• Journal of Microbiology and Biotechnology
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• 제11권6호
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• pp.1115-1119
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• 2001

Eight Escherichia coli cells with aerobic growth deflects were isolated by the insertion of ${\lambda}placMu53$, a hybrid bacteriophage of ${\lambda}$ and Mu, which created transcriptional fusion to lacZY. Two of these mutants, CLIO and CLl2, were irradiated with UV to obtain specialized transducing phages. The phages that took out the neighboring chromosomal DNA of the related gene responsible for deflective aerobic growth were identified. The in vivo cloned chromosomal sequence revealed that the mutated gene of CLIO was located at min 34.5 on the Escherichia coli linkage map and 1,599,515 on the physical map. The physical map indicated that there were 7 cistrons in the operon. We named this operon oxg10. The promoter sequence of oxg10 exhibited a possible binding site far SoxS, a transcriptional regulator that activates the transcription of various SoxRS regulon genes. Transferring the oxg10:: ${\lambda}placMu53$ mutation into the wild-type strain, RZ4500, resulted in the inhibition of normal aerobic growth, while the salute mutation in strain MO inhibited aerobic cell growth completely. The full operon sequences of oxg10 were cloned from the Excherichia coli genomic library. The mutated gene of CLl2 was identified to be a sucA gene encoding the ${\alpha}$-ketoglutarate dehydrogenase El component in the TCA cycle.

• Journal of Microbiology and Biotechnology
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• 제14권3호
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• pp.483-489
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• 2004

Pseudomonas sp. S-47 is a bacterium capable of degrading benzoate as well as 4-chlorobenzoate (4CBA). Benzoate and 4CBA are known to be degraded via a meta-cleavage pathway characterized by a series of enzymes encoded by xyl genes. The meta-cleavage pathway operon in Pseudomonas sp. S-47 encodes a set of enzymes which transform benzoate and 4CBA into TCA cycle intermediates via the meta-cleavage of (4-chloro )catechol to produce pyruvate and acetyl-CoA. In the current study, the meta-pathway gene cluster was cloned from the chromosomal DNA of S-47 strain to obtain pCS1, which included the degradation activities for 4CBA and catechol. The genetic organization of the operon was then examined by cloning the meta-pathway genes into a pBluescript SKII(+) vector. As such, the meta-pathway operon from Pseudomonas sp. S-47 was found to contain 13 genes in the order of xylXYZLTEGFlQKIH. The two regulatory genes, xylS and xylR, that control the expression of the meta-pathway operon, were located adjacently downstream of the meta-pathway operon. The xyl genes from strain S-47 exhibited a high nucleoside sequence homology to those from Pseudomonas putida mt-2, except for the xylJQK genes, which were more homologous to the corresponding three genes from P. stutzeri AN10. One open reading frame was found between the xylH and xylS genes, which may playa role of a transposase. Accordingly, the current results suggest that the xyl gene cluster in Pseudomonas sp. S-47 responsible for the complete degradation of benzoate was recombined with the corresponding genes from P. putida mt-2 and P. stutzeri AN10.

• Journal of Microbiology and Biotechnology
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• 제16권6호
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• pp.870-879
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• 2006

The present study describes the formation of succinic acid by a nonvirulent, highly osmotolerant Klebsiella pneumoniae strain SAP (succinic acid producer), its profile of metabolites, and enzymes of the succinate production pathway. The strain produced succinate along with other metabolites such as lactate, acetate, and ethanol under aerobic as well as anaerobic growth conditions. The yield of succinate was higher in the presence of $MgCO_3$ under $N_2$ atmosphere as compared with that under $CO_2$ atmosphere. Analysis of intracellular metabolites showed the presence of a smaller PEP pool than that of pyruvate. Oxaloacetate, citrate, and $\alpha$-ketoglutarate pools were considerably larger than those of isocitrate and fumarate. In order to understand the synthesis of succinate, the enzymes involved in end-product formation were studied. Levels of phosphoenolpyruvate carboxykinase, fumarate reductase, pyruvate kinase, and acetate kinase were higher under anaerobic growth conditions. Based on the profiles of the metabolites and enzymes, it was concluded that the synthesis of succinate took place via oxaloacetate, malate, and fumarate in the strain under anaerobic growth conditions. The strain SAP showed potential for the bioconversion of fumarate to succinate under $N_2$ atmosphere in the presence of $MgCO_3$. At an initial fumarate concentration of 10 g/l, 7.1 g/l fumarate was converted to 7 g/l succinate with a molar conversion efficiency of 97.3%. The conversion efficiency and succinate yield were increased in the presence of glucose. Cells grown on fumarate contained an 18-fold higher fumarate reductase activity as compared with the activity obtained when grown on glucose.

