• KSBB Journal
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• v.13 no.3
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• pp.325-330
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• 1998

The methylotrophic yeast, Pichia pastoris, is known to be a potential host to offer many advantages for production of recombinant proteins. Fermentation parameters were optimized to enhance the heterologous ${\beta}$-galactosidase productivity with P. pastoris. Optimum concentration of methanol, used as inducer, was observed to be 8 g/L and the extent of repression of AOX1 promoter by glycerol was lower than by glucose. The degradation of the gene product ${\beta}$-galactosidase by protease was inhibited as the pH increased from 5 to 8 and the yeast extract(1%) as nitrogen source increased expression level 4 times higher compared to yeast nitrogen base(1%) as nitrogen source increased expression level 4 times higher compared to yeast nitrogen base(1%). Induction method, in which methanol is just added to fermentation medium without centrifugation, was found to be as much effective as the one with centrifugation.

• Journal of Microbiology and Biotechnology
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• v.12 no.3
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• pp.490-496
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• 2002

The production of E. coli WC7 phytase from a recombinant E. coli strain was optimized using a statistical experimental design approach. Two-level complete factorial designs with seven variables were used for the media optimization. In the first optimization step, the influence of disodium succinate, yeast extract, $K_2HPO_4,\;NH_4H_2PO_4,\;MgSO_4$, NaCl, and trace elements on phytase production was evaluated. As a result, disodium succinate, yeast extract, $NH_4H_2PO_4$, NaCl, and the trace elements were found to have a positive influence on the phytase production, while $K_2HPO_4\;and\;MgSO_4$ had a negative influence. In the second step, the concentrations of disodium succinate and yeast extract were further optimized using central composite designs. The maximum phytase activity obtained was 234 U/ml using 15.9 g/1 disodium succinate, 20 g/1 yeast extract, 5 g/1 K_2HPO_4,\;10 g/1 NH_4H_2PO_4,\;1.5 g/1 MgSO_4$, 4 g/1 NaCl, and 1.5 m1/1 trace elements, which was about a 14-fold increase in comparison with that obtained using the basal medium.

• Korean Journal of Microbiology
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• v.24 no.4
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• pp.357-363
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• 1986

Synthesis of threonine and methionine in yeast, Saccharomyces cerevisiae shares a common pathway from aspartate via homoserine. HOM6 gene encodes homoserine dehydrogenase (HSDH) which catalyzes the inter-conversion of beta-aspartate semialdehyde and homoserine. The level of HSDH is under methionine specific control. A recombinant plasmid (pEK1: 13.3kb), containing HOM6 gene, has been isolated and cloned into E. coli by complenemtary transformation of a homoserine auxotrophic yeast strain M-20-20D (hom6, trp1, ura3) to a prototrophic M20-20D/pEK1, using a library of yeast genomic DNA fragments in a yeast centromeric plasmid, YCp50(8.0kb). Isolation of HOM6has been primarily confirmed by retransformation of the original yeast strain M20-20D, using the recombinant plasmid DNA which was extracted from M20-20D/pEK1 and subsequently amplified in E. coli. Eleven cleavage sites in the insery (5.3kb) have been localized through fragment analysis for 8 restriction endonucleases; Bgl II(2 site), Bgl II(1), Cla I(3), Eco RI(1), Hind III(2), Kpn I (1), Pvu II(1) and Xho I(1).

• Toxicological Research
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• v.22 no.1
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• pp.15-22
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• 2006

Many of endocrine disrupting chemicals induce effects via interaction with hormone receptors and responsive elements in target cells. We investigated endocrine disrupting effects of some food additives and contaminants including BHA, BHT, ethoxyquin, propionic acid, sorbic acid, benzoic acid, CPM, aflatoxin B1, cadmium chloride, genistein, TCDD and PCBs in yeast transformants expressing human steroid hormone receptors along with steroid responsive elements. The response limit of genetically recombinant yeast to $17{\beta}$-estradiol, testosterone and progesterone was $1{\times}10^{-16},\;1{\times}10^{-12}\;and\;1{\times}10^{-13}M$, respectively. BHT induced weak transcriptional activity in estrogen sensitive yeast, while BHA and sorbic acid interacted weakly with androgen receptor/responsive element. CPM induced transcriptional activities in all types of yeasts sensitive to steroid hormones. Zearalenone and genistein induced high transcriptional activation in estrogen sensitive yeast with relative potencies almost $10^8$ folds lower than $17{\beta}$-estradiol. TCDD induced transcriptional activation weakly in estrogen- and progesterone- sensitive yeasts. This study elucidated that recombinant yeast is a sensitive and high-throughput system and can be used for the direct assessment on chemical interactions with steroid receptors and responsive elements. Also, the present study raises the requirement of evaluation on the endocrine disrupting effects of BHT, BHA, sorbic acid, CPM and TCDD for their transcription activity in yeast screening system though weak in intensity.

• Biotechnology and Bioprocess Engineering:BBE
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• v.6 no.3
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• pp.173-178
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• 2001

Food yeast, Saccharomyces cerevisiae, is a safe organism with a long history of use for the production of biomass rich in high quality proteins and vitamins. AmA1, a seed storage albumin from Amaranthus hypochondriacus, has a well-balanced amino acid composition and high levels of essential amino acids and offers the possibility of further improving food animal feed additives. In order to find an effective means of expressing AmA1 in yeast, the gene was cloned into an episomal shuttle vector. Four different promoters were tested: the glyceraldehyde-3-phosphate dehydrogenase promoter, galactose dehydrogenase 10 promoter, alcohol dehydrogenase II promoter, and a hybrid ADH2-GPD promoter. The recombinant AmA1 genes were then introduced into the yeast Saccharomyces cerevisiae 2805. Northern and Western blot analyses of the yeast under appropriate conditions revealed that AmA1 was expressed by all four promoters at varying levels. An enzyme-linked immunosorbent assay demonstrated that the amount of AmA1 protein in the recombinant yeast was 1.3-4.3% of the total soluble proteins. The highest expression level was obtained from the hybrid ADH2-GPD promoter.

