

• Microbiology and Biotechnology Letters
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• v.17 no.5
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• pp.454-460
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• 1989

Antibiotics resistance genes both in natural bacterial isolates and the genetically cloned bacteria were comparatively studied for their transfer frequencies by the method of conjugation in several different water environments. The Kmr genes in both kinds of bacteria were transferred more frequently in autoclaved wastewater of laboratory environment than in natural river water, but in Luria Bertani (LB) broth medium under the laboratory conditions the transfer frequences of the genes were much higher than in the autoclaved wastewater. The transfer frequencies at 2$0^{\circ}C$ and 3$0^{\circ}C$ were not much different in any water environments. The Km$^{${\gamma}$}$ genes of the genetically cloned bacteria and the natural isolates were transferred at the same frequency both in natural river water and in the autoclaved wastewater of laboratory environment, but in LB broth under laboratory conditions the transfer frequencies were lowered by 10$^{-3}$ to 10$^{-4}$ in the genetically cloned cells than the natural isolates. When donors of different cloned cells were conjugated with recipient of a natural isolates, the Km$^{${\gamma}$}$ genes of different donor cells were transferred at the about same frequency, but the same donor of the cloned cell were conjugated with recipients of different natural isolates, the transfer of Km$^{${\gamma}$}$ gene of the cloned cell showed some differences of 101 to 102 in frequency.

• KSBB Journal
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• v.15 no.2
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• pp.174-180
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• 2000

Pichia pastoris and Hansenula polymorpha, the methylotrophic yeasts have been widely used as a host for the production of e eudaryotic proteins due to the advantages related to their inherited characters. This paper describes the method to enhance t the productivity of recombinant proteins by P. pastoris and H. po$\psi$morpha. In the production of recombinant proteins using a f fed batch fermentation system, the effects of specific growth rate on the specific expression rate of re$\infty$mbinant proteins w were studied. In both species, the expression system of recombinant proteins using the fed batch fermentation was optimezed.

• YAKHAK HOEJI
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• v.29 no.6
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• pp.137-146
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• 1985

본고에서는 빌효산물을 개발하는 초기단계에서 발효학이 관여하여야 할 분야를 고찰함에 있어 종래방법에 비해 Rec.DNA기술에 의해 개발균주를 이용할 때 공통점과 아울러 차이점등을 살펴보기로 한다.

• Korean Journal of Microbiology
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• v.28 no.3
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• pp.192-198
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• 1990

To determine whether the muc region of pKM101 and its mutant pSL4 is sufficient for the expression of these phenotypes, muc regions of pKM101 and pSL4 were subcloned onto the highcopy number vector pKB354 and were selected the cloned pJB200 and pJB210. The recombinant plasmids pJB200 and pJB210 were introduced into umu $C36^{-}$ uvr $A6^{-}$ (TK610) strain and determined the protection effect and mutagenecity for UV and MMS. The protection effect and mutagenecity of umu $C36^{-}$ uvr $A6^{-}$ (TK610) were supressed by muc gene of the recombinant plasmids. The muc gene of pSL4 has higher effect than that of pKM101. The recombiant plasmid pJB210(inclued muc gene of pKM101) did not affect uv-mutagenesis in the $recA^{-}$ (JC2926) mutant.

• Korean Journal of Microbiology
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• v.52 no.2
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• pp.220-225
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• 2016

To improve antioxidant glutathione (GSH) content and saccharification ability in sake yeasts of Saccharomyces cerevisiae, the ${\gamma}$-glutamylcysteine synthetase gene (GSH1) from S. cerevisiae, glucoamylase gene (GAM1) and ${\alpha}$-amylase gene (AMY) from Debaryomyces occidentalis were co-expressed in sake yeasts for manufacturing a refreshing alcoholic beverage abundant in GSH from rice starch. The extracellular GSH content of the recombinant sake yeasts increased 1.5-fold relative to the parental wide-type strain. The saccharification ability by glucoamylase of the new yeast strain expressing both GAM1 and AMY genes was 2-fold higher than that of the yeast strain expressing only GAM1 gene when grown in the culture medium containing 2% (w/v) rice starch. It generated 11% (v/v) ethanol from 20% (w/v) rice starch and consumed up to 90% of the starch content after 7 days of fermentation.