• 미생물학회지
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• 제32권2호
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• pp.109-114
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• 1994

대장균에서 두 가지 다른 아미노산의 생합성에 관여하는 serC 유전자와 araA유전자는 혼합 operon을 이루고 있다. SerC-aroA 혼합 operon의 발현 조절 현상을 serC-aroA-lacZ fusion plasmid pWH2를 이용하여 측정하였다. serC-aroA 혼합 operon의 발현은 L-tyrosine, L=phenulalanine 및 L-tryptophan 등 방향족 아미노산들에 의하여 억제되었다. 방향족 아미노산에 의한 억제 효과는 $tyrR^-$ 균주 혹은 $trpT^-$ 균주에서는 감소하였다. 또한, 방향족 아미노산은 cyclic AMP에 의한 이 operon의 발현 상승 효과를 감소시키기도 하였다. 이들 결과로부터 대장균 serC-aroA 혼합 operon의 발현은 방향종 아미노산들에 의해 억제된다고 추정하였다.

• Journal of Microbiology and Biotechnology
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• 제10권6호
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• pp.789-796
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• 2000

In order to analyze the effects of tktA, $aroF^{FBR}$, and aroL expression in a tryptophan-producing Escherichia coli, a series of plasmids carrying the genes were constructed. Introduction of tktA, $aroF^{FBR}$, and aroL into the E. coli strain resulted in approximately 10-20 fold increase in the activities of transketolase, the feedback inhibition-resistant 3-deoxy-D-arabinoheptulsonate-7-phosphate synthase, and shikimate kinase. Expression of $aroF^{FBR}$ in the aroB mutant strain of E. coli resulted in the accumulation of 10 mM of 3-deoxy-D-arabinoheptulsonate-7-phosphate (DAHP) in the medium. Simultaneous expression of tktA and $aroF^{FBR}$ in the strain further increased the amount of excreted DAHP to 20 mM. In contrast, the mutant strain which has no gene introduced accumulated 0.5 mM of DAHP. However, the expression of tktA and $aroF^{FBR}$ in a tryptophan-producing E. coli strain did not lead to the increased production of tryptophan, but instead, a significant amount of shikimate, which is an intermediate in the tryptophan biosynthetic pathway, was excreted to the growth medium. Despite the fact that additional expression of shikimate kinase in the strain could possibly remove 90% of excreted shikimate to 0.1 mM, the amount of tryptophan produced was still unchanged. Removing shikimate using a cloned aroL gene caused the excretion of glutamate, which suggests disturbed central carbon metabolism. However, when cultivated in a complex medium, the strain expressing tktA, $aroF^{FBR}$, and aroL produced more tryptophan than the parental strain. These data indicate that additional rate-limiting steps are present in the tryptophan biosynthetic pathway, and the carbon flow to the terminal pathway is strictly regulated. Expressing tktA in E. coli cells appeared to impose a great metabolic burden to the cells as evidenced by retarded cell growth in the defined medium. Recombinant E. coli strains harboring plasmids which carry the tktA gene showed a tendency to segregate their plasmids almost completely within 24h.

• ETRI Journal
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• 제33권1호
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• pp.39-49
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• 2011

A new structure learning approach for Bayesian networks based on asexual reproduction optimization (ARO) is proposed in this paper. ARO can be considered an evolutionary-based algorithm that mathematically models the budding mechanism of asexual reproduction. In ARO, a parent produces a bud through a reproduction operator; thereafter, the parent and its bud compete to survive according to a performance index obtained from the underlying objective function of the optimization problem: This leads to the fitter individual. The convergence measure of ARO is analyzed. The proposed method is applied to real-world and benchmark applications, while its effectiveness is demonstrated through computer simulations. Results of simulations show that ARO outperforms genetic algorithm (GA) because ARO results in a good structure and fast convergence rate in comparison with GA.

• Journal of Microbiology and Biotechnology
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• 제24권10호
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• pp.1421-1426
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• 2014

Although a large number of AroA enzymes (EPSPS: 5-enopyruvylshikimate-3-phosphate synthase) have been identified, cloned, and tested for glyphosate resistance, only two AroA variants, derived from Agrobacterium tumefaciens strain CP4 and Zea mays, have been utilized to produce the commercial glyphosate-resistant crops. Here, we have used a PCR-based twostep DNA synthesis method to synthesize an aroA gene ($aroA_{A.\;metalliredigens}$) from Alkaliphilus metalliredigens, encoding a new EPSPS. Furthermore, transgenic Arabidopsis with the new $aroA_{A.\;metalliredigens}$ gene was obtained to confirm the potential of the novel aroA gene in developing glyphosate-resistant crops.

