• KSBB Journal
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• v.16 no.1
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• pp.1-10
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• 2001

Escherichia coli has been used as an expression work horse for foreign genes. This article summarized recent development in genetic engineering techniques for overproduction of medical proteins and industrial enzymes. Special emphasis was placed upon research activities concerning folding and refolding of inclusion bodies at genetic and fermentation levels. Plasmid and mRNA stabilization, development of strong inducible promoters, modification of translational elements and reduction of rpoteolytic degradation were carried out to elevate an expression level of a target protein. Optimization of culture conditions, improvement of denaturation and renaturation steps and coexpression of molecular chaperones or foldase were accomplished to produce active proteins in soluble form. Fusion protein systems with selective separation and surface display technology were also performed in an effort to make the E. coli expression system more effective and versatile.

• Food Science and Biotechnology
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• v.17 no.6
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• pp.1365-1367
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• 2008

The antibacterial activity of chitosan against Escherichia coli was investigated in the presence of NaCl, sucrose, and ethanol to assess the potential use of chitosan as a biopreservative in food products containing these components. The inhibitory activity of chitosan decreased slightly upon the addition of NaCl and sucrose, respectively to culture broth containing 100 ppm of chitosan (Mw 3,000), while the addition of ethanol enhanced the inhibitory activity of chitosan on growing cells. The addition of these components to non-growing cells prior to chitosan treatment demonstrated that NaCl protected the cells from the inhibitory activity of chitosan, while sucrose had no effect. Ethanol addition to non-growing cells increased cell death by chitosan treatment. Finally, binding of fluorescein isothiocyanate (FITC)-labeled chitosan to E. coli was measured in the presence of the food components. The FITC-labeled chitosan binding to cells decreased upon NaCl addition, was not affected by sucrose, and increased following treatment with ethanol.

• Herbal Formula Science
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• v.8 no.1
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• pp.299-318
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• 2000

In order to study on SOD like activity and Electric donor ability of Hedyotis diffusa $W_{ILLD}$, testing antibacterial effect on E. coli O157 which causes infectious inflammatory disease; measunng inhibitory effect on tyrosinase which stimulates melanin formation; and measuring the influence of it to SOD like activity, DPPH and TBARS which are related to ageing and carcinogenesis. The results of these experiments, are as follows. 1. In the antibacterial experiment with the extract of Hedyotis diffusa $W_{ILLD}$ on E. coli O157, there was no change in proliferation from the beginning of culture to two hours after, but proliferation inhibiting effect on E. coli O157 was detected from three hours after the beginning of culture. 2. Tyrosinase inhibitory effect was measured as $0.39{\pm}0.026%$. Compared with the control group, the effect was very slight. 3. The SOD like activity of the extract of Hedyotis diffusa $W_{ILLD}$. was measured as 21.33${\pm}$ 4.264%. Compared with the extracts of several other herbs, it was much more significant. 4. The DPPH of the extract of Hedyotis diffusa $W_{ILLD}$. was $53.3{\pm}0.91%$ when 0.02g was used and $83.5{\pm}1.82% $ when 0.05g was used. The result when 0.05g was used was more significant than 0.02g was used. 5. The TBARS of the extract of Hedyotis diffusa $W_{ILLD}$. was measured as 0.724 ${\pm}$0.O04MDAppm. Compared with the extracts of several other herbs, the result was more significant. From these results, we found that Hedyotis diffusa $W_{ILLD}$. can be used in therapy of dysentery with bloody stool and fever which is caused by infection with E. coli O157 and that it can be also used effectively as age resister or anticarcinogen.

• Journal of Life Science
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• v.25 no.2
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• pp.130-135
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• 2015

When the alginate lyase gene (aly) from Pseudoalteromonas elyakovii IAM 14594 was expressed in E. coli, most of the gene product expressed was produced as aggregated insoluble particles known as inclusion bodies. In order to produce with an elevated level of a soluble and active form of alginate lyase in E. coli, the hyperthermophilic chaperonins (ApCpnA and ApCpnB) from archaeon Aeropyrum pernix K1 were employed as the coexpression partners. At $25^{\circ}C$ culture temperature, the level of alginate lyase activity was increased from 10.1 unit/g-soluble protein in aly single expression to 83.1 unit/g-soluble protein by coexpressing with ApCpnA and to 100.3 unit/g-soluble protein by coexpressing with ApCpnB. This results indicate that the coexpression of aly with ApCpnA and ApCpnB revealed a marked enhancement, about 8~10 fold, in the production of alginate lyase as a soluble and active form. Based on the results of various examinations on the expression variables, the optimal conditions for the maximal production of alginate lyase were determined as 1.0 mM IPTG for the inducer concentration, $25^{\circ}C$ for the culture temperature after IPTG induction, and ApCpnB for the coexpression partner. The coexpression set in the present report may be useful in the industrial production of functionally or medically important recombinant proteins in E. coli.