• Journal of Microbiology and Biotechnology
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• 제23권11호
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• pp.1586-1597
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• 2013

Energy-efficient metabolic responses were often noted in high-productive cultures. To better understand these metabolic responses, an investigation into the relationship between metabolic responses and energy regulation was conducted via a comparative analysis among cultures with different energy source supplies. Both glycolysis and glutaminolysis were studied through the kinetic analyses of major extracellular metabolites concerning the fast and slow cell growth stages, respectively, as well as the time-course profiles of intracellular metabolites. In three cultures showing distinct antibody productivities, the amino acid metabolism and energy state were further examined. Both the transition of lactate from production to consumption and steady intracellular pools of pyruvate and lactate were observed to be correlated with efficient energy regulation. In addition, an efficient utilization of amino acids as the replenishment for the TCA cycle was also found in the cultures with upregulated energy metabolism. It was further revealed that the inefficient energy regulation would cause low cell productivity based on the comparative analysis of cell growth and productivity in cultures having distinct energy regulation.

• Journal of Plant Biology
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• 제17권4호
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• pp.143-156
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• 1974

The purpose of this experiment was to study one side of germination physiology based on that protein profiles and protease relating to protein metabolism, that peroxidase, catalase, $\alpha$-amylase, $\beta$-amylase, and malate dehydrogenase involved in the carbohydrate metabolism of seed germination. All these experiments were divided into the two groups with and without acetone treatment, and were carried out. The protein bands of each germinating stage between the groups treated with and without acetone showed certain basic pattern in polyacrylamide gel disc electrophoresis. However, there was a little difference in the number of protein band, optical density, and migration velocity between two groups. The isozyme bands of peroxidase, and catalase between two groups in polyacrylamide gel disc electrophoresis did not show the numeral difference, but the optical density of certain germinating stage treated with acetone was higher than the group untreated with it and it showed their enzyme activity. The $\alpha$-amylase and $\beta$-amylase activities which involved in starch metabolism of seed germination were higher in the treated group than the other. On one hand, the protease activity of hydrolase occurred in the seeds for germination was also higher, more or less in the treated group than in the other. The isozyme band pattern of malate dehydrogenase in TCA cycle of energy metabolism pathway was very different between two groups growing for 72 hours with and without acetone treatment in cellulose acetate electrophoresis. It indicated that two isozyme bands of malate dehydrogenase was high. Consequently these experimental results mentioned above indicated that acetone treatment before sowing had an effect on dissolving certain complexed lipid substance involved in the seed coats, the activity of carbohydrate hydrolase increased with water absorption which was most comfortable in its germination, dissolved glycerin and fatty acid became certain energy source, and they stimulated the acceleration of respiration metabolism.

• Asian-Australasian Journal of Animal Sciences
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• 제19권7호
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• pp.1033-1039
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• 2006

The metabolic response of Labeo rohita to thermal acclimation was assessed. Advanced fingerlings of L. rohita (average weight $31{\pm}1.4g$) were acclimated to 31, 33 and $36^{\circ}C$ compared with ambient temperatures ($26^{\circ}C$) for 30 days and different enzymes associated with stress response were estimated. Glycolytic enzyme-Lactate dehydrogenase, (LDH, E.C.1.1.1.27), TCA cycle enzyme-Malate dehydrogenase (MDH, E.C.1.1.1.37), Protein metabolizing enzymes-Aspartate amino transferase (AST, E.C.2.6.1.1) and Alanine amino transferase (ALT, E.C.2.6.1.2) of liver, gill and muscle, Gluconeogenic enzymes-Fructose 1,6 Bi phosphatase (FBPase, E.C. 3.1.3.11) and Glucose 6 phosphatase (G6Pase, E.C. 3.1.3.9) of liver and kidney were significantly (p<0.05) different with increasing acclimation temperatures. Heat Shock Protein-70 (HSP-70) was expressed in increasing intensity at 31, 33 and $36^{\circ}C$ but was not expressed at $26^{\circ}C$. Results suggest that higher acclimation temperatures enhance metabolism and L. rohita maintains homeostasis between $26-36^{\circ}C$ via an acclimation episode. Such adaptation appears to be facilitated by resorting to gluconeogenic and glycogenolytic pathways for energy mobilization and induction of HSPs.