• BMB Reports
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• 제28권1호
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• pp.21-26
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• 1995

The Escherichia coli mixed-function serC-aroA operon encodes biosynthethic enzymes for unrelated pathways leading to the syntheses of serine and aromatic amino acids. It has been proposed that the operon is expressed in a cAMP-dependent manner. In this work experiments were performed to investigate the cAMP-dependent expression of the operon. Exogenous cAMP increased ${\beta}$-galactosidase synthesis in the $cya^+$ and cya strains harboring the serC-aroA-lac fusion plasmid. This enhancement was more dramatic in the $cya^-$ strain grown in a minimal medium. In a dot blot assay the serC-aroA mRNA content increased in a concentration-dependent pattern after the addition of exogenous cAMP. The activity of phosphoserine aminotransferase, encoded by the serC gene, apparently increased in E. coli cells after the addition of cAMP. All results obtained confirmed that the expression of the E. coli serC-aroA operon is positively regulated by cAMP at the level of transcription.

• Journal of Microbiology and Biotechnology
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• 제24권9호
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• pp.1162-1169
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• 2014

Glyphosate is the active component of the top-selling herbicide, the phytotoxicity of which is due to its inhibition of the shikimic acid pathway. 5-Enolpyruvylshikimate-3-phosphate synthase (EPSPS) is a key enzyme in the shikimic acid pathway. Glyphosate tolerance in plants can be achieved by the expression of a glyphosate-insensitive aroA gene (EPSPS). In this study, we used a PCR-based two-step DNA synthesis method to synthesize a new aroA gene ($aroA_{R.\;leguminosarum}$) from Rhizobium leguminosarum. In vitro glyphosate sensitivity assays showed that $aroA_{R.\;leguminosarum}$ is glyphosate tolerant. The new gene was then expressed in E. coli and key kinetic values of the purified enzyme were determined. Furthermore, we transformed the aroA gene into Arabidopsis thaliana by the floral dip method. Transgenic Arabidopsis with the $aroA_{R.\;leguminosarum}$ gene was obtained to prove its potential use in developing glyphosate-resistant crops.

• Journal of Microbiology and Biotechnology
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• 제33권10호
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• pp.1370-1375
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• 2023

In this study, we aimed to enhance the accumulation of chorismate (CHR) and anthranilate (ANT), key intermediates in the shikimate pathway, by modifying a shikimate over-producing recombinant strain of Corynebacterium glutamicum [19]. To achieve this, we utilized a CRISPR-driven genome engineering approach to compensate for the deletion of shikimate kinase (AroK) as well as ANT synthases (TrpEG) and ANT phosphoribosyltransferase (TrpD). In addition, we inhibited the CHR metabolic pathway to induce CHR accumulation. Further, to optimize the shikimate pathway, we overexpressed feedback inhibition-resistant Escherichia coli AroG and AroH genes, as well as C. glutamicum AroF and AroB genes. We also overexpressed QsuC and substituted shikimate dehydrogenase (AroE). In parallel, we optimized the carbon metabolism pathway by deleting the gntR family transcriptional regulator (IolR) and overexpressing polyphosphate/ATP-dependent glucokinase (PpgK) and glucose kinase (Glk). Moreover, acetate kinase (Ack) and phosphotransacetylase (Pta) were eliminated. Through our CRISPR-driven genome re-design approach, we successfully generated C. glutamicum cell factories capable of producing up to 0.48 g/l and 0.9 g/l of CHR and ANT in 1.3 ml miniature culture systems, respectively. These findings highlight the efficacy of our rational cell factory design strategy in C. glutamicum, which provides a robust platform technology for developing high-producing strains that synthesize valuable aromatic compounds, particularly those derived from the shikimate pathway metabolites.

• Journal of Microbiology and Biotechnology
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• 제31권9호
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• pp.1305-1310
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• 2021

Shikimate is a key high-demand metabolite for synthesizing valuable antiviral drugs, such as the anti-influenza drug, oseltamivir (Tamiflu). Microbial-based strategies for shikimate production have been developed to overcome the unstable and expensive supply of shikimate derived from traditional plant extraction processes. In this study, a microbial cell factory using Corynebacterium glutamicum was designed to overproduce shikimate in a fed-batch culture system. First, the shikimate kinase gene (aroK) responsible for converting shikimate to the next step was disrupted to facilitate the accumulation of shikimate. Several genes encoding the shikimate bypass route, such as dehydroshikimate dehydratase (QsuB), pyruvate kinase (Pyk1), and quinate/shikimate dehydrogenase (QsuD), were disrupted sequentially. An artificial operon containing several shikimate pathway genes, including aroE, aroB, aroF, and aroG were overexpressed to maximize the glucose uptake and intermediate flux. The rationally designed shikimate-overproducing C. glutamicum strain grown in an optimized medium produced approximately 37.3 g/l of shikimate in 7-L fed-batch fermentation. Overall, rational cell factory design and culture process optimization for the microbial-based production of shikimate will play a key role in complementing traditional plant-derived shikimate production processes.