• KSBB Journal
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• v.19 no.4
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• pp.327-330
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• 2004

An inducible expression system of poly[(R)-3-hydroxybutyrate] (PHB) depolymerization was established in metabolically engineered Escherichia coli with the PHB biosynthesis genes. The Ralstonia eutropha PHB depolymerase gene was cloned in a vector system containing the PHB biosynthesis genes and expressed under inducible promoter. Recombinant E. coli harboring the PHB biosynthesis genes and depolymerase gene was first cultured for the accumulation of PHB, and then the depolymerase was expressed resulting in the degradation of accumulated PHB into (R)-3-hydroxybutyric acid (R3HB). R3HB could be produced with the concentration of 7.6 g/L in flask culture. Two different PHB biosynthesis genes from Alcaligenes latus and R. eutropha were compared for the production of R3HB. This strategy can be used for the production of enantiomerically pure (R)-hydroxycarboxylic acids with high concentration.

• Journal of Microbiology and Biotechnology
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• v.19 no.10
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• pp.1127-1134
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• 2009

Escherichia coli excretes acetate during aerobic growth on glycolytic carbon sources, which has been explained as an overflow metabolism when the carbon flux into the cell exceeds the capacity of central metabolic pathways. Nonacetogenic growth of E. coli on gluconeogenic carbon sources like succinate or in carbon-limited slow growth conditions is believed an evidence for the explanation. However, we found that a strain defected in the acs (acetyl Co-A synthetase) gene, the product of which is involved in scavenging acetate, accumulated acetate even in succinate medium and in carbon-limited low growth rate condition, where as its isogenic parental strain did not. The acs promoter was inducible in noncatabolite repression condition, whereas the expression of the ackA-pta operon encoding acetate kinase and phosphotransacetylase for acetate synthesis was constitutive. Results in this study suggest that E. coli excretes and scavenges acetate simultaneously in the carbon-limited low growth condition and in nonacetogenic carbon source, and the activity of the acetate consumption pathway directly affects the accumulation level of acetate in the culture broth.

• Journal of Veterinary Clinics
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• v.34 no.3
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• pp.213-217
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• 2017

A 1-year and 8-month-old male, thoroughbred horse showed fever ($39.8^{\circ}C$), cardiac murmur, tachycardia up to 80 beats/min, anorexia, depression and lameness for about 2 months. The dead horse was referred to pathology laboratory at the College of Veterinary Medicine in Jeju National University. At necropsy, Severe protruding multiple rough cauliflower-like yellowish red nodules ranged $5{\sim}6{\times}2{\sim}3cm$ in size were attached on the mitral valve of the left heart. A yellowish red long stick-shaped thrombus $15{\times}3.5{\times}1.5cm$ in size was also present inside the right ventricle. Multifocal infarcts were scattered in the myocardium and renal cortex. Histopathologic examination revealed that morphologic diagnosis were vegetative endocarditis, thrombus in right ventricle, infarcts in myocardium and kidney, pulmonary congestion and edema, and splenic congestion. The isolated bacteria from vegetative lesions and thrombus were confirmed as Escherichia (E.) coli based on the bacterial culture and VITEK 2 system. Based on the gross and histopathologic features, and bacterial test, this case was diagnosed as vegetative endocarditis with thrombus formation associated by E. coli in a thoroughbred horse.

• Pediatric Infection and Vaccine
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• v.10 no.2
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• pp.167-177
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• 2003

