• Korean Journal of Microbiology
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• v.23 no.1
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• pp.9-12
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• 1985

As a part of the host cell development for a amplified recombinant trp operon, $aroP^-$ mutation was introduced in a E. coli host strain. $aroP^-$ mutation was induced by transposon Tn10 and transduced into the E. coli host cell by bacteriophage P1Kc. The effect of $aroP^-$ mutation on the excretion of tryptophan in E. coli $trpR^{-ts}/ColE_1 -trp^+$ cells was investigated. Mutant lacking the general aromatic transport system was resistant to ${\beta}-2-thienylalanine\;(2{\times}10^{-4}\;M)$, p-fluorophenylalanine $(2{\times}10^{-4}M)$, or 5-methyltryptophan $(2{\times}10^{-4}\;M.)[^3H]-tryptophan$ uptake of the $aroP^-$ mutant strain was reduced considerably as compared with $aroP^+$ counterpart. The rate of $[^3H]-tryptophan$ uptake of the $aroP^-$ mutant strain treated with $NaN_3(3{\times}10^{-2}\;M)$ was much less affected than that of $aroP^+$ counterpart. The $aroP^-$ transductants increased the tryptophan excretion from E. coli $trpR^{-ts}/ColE_1 -trp^+$ four times more than $aroP^+$ counterpart.

• Korean Journal of Microbiology
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• v.36 no.1
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• pp.46-51
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• 2000

Salmonella typhi is one of important causes of human enteric infections. S. typhi KNIH100 was isolated from a patient of typhoid fever in Korea. We cloned a 5.0 kb SalⅠ fragment containing the aroA gene encoding a 5-enolpyruvylshikimate-3-phosphate synthetase from chromosomal DNA of this strain. This recombinant plasmid was named pSAL80. E. coli CGSC2829, an aroA- mutant, was not grown on the M9 minimal medium but E. coli CGSC2829 (pSAL80) was grown on the M9 minimal medium. The aroA gene was composed of 1,284 base pairs with ATG initiation codon and TAA termination codon. Sequence comparison of the aroA gene exhibited 99%, 98%, and 77% identity with those of S. typhi Ty2, S. typhimurium, and E. coli respectively. As in the cases of Shigella sonnei and E. coli, the serC and aroA genes lie in a single operonic structure.

• Korean Journal of Microbiology
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• v.32 no.2
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• pp.109-114
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• 1994

The Escherichia coli aroA and serC genes constitute a mixed-function operon which involves in two different amino acid biosynthetic pathways. The regulation of expression of serC-aroA operon was evaluated through the use of a serC-araA-lacZ fusion plasmid pWH2. The expression of the serC-aroA operon was decreased by aromatic amino acids such as tyrosine, tryptophan, and phenylalanine. The repressible effects were diminished in E. coli tyrR of trpR strain, indicating the involvemnt of TyrR of TrpR protein in the repression. Tyrosine was competitie with cAMP in the influence on the expression of the serC-AroA operon. From these data, it was suggested that the serC-aroA operon is controlled by aromatic amino acids in a negative manner.

• Korean Journal of Microbiology
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• v.36 no.3
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• pp.187-191
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• 2000

Sulmonella gyhi is one of important causes of human enteric infections. S @phi I

• Journal of Microbiology and Biotechnology
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• v.34 no.4
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• pp.969-977
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• 2024

Indigo is a valuable, natural blue dye that has been used for centuries in the textile industry. The large-scale commercial production of indigo relies on its extraction from plants and chemical synthesis. Studies are being conducted to develop methods for environment-friendly and sustainable production of indigo using genetically engineered microbes. Here, to enhance the yield of bioindigo from an E. coli whole-cell system containing tryptophanase (TnaA) and flavin-containing monooxygenase (FMO), we evaluated tryptophan transporters to improve the transport of aromatic compounds, such as indole and tryptophan, which are not easily soluble and passable through cell walls. Among the three transporters, Mtr, AroP, and TnaB, AroP enhanced indigo production the most. The combination of each transporter with AroP was also evaluated, and the combination of AroP and TnaB showed the best performance compared to the single transporters and two transporters. Bioindigo production was then optimized by examining the culture medium, temperature, isopropyl β-D-1-thiogalactopyranoside concentration, shaking speed (rpm), and pH. The novel strain containing aroP and tnaB plasmid with tnaA and FMO produced 8.77 mM (2.3 g/l) of bioindigo after 66 h of culture. The produced bioindigo was further recovered using a simple method and used as a watercolor dye, showing good mixing with other colors and color retention for a relatively long time. This study presents an effective strategy for enhancing indigo production using a combination of transporters.

• Korean Journal of Microbiology
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• v.28 no.2
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• pp.169-173
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• 1990

For the overproduction of L-phenylalanine using Escherichia coli, the authors constructed various recombinant plasmids including pMW 10, pMW 11 and pMW 12. The $aroF{FR}$ and $pheA^{FR}$ genes for the production of L-phenylalanine were isolated from Escherichia coli MWEC 101-5 strains. The productivity and atability of Escherichia coli regulatory mutants containing recombinant plasmids were investigated to evaluate the efficiency of the $aroF^{FR}$ and $pheA^{FR}$ genes. The MWEC 101-5/pMW 11 strain produced 24.3g/l of L-phenylalanine while its stability was 73.8 percent. The specific activity of prephenate dehydratase in the MWEC 101-5/pMW 11 strain increased by 26-fold compared with that of Escherichia coli K-12.