• 농업과학연구
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• 제44권1호
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• pp.30-39
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• 2017

This study was performed to produce succinic acid from biomass by developing mutants of Cellulomonas flavigena in which the succinate dehydrogenase gene (sdh) is deleted. For development of succinate producing mutants, the upstream and downstream regions of sdh gene from C. flavigena and antibiotic resistance gene (neo, bla) were inserted into pKC1139, and the recombinant plasmids were transformed into Escherichia coli ET12567/pUZ8002 which is a donor strain for conjugation. C. flavigena was conjugated with the transformed E. coli ET12567/pUZ8002 to induce the deletion of sdh in chromosome of this bacteria by double-crossover recombination. Two mutants (C. flavigena H-1 and H-2), in which sdh gene was deleted in the chromosome, were constructed and confirmed by PCR. To estimate the production of succinic acid by the two mutants when the culture broth was fermented with biomass such as CMC, xylan, locust gum, and rapeseed straw; the culture broth was analyzed by HPLC analysis. The succinic acid in the culture broth was not detected as a fermentation products of all biomass. One of the reasons for this may be the conversion of succinic acid to fumaric acid by sdh genes (Cfla_1014 - Cfla_1017 or Cfla_1916 - Cfla_1918) which remained in the chromosomal DNA of C. flavigena H-1 and H-2. The other reason could be the conversion of succinyl-CoA to other metabolites by enzymes related to the bypass pathway of TCA cycle.

• Journal of Plant Biology
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• 제17권2호
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• pp.69-83
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• 1974

To elucidate the relationship between chemical structure and biological activity of alantolactone, and also to investigate the relationship between the growth of cells and the respiration of Chlorella pyrenoidosa affected by alantolactone, alantolactone and isoalantolactone were isolated from Inula helenium L., and di-, and tetrahydroalantolactones were prepared by the hydrogenation. At a concentration of 5$\times$10-5M alantolactone, the growth rate of Chlorella was greatly reduced. The viability of cells was also reduced over 50% within 2 hr at a concentration of 2.5$\times$10-4M alantolactone. However, oxygen uptake was increased by 20% over 3 hr. And 14CO2 production from glucose-1-14C, glucose-6-14C and 14C-acetate-U.L. was also increased by alantolactone. Biological activityof alantolactone was significantly reduced by cysteine, reduced glutathione or cystine but not by tryptophan or histidine. It was detected by spectrophotometrically and by TLC that alantolactone was also reacted with thiols except cystine. The solution of alantolactone reached with thiol gave the UV absorption spectrum of $\alpha$-saturated ${\gamma}$-lactone, and most of SH groups were disappeared by the addition reaction. From the reaction mixture of alantolactone and cysteine, a lactone adduct was isolated and purified. Isoalantolactone had shown similar activity as alantolactone, however, it was appeared that di-, and tetrahydroalantolactones were not only inactive biologically but also in vitro. It was concluded that there was no correlationship between increased respiration rate and mortality of Chlorella. During the respiration TCA cycle was activated, however it was uncertain that the activation of EMP or HMP was also appeared. Alantolactone and isoalantolactone were biologically active compounds but others were inactive. The reactivity of $\alpha$-methylene ${\gamma}$-lactone moiety toward SH group was principally responsible for its biological activity in sesquiterpene lactones.

• The Korean Journal of Physiology
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• 제1권1호
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• pp.33-41
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• 1967

