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Ko H.-J.,Kangwon National University | Lee H.Y.,Seowon University | Park D.-S.,Functional food and Nutrition Division | Chung H.-C.,Newtree Co. | And 2 more authors.
Biomolecules and Therapeutics | Year: 2013

Codonopsis lanceolata (Campanulaceae) traditionally have been used as a tonic and to treat patients with lung abscesses. Recently, it was proposed that the extract and some compounds isolated from C. lanceolata reversed scopolamine-induced memory and learning deficits. The purpose of this study was to evaluate the improvement of cognitive enhancing effect of C. lanceolata by steam and fermentation process in scopolamine-induced memory impairment mice models by passive avoidance test and Morris water maze test. The extract of C. lanceolata or the extract of steamed and fermented C. lanceolata (SFCE) was orally administered to male mice at the doses of 100 and 300 mg/kg body weight. As a result, mice treated with steamed and fermented C. lanceolata extract (SFCE) (300 mg/kg body weight, p.o.) showed shorter escape latencies than those with C. lanceolata extract or the scopolamine-administered group in Morris water maze test. Also, it exerted longer step-through latency time than scopolamine treated group in passive avoidance test. Furthermore, neuroprotective effect of SFCE on glutamate-induced cytotoxicity was assessed in HT22 cells. Only SFCE-treated cells showed significant protection at 500 μg/ml. Interestingly, steamed C. lanceolata with fermentation contained more phenolic acid including gallic acid and vanillic acid than original C. lanceolata. Collectively, these results suggest that steam and fermentation process of C. lanceolata increased cognitive enhancing activity related to the memory processes and neuroprotective effect than original C. lanceolata. © 2013 The Korean Society of Applied Pharmacology.

Honga K.-K.,Chalmers University of Technology | Honga K.-K.,Research Institute of Biotechnology | Nielsen J.,Chalmers University of Technology | Nielsen J.,Technical University of Denmark
Applied and Environmental Microbiology | Year: 2012

In a previous study, system level analysis of adaptively evolved yeast mutants showing improved galactose utilization revealed relevant mutations. The governing mutations were suggested to be in the Ras/PKA signaling pathway and ergosterol metabolism. Here, site-directed mutants having one of the mutations RAS2Lys77, RAS2Tyr112, and ERG5Pro370 were constructed and evaluated. The mutants were also combined with overexpression of PGM2, earlier proved as a beneficial target for galactose utilization. The constructed strains were analyzed for their gross phenotype, transcriptome and targeted metabolites, and the results were compared to those obtained from reference strains and the evolved strains. The RAS2Lys77 mutation resulted in the highest specific galactose uptake rate among all of the strains with an increased maximum specific growth rate on galactose. The RAS2Tyr112 mutation also improved the specific galactose uptake rate and also resulted in many transcriptional changes, including ergosterol metabolism. The ERG5Pro370 mutation only showed a small improvement, but when it was combined with PGM2 overexpression, the phenotype was almost the same as that of the evolved mutants. Combination of the RAS2 mutations with PGM2 overexpression also led to a complete recovery of the adaptive phenotype in galactose utilization. Recovery of the gross phenotype by the reconstructed mutants was achieved with much fewer changes in the genome and transcriptome than for the evolved mutants. Our study demonstrates how the identification of specific mutations by systems biology can direct new metabolic engineering strategies for improving galactose utilization by yeast. © 2012, American Society for Microbiology.

Hong K.-K.,Chalmers University of Technology | Hong K.-K.,Research Institute of Biotechnology | Nielsen J.,Chalmers University of Technology | Nielsen J.,Technical University of Denmark
Metabolic Engineering | Year: 2013

Adaptive evolution offers many opportunities in metabolic engineering; however, several constraints still exist as evolutionary trade-offs may impose collateral cost to obtain new traits. The application of adaptive evolution for strains development could be further improved by elucidating the molecular mechanisms. In this study, adaptively evolved yeast mutants with improved galactose utilization ability showed impaired glucose utilization. The molecular genetic basis of this trade-off was investigated using a systems biology approach. Transcriptional and metabolic changes resulting from the improvement of galactose utilization were found maintained during growth on glucose. Moreover, glucose repression related genes showed conserved expression patterns during growth on both sugars. Mutations in the RAS2 gene that were identified as beneficial for galactose utilization in evolved mutants exhibited significant correlation with attenuation of glucose utilization. These results indicate that antagonistic pleiotropy is the dominant mechanism in the observed trade-off, and it is likely realized by changes in glucose signaling. © 2013 Elsevier Inc.

Park C.,Sogang University | Yun S.,Sogang University | Yun S.,Research Institute of Biotechnology | Lee S.Y.,Korea Advanced Institute of Science and Technology | And 2 more authors.
Applied Biochemistry and Biotechnology | Year: 2012

The global pool of intracellular metabolites is a reflection of all the metabolic functions of an organism. In the absence of in situ methods capable of directly measuring metabolite pools, intracellular metabolite measurements need to be performed after an extraction procedure. In this study, we evaluated the optimization of technologies for generation of a global metabolomics profile for intracellular metabolites in Klebsiella oxytoca. Intracellular metabolites of K. oxytoca were extracted at the early stationary phase using six different common extraction procedures, including cold methanol, boiling ethanol, methanol/chloroform combinations, hot water, potassium hydroxide, and perchloric acid. The metabolites were subsequently collected for further analysis, and intracellular metabolite concentration profiles were generated using ultra-performance liquid chromatography/ quadrupole time-of-flight mass spectrometry. During analysis, the stability of metabolites extracted using cold methanol was clearly higher than that obtained by other extraction methods. For the majority of metabolites, extracts generated in this manner exhibited the greatest recovery, with high reproducibility. Therefore, the use of cold ethanol was the best extraction method for attaining a metabolic profile. However, in another parallel extraction method, perchloric acid may also be required to maximize the range of metabolites recovered, particularly to extract glucose 1-phosphate and NADPH. © Springer Science+Business Media, LLC 2012.

Park C.,Sogang University | Lu M.,Sogang University | Yun S.,Sogang University | Yun S.,Research Institute of Biotechnology | And 2 more authors.
Bioprocess and Biosystems Engineering | Year: 2013

The efficiency of the bioconversion process and the achievable end-product concentration decides the economic feasibility of microbial 2,3-butanediol (2,3-BDO) production. In 2,3-BDO production, optimization of culture condition is required for cell growth and metabolism. Also, the pH is an important factor that influences microbial performance. For different microorganisms and substrates, it has been shown that the distribution of the metabolites in 2,3-BDO fermentation is greatly affected by pH, and the optimum pH for 2,3-BDO production seems dependently linked to the particular strain and the substrate employed. Quantification analysis of intracellular metabolites and metabolic flux analysis (MFA) were used to investigate the effect of pH on the Klebsiella oxytoca producing 2,3-BDO and other organic acids. The main objectives of MFA are the estimation of intracellular metabolic fluxes and the identification of rate-limiting step and the key enzymes. This study was conducted under continuous aerobic conditions at different dilution rates (0.1, 0.2, and 0.3 h-1) and different pH values (pH 5.5 and 7.0) for the steady-state experimental data. In order to obtain the flux distribution, the extracellular specific rates were calculated from the experimental data using the metabolic network model of K. oxytoca. Intracellular metabolite concentration profiles were generated using ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry. © 2013 Springer-Verlag Berlin Heidelberg.

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