• Journal of Microbiology and Biotechnology
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• v.22 no.6
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• pp.856-865
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• 2012

We describe the construction and immunobiological properties of a novel whooping cough vaccine candidate, in which the aroQ gene, encoding 3-dehydroquinase, was deleted by insertional inactivation using the kanamycin resistance gene cassette and allelic exchange using a Bordetella suicide vector. The aroQ B. pertussis mutant required supplementation of media to grow but failed to grow on an unsupplemented medium. The aroQ B. pertussis mutant was undetectable in the trachea and lungs of mice at days 6 and 12 post-infection, respectively. Antigen-specific antibody isotypes IgG1 and IgG2a, were produced, and cell-mediated immunity [CMI], using interleukin-2 and interferon-gamma as indirect indicators, was induced in mice vaccinated with the aroQ B. pertussis vaccine candidate, which were substantially enhanced upon second exposure to virulent B. pertussis. Interleukin-12 was also produced in the aroQ B. pertussis-vaccinated mice. On the other hand, neither IgG2a nor CMI-indicator cytokines were produced in DTaP-vaccinated mice, although the CMI-indicator cytokines became detectable post-challenge with virulent B. pertussis. Intranasal immunization with one dose of the aroQ B. pertussis mutant protected vaccinated mice against an intranasal challenge infection, with no pathogen being detected in the lungs of immunized mice by day 7 post-challenge. B. pertussis aroQ thus constitutes a safe, non-reverting, metabolite-deficient vaccine candidate that induces both humoral and cell-mediated immune responses with potential for use as a single-dose vaccine in adolescents and adults, in the first instance, with a view to disrupting the transmission cycle of whooping cough to infants and the community.

• Proceedings of the Microbiological Society of Korea Conference
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• 2000.05a
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• pp.58-65
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• 2000

Diarrheal diseases are a major cause of both illness and death in developing countries and are caused by rotavirus, Shigella spp., Salmonella spp., enterotoxigenic Escherichia coli (ETEC), and Vibrio spp. In this study, for the development of vaccine against diarrheal diseases caused by Shigella sonei, Salmonella typhimurium, E. coli O157, and Vibrio cholerae, cloning and nucleotide sequence analysis of genes and characteristics of their gene products in E. coli were performed. For construction of attenuated strain of S. sonnei KNIH104 and Salmonella typhimurium KNIH100, the aroA genes were cloned, respectively. The recombinant plasmid $_pJP{\Delta}A45$ containing aroA deleted region and suicide vector $(_pJP5603)$ was constructed. The aroA gene deleted mutants were constructed using this recombinant plasmid. For cloning gene encoding antigenic region of E. coli O157 KNIH317, the O-antigen synthesis gene cluster and sit gene was cloned. The E. coli XL1-Blue cells harboring this recombinant plasmid showed cytotoxicity in Vero cells. The ctx gene was cloned for tile purpose of antigenic region against V. cholerae KNIH002. Sequence analysis confirmed that the virulence gene cassette was consisted of ace, zot, ctxA and ctxB genes.

• Korean Journal of Microbiology
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• v.33 no.1
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• pp.1-6
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• 1997

5-Enolpyruvylshikimate 3-phosphate(EPSP) synthase is the sixth enzyme of the shikimate pathway that synthesizes aromatic amino acids. The enzyme is a primary target for the glyphos'lte which is a broad-spectrum and environmetally safe herbicide. As a first step toward development of glyphpsate-resistant EPSP synthase, the EPSP synthase gene(aroA) was amplified by polymerase chain reaction and cluned into pET-25b vector. In this construct. designated pET-aro, the aroA gene is expressed under control of strong T7 promoter. and the EPSP synthase is produced as a fusion protein with pelB leader at N-terminus and HSV-tag and His-tag at C-terminus. When the pET-aro clone was induced to produce the enzyme, it was found that the EPSP synthase was successfully exported to peri plasmic space. The periplasmic transport was greatly dependent on the induction temperatures. Among the induction temperatures examined($25^{\circ}C$, $30^{\circ}C$, $34^{\circ}C$ and $37^{\circ}C$). induction at $34^{\circ}C$ gave rise to maximal periplasmic transport. The recomhinant EPSP synthase could have been purified hy $Ni^{2+}$ -affinity chromatography using the His-tag. and detected hy anti-HSV -tag antibody. The recombinant EPSP synthase also hound to phosphocellulose resin and was eluted hy shikimate 3-phosphate and phosphoenolpyruvate. as expected. The recombinant EPSP synthase purified from phosphocellulose resin showed typical EPSP synthase activity.

• Toxicological Research
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• v.11 no.1
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• pp.31-36
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• 1995

Cyclophosphamide (CP) must be enzymatically activated by cytochrome P450(CYP)-linked mixed-function oxidation pathway to be either mutagenic or teratogenic. Influences of alterations in hepatic mixed-function oxidase acitivity and glutathione (GSH) content on the embryotoxicity of CP were studied in rat whole embryo culture system. The embryotoxicity of CP was compared using rat S-9 fraction (S-9) pretreated with chemicals inducing different CYP isozymes, acetone (ACE), Aroclor 1254 (ARO), $\beta$-naphthoflavone (NAF) and phenobarbital (PHE). When 10.5 day embryos were cultured in the immediately centrifuged rat serum for 48 hrs using general gas char{ging schedule, CP$(40{\mu}g/ml)$ with S-9 induced by either NAF or PHE increased the incidence of realformations and significantly decreased embryonic growth compared with the non-induced S-9 group. ACE or ARO induced S-9 group showed no significant difference in embryonic growth. These data suggest that PB and/or NAF inducible CYP isoenzymes are mainly involved in the activation of CP. To examine the effect of GSH on the embryotoxicity of CP, 10.5 day embryos were exposed to CP and S-9 after preincubation with 10 mM of GSH for 3 hrs. In the GSH pretreated group the growth of embryos increased significantly compared with that of the untreated group, suggesting that GSH may protect embryos in culture from some toxic effects of CP.