The metabolic patterns of C-1 and C-6-carbon atoms of glucose were observed in the tissue homogenates of the Ehrlich ascites tumor tissue which was incubated for 3 hours in the Dubnuff metabolic shaking incubator. $C^{14}-1-and\;C^{14}-6-glucose$ were used as tracers. The glucose media in which tissue homogenate was incubated was kept at a concentration of 200mg% glucose of carrier and appropriate amount of $C^{14}-1-or\;C^{14}-6-tracer$. At the end of 3 hour incubation, respiratory $CO_2$ samples trapped by alkaline which is placed in the tenter well of incubation flask were analyzed for the total $CO_2$ production rates and their radioactivities. The tissue homogenate samples after incubation were analyzed for their concentrations of glucose, lactate, pyruvate and glycogen and calculations were made on the glucose consumption rate, pyruvate and lactate accumulation rates. The following results were obtained. Data obtained in each group are as follows: 1. In the tissue homogenate, which was incubated with $C^{14}-1-glucose as a substrate, total $CO_2$ production rate averaged $19.0{\pm}5.0{\mu}M/hr/gm$ and the mean specific activity of respiratory $CO_2$ was $840{\pm}296\;cpm/mgC.$ Relative specific activity (RSA) which means the fraction of $CO_2$ derived from medium $C^{14}-1-glucose$ to total $CO_2$ production rate was calculated by ratio of SA of respiratory $CO_2$ and medium $C^{14}-1-glucose.$ RSA was $14.3{\pm}5.0%,$ Accordingly actual $CO_2$ production rate from medium $C^{14}-1-glucose$ showed a mean value of $2.79{\pm}1.35\;{\mu}m$ of which amount was equivalent to the mean value of total glucose consumption rate $(RGDco_2)$, namely, $5.1{\pm}1.3%.$ Lactate and pyruvate appearance rates averaged $7.13{\pm}1.26\;and\;0.21{\pm}0.02{\mu}M/hr/gm,$ respectively. Assuming that these 3 carbon compounds appeared in the medium were derived from glucose, calculations were made that relative glucose disappearance rate into lactate $(RGD_L)$ was $38.0{\pm}5.4%\;and\;RGD_P$ was $1.23{\pm}0.03%.$ Therefore, about 43.3% of the total glucose consumed were accounted for by conversion into the respiratory $CO_2$, lactate and pyruvate. 2. In the second group, which was incubated with $C^{14}-1-glucose$ as a substrate, glucose consumption rate, lactate and pyruvate appearance rates showed almost the same order as the values of the $C^{14}-1-glucose$ substrate group. However, RSA was remarkably decreased showing a mean value of $1.02{\pm}0.13%.$ This fact means that the C-6 carbon of glucose take the minor part in the oxidative metabolism of glucose. The glycogen level in both substrate tissue homogenate showed less than 0.3% of tissue weight. These low value suggested that there was an inhibition of carbohydrate synthesis in the Ehrlich ascites tumor tissue. 3. The catabolic pathway of glucose in the tumor tissue were analyzed on the basis of Bloom's principle from the values of RSA. It was found that in the tumor tissue more than 90% of $CO_2$ derived from glucose were oxidized via the alternate pathway other than principal EMP-TCA cycle such as hexose monophosphate pathway (HMP). From the data described above, it was assumed that in the Ehrlich tumor tissue anaerobic glycolysis proceeds normally although, the oxidation of products of anaerobic glycolysis via the TCA cycle is inhibited resulting in the accumulation of lactate and almost all of oxidative energy from glucose is released by oxidative pathway such as HMP.

• KSBB Journal
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• 제24권1호
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• pp.30-40
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• 2009

Aspergillus terreus에 의한 이타콘산 생산 발효공정에서 생산균주의 성장을 어느 정도 제한시킴으로써 배양생리적 특성이 이타콘산 생합성 쪽으로 치우치도록 통계적 방법을 적용하여 itaconic acid의 생산배지 조성을 최적화하는 연구를 수행하였다. 이타콘산은 TCA회로를 거쳐 합성된 cis-aconitic acid의 디카르복실화 반응에 의해 생합성되는 고부가 화학원료물질이다. 우선 One factor at a time (OFAT) 방법을 이용하여 이타콘산의 생산성 증가에 크게 영향을 미치는 중요한 탄소원들로 sucrose, glucose, fructose와 soluble starch를 확인할 수 있었고, 질소원들로는 cottonseed flour와 soybean meal을 찾을 수 있었다. Fractional factorial design을 통하여 이들 6가지 요인들 간의 상호작용의 정도를 확인한 결과 sucrose와 cottonseed flour간의 상호작용의 정도가 가장 컸고, 나머지 요인들 간의 상호작용의 정도는 작거나 혹은 이타콘산 생산에 오히려 부정적인 결과를 나타냈다. 또한 full factorial design (FFD) 실험을 통해 생산배지에 $KH_2PO_4$와 $MgSO_4$가 과량 첨가되면 이타콘산의 생산성이 심각하게 저해됨을 알 수 있었다. FFD의 1차모델식을 근간으로 하여 최급상승법 (steepest ascent method, SAM)을 적용하여 sucrose, cottonseed flour, $KH_2PO_4$ 및 $MgSO_4$의 최적 농도로 향하는 가장 가파른 기울기를 구함으로써, 신속하고 효율적으로 최적 농도지점에 대한 정보를 얻을 수 있었다. SAM이 제시해주는 농도 부근에서 반응표면분석 (response surface method, RSM)을 적용하여 각 배지성분의 농도를 최적화시키기 위해, 2개의 중요한 요인인 sucrose와 cottonseed flour를 이용하여 중심합성계획 (central composite design, CCD) 실험을 수행하였다. 그 결과 이타콘산의 최적 배지 조건은 sucrose 90.4 g/L, cottonseed flour 53.8 g/L인 것으로 관찰되었고, 이 농도에서 이타콘산의 생산성은 초기 사용된 배지에서의 생산성에 비해 약 7배 증가한 4360 mg/l로 나타났다. 이로부터 탄소원 (C)으로 사용한 sucrose와 질소원 (N)으로 사용한 cottonseed flour 간의 C/N 비율이 이타콘산의 생산성에 큰 영향을 미친다는 것을 확인할수 있었다.