• Korean Journal of Microbiology
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• v.30 no.5
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• pp.403-409
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• 1992

For the development of a glucanolytic yeast strain. the seceretion of endo-1.4-$\beta$-D-glucanase (CMCase) of Bacillus subtilis was performed in yeast using glucoamylase gene (STA1) of Saccharomyces diastaticus. A 1.7 kb-DNA fragment of STA1 gene containing authentic promoter, signal sequence, threonine serine-rich (TS) region and N-terminal region (98 amino acids) of mature glucoamylase was ligated to YEp 24. E. coli-yeast shuttle vector. And then. CMCase structural gene of B. subtilis was fused in frame with the 1.7 kb-DNA fragment of STA1 gene, resulting in recombinant plasmid pYES('24. Yeast transformant harboring pYESC24 had no CMCase activity. So. we deleted TS region and N-terminal region of mature glucoamylase existing between signal sequence and CMCase structural gene in pYESC24. consequently constructed recombinant plasmid pYESC11. The yeast transformed with the newly constructed recombinant plasmid pYESC11 efficiently secreted CMCase to extracellular medium. After 4 days culture. total CMCase activity of this transformant was 44.7 units/ml and over 93% of total CMCase activity was detected in culture supernatant.

• Journal of Life Science
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• v.20 no.6
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• pp.934-939
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• 2010

We have previously reported recombinant productions of bacteriocins using yeast expression plasmid pAUR123, which contains the alcohol dehydrogenase (ADH) promoter, in Saccharomyces cerevisiae cells and their antibacterial activities. In order to improve the antibacterial activities of bacteriocidal yeast cells, a strong glyceraldehyde phosphate dehydrogenase (GPD) promoter gene of S. cerevisiae was amplified and inserted upstream into bacteriocin genes such as the OR-7, Subpeptin JM4-A or JM4-B gene in the corresponding recombinant yeast plasmid. Yeast cells transformed by the recombinant plasmid containing the GPD promoter represented higher antibacterial activities against both Gram positive B. subtilis and Gram negative E. coli cells compared to those transformed by the corresponding recombinant plasmid containing the ADH promoter. Thus, yeast cells harboring the recombinant plasmid containing the GPD promoter constructed in this study could be applied in the food preservative or animal feed industries.

• Microbiology and Biotechnology Letters
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• v.14 no.6
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• pp.455-460
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• 1986

Dane particle was prepared from the plasma of chronic HBsAg carrier with high levels of HBsAg activity. DNA extracted front Dane particle core after a DNA polymerase reaction with $\alpha$-($^{32}$P) dNTP, was identified as HBV DNA by liquid scintillation counter and agarose gel electrophoresis-G.M. counting. To produce Hepatitis B surface antigen for use as a vaccine against Hepatitis B virus infection, yeast strains harboring recombinant plasmid with Apase promoter was used. Recombinant plasmid was construced from pHBV 130 and pAN 82, transformed into E coli, and then transferred into yeast strains. HBsAg was produced by derepression in Burkholder minimal medium with controlled inorganic phosphate concentration. The kinetics of HBsAg production was also investigated. Total HBsAg activity increased rapidly between 3 and 6 hours after transfer to phosphate-free medium and reached a maximum at around 9th hour. The transfer into phosphate-free medium after 6 hours in high phosphate cell growth medium gave maximum activity.

• Journal of Food Hygiene and Safety
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• v.24 no.2
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• pp.124-127
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• 2009

The aim of this study is to evaluate the estrogenic activity of Cucurbita pepo seed extract which includes ${\beta}-sitosterol$ and other phytosterols. Sample was extracted from Cucurbita pepo seed by supercritical carbondioxide method and resuspended with ethanol. Estrogenic activity was measured by recombinant yeast assay which detects estrogenic activity using recombinant yeast with high level of estrogenic receptor. However, estrogenic activity of pumpkin seed extract was not found in this study. Based on this data, pumpkin seed extract will not cause estrogenic disturbance.

• Environmental Analysis Health and Toxicology
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• v.29
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• pp.19.1-19.10
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• 2014

Objectives Lysosome is the cell-organelle which is commonly used as biomonitoring tool in environmental pollution. In this study, the lysosomal proteomic of the yeast Saccharomyces cerevisiae was analyzed for utilization in the detection of toxic substances in mine water samples. Methods This work informs the expression of lysosomal proteomic in yeast in response with toxic chemicals, such as sodium meta-arsenite and tetracycline, for screening specific biomarkers. After that, a recombinant yeast contained this biomarker were constructed for toxic detection in pure toxic chemicals and mine water samples. Results Each chemical had an optimal dose at which the fluorescent protein intensity reached the peak. In the case of water samples, the yeast showed the response with sample 1, 3, 4, and 5; whereas there is no response with sample 2, 6, and 7. Conclusions The recombinant yeast showed a high ability of toxic detection in response with several chemicals such as heavy metals and pharmaceuticals. In the case of mine water samples, the response varied depending on the sample content.