• 한국동물학회:학술대회논문집
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• 한국동물학회 1998년도 한국생물과학협회 학술발표대회
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• pp.335.2-335
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• 1998

No Abstract, See Full Text

• 미생물학회지
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• 제41권1호
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• pp.24-37
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• 2005

국내 사육 하우 염소, 돼지, 개, 닭 및 마우스 등을 포함한 각종 동물과 사람환자에서 분리한 총 116주의 S. aureus 분리주에 대해서 5종의 PCR 기법을 적용하여 분자역학적 특성을 분석하였다. 먼저 종특이 유전자(SSG: aroA, coa, nuc 및 spa-gene)의 다양성을 PCR 기법으로 조사하였고, 또한 약제내성의 MRSA 균주의 분포양상과 내독소(Enterotoxin)산생유전자(SE)의 분포양상에 대해서도 조사하였다. 그리고 이 연궁선 수행한 5종의 PCR 기법 들이 생산한 개별성적을 객관적으로 비교하여, 가장 신뢰도 높고 효율적 PCR기법을 선발하였다. 먼저 PCR기법을 적용한 SSG의 분포양상 조사에서는 $nuc-gene\;(100\%)$, $spa-gene\;(91.4\%)$, $coa-gene\;(87.9\%)$, 및 $aroA-gene\;(26.7\%)$의 빈도로 조사되었다. 그리고 aroA와 coa-gene PCR 증폭산물에 대한 RsaI과 AluI 효소로 소화시킨 RELP 성적에서 coa-Bene PCR-RFLP에서는 모두 10 type의 con-type이 동정되었고, 그중 coa-3 type (809 bp)이 닭, 마우스, MRSA 및 사람유래 균주를 포함한 총 36주 $(33.0\%)$로서 가장 대표적인 genotype으로 결정되었다. 반면에 aroA-gene PCR-RELP에서는 단지 7 type의 genotype만이 산생되어 대조를 보였고, 17주 $(73.9\%)$가 대표적인 그룹으로 분류되었다. 한편 spa-gene PCR에서는 총 11 type의 spa-type이 확인되었고, 그중 spa-7 type (9 repeats; 263 bp)이 한우, 닭, 개, 염소, 마우스 및 MRSA 균주 등을 포함한 총 39주 $(34.8\%)$로서 가장 대표적인 genotype으로 결정되었다. 또한 총 116주에 대해 MRSA 균주의신속한 검출을 위해서 mecA-gene PCR을 적용하였던 바, 단지 사람유래의 14주 $(12.1\%)$에서만 양성의 증폭산물 (533 bp)이 검출되었고, 이들 증폭된 PCR산물의 유전학적 검증을 위해 HhaI으로 소화시켰던바 14주 모두 $(100\%)$가 알려진 2개의 DNA band (332 & 201 bp)로 양분되어 유전자수준에서 MRSA 양성균주로 검증되었다. 한편 내독소 산생유전자 (SE-gene)는 multiplex-PCR기법으로 조사하였으며 sea-gene $(63.7\%)$이 가장 지배적이었고, 이어서 seb-gene $(10.0\%)$ 및 sec-gene $(7.2\%)$의 순서였다. 특히 sea+sec의 2종의 SE genes를 보유한 균주도 11주로서 가장 많았으며 그 외에도 sea+seb (2주)및 seb+see (1주) genes를 보유한 균주도 함께 검출되었다. 최종적으로 5종의 PCR기법들이 생산한 성적들을 수치 (SID)로 변환하여 객관적으로 감별능력 (DA)을 비교하였던바, aroA-gene PCR-RFLP는 가장 저조한 DA [SID=0.462]을 나타내었고, 반면에 coa-gene PCR-RELP는 5종의 분석기법 중에서 가장 신뢰도 높은 DA [SID=0.894]을 발현하였다. 따라서 현재의 연구결과con-gene PCR-RELP기법이 사람을 포함한 다양한 동물종유래 S. aureus 균주들의 유전학적 분석목적에 가장 신뢰도 높고 감별능력이 뛰어난 분석기법으로 선발되었다.