Purpose : To know the trends of antimicrobial susceptibility is critical for antimicrobial treatment. We studied the organisms isolated from blood, urine, stool, and cerebrospinal fluid from 1997 to 2001 to reveal the trends of their antimicrobial susceptibility. Methods : We conducted a retrospective study with isolates obtained from 0~18 year old outpatients and inpatients from 1997 to 2001 at Department of Pediatrics, Hanil general hospital. We gathered the data through the laboratory test files and the origin of microorganisms cultured from blood, urine, stool and cerebrospinal fluid and their antimicrobial susceptibility. Results : Microorganisms were isolated from 226(3.3%) out of 6,974 blood cultures, 365 (8.0%) out of 4,549 urine cultures, 50(1.9%) out of 2,593 stool cultures and 9(1.4%) in 655 cerebrospinal fluid cultures. The most frequently isolated organisms from blood cultures was Staphylococcus epidermidis(33.5%) which was followed by Staphylococcus aureus(19.7%), Escherichia coli(13.8%), and Burkholderia cepacia(9.0%). Among the urine cultures, E. coli was the most common(74.7%) which was followed by Group D Enterococcus(11.3%), Klebsiella pneumoniae(7.1%) and Proteus mirabilis(2.5%). The positive stool cultures all yield Salmonella species. Group D Salmonella was obtained most frequently. Among the positive cerebrospinal fluid cultures, Group B Streptococcus was isolated most frequently. Among the 40 cases of S. aureus in blood cultures, 27 cases were methicillin-resistant. The rates of susceptibility for amikacin, ceftizoxime and ceftriaxone of E. coli isolated from blood cultures were 80%, 100% and 60% in 1997 and 60%, 80% and 60% in 2001. The rates of susceptibility for amikacin, ceftizoxime and ceftriaxone of K. pnumoniae isolated from urine cultures. were 80%, 100% and 80% in 1997 and 50%, 83% and 50% in 2001 Enterococcus was isolated from 6.7% to 15.8% and vancomycin-resistant Enterococcus was observed in 17% of Group D Enterococcus isolated from urine cultures. The rates of susceptibility for amikacin, ceftizoxime and ceftriaxone of Group D Salmonella were 96%, 96% and 92% during the study period. Conclusion : Among the blood cultures S. epidermidis, S. aureus, E. coli and B. cepacia were isolated in order of frequency and among the urine cultures E. coli, Group D Enterococcus, K. pneumoniae and P. mirabilis were isolated in order of frequency. During the study period there was no big difference in major organisms isolated from blood and urine. The methicillin-resistant S. aureus was observed in 67% of S. aureus isolated from blood cultures but vancomycin-reistant S. aureus or vancomycin intermediate resistant S. aureus was not observed. The rates of susceptibility to amikacin and the third generation cephalosporin of E. coli isolated from blood cultures and K. pneumoniae from urine cultures have decreased. The isolation rates of Group D Enterococcus and vancomycin resistant Enterococcus have increased.

• 한국생물공학회:학술대회논문집
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• 2001.11a
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• pp.758-761
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• 2001

In this study, the nar promoter of E. coli was characterized to see whether the nar promoter cloned onto pBBR122 can be used as an expression promoter of gram negative microbes. For this purpose, a plasmid with lacZ gene expressing ${\beta}-galactosidase$ instead of the structural genes of nar operon in a gram negative host strain(Agrobacterium.tumefaciens) was used to simplify an assay of induction of the nar promoter. The following effects were investigated to find optimal conditions: methods of inducing the nar promoter, optimal nitrate concentration, maximally inducing the nar promoter, the amount of expressed ${\beta}-galactosidase$ and induction ratio(specific ${\beta}-galactosidase$ activity after maximal induction/specific ${\beta}-galactosidase$ activity before induction). The following results were obtained from the experiments: the growth of Agrobacterium with E.coli nar promoter was not much affected by nitrate concentration in the shake-flask; induction of nar promoter was optimal when Agrobacterium was grown in the presence of 1% nitrate ion at the beginning of culture and when overnight culture was completely grown in the shake-flask before being transferred to other shake-flask; the amount of ${\beta}-galactosidase$ per cell and per medium volume was maximal when Agrobacterium was grown under aerobic condition to $OD_{600}$ of 1.7; then the nar promoter was induced under microaerobic and anaerobic condition made by lowering dissolved oxygen level(DO). After 2-3h of induction in the YEP medium selected as a main culture medium, the specific ${\beta}-galactosidase$ activity became about 17,000 Miller units in the fermentor cluture.

• Journal of Microbiology and Biotechnology
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• v.28 no.12
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• pp.2079-2094
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• 2018

Sunflower trypsin inhibitor (SFTI) is a 14-amino-acid bicyclic peptide that contains a single internal disulfide bond. We initially constructed chimeras of SFTI with N-terminal secretion signals from the Escherichia coli OmpA and Pseudomonas aeruginosa ToxA, but only detected small amounts of protease inhibition resulting from these constructs. A substantially higher degree of protease inhibition was detected from a C-terminal SFTI fusion with E. coli YebF, which radiated more than a centimeter from an individual colony of E. coli using a culture-based inhibitor assay. Inhibitory activity was further improved in YebF-SFTI fusions by the addition of a trypsin cleavage signal immediately upstream of SFTI, and resulted in production of a 14-amino-acid, disulfide-bonded SFTI free in the culture supernatant. To assess the potential of the secreted SFTI to protect the ability of a cytotoxic protein to kill tumor cells, we utilized a tumor-selective form of the Pseudomonas ToxA (OTG-PE38K) alone and expressed as a polycistronic construct with YebF-SFTI in the tumor-targeted Salmonella VNP20009. When we assessed the ability of toxin-containing culture supernatants to kill MDA-MB-468 breast cancer cells, the untreated OTG-PE38K was able to eliminate all detectable tumor cells, while pretreatment with trypsin resulted in the complete loss of anticancer cytotoxicity. However, when OTG-PE38K was co-expressed with YebF-SFTI, cytotoxicity was completely retained in the presence of trypsin. These data demonstrate SFTI chimeras are secreted in a functional form and that co-expression of protease inhibitors with therapeutic proteins by tumor-targeted bacteria has the potential to enhance the activity of therapeutic proteins by suppressing their degradation within a proteolytic environment.