• Microbiology and Biotechnology Letters
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• v.29 no.4
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• pp.206-211
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• 2001

In order to overexpress constitutively the Kluyveromyces marxianus exoinulinase gene (INUI) in Saccharomyces cerevisiae, four episomal expression systems employing GAPDH, ADHI, PGK and ENOI promoters were constructed as p YIGP aADHI -INU, pPGK-INU, and pENOI- INU plasmids respectively, When S cereviais transformants harboring each plasmid were batchwisely cultivated in the fermentor containing 5% glucose medium no significant differences in the cell growth are observed How- ever the experession level of exoinulinase and plasmid stability showed a strong dependency on the promoter employed. The expression levels of exoinulinase were about 1.70 unit/ml for GAPDH promoter 1.67 unit/ml for PGK promoter, 1.29 unit /ml for ADH1 promoter, and 0.80 unit/ml for ENOl promoter. The plasmid stabilites were maintaines above 80% in all experession systems. except the GAPDH promoter system of 55%, Based on the plas- mid stability and expression level of exoinulinase the ADHl and PGK promoter system were selected for the fed - batch culture to overproduce exoinulinase By the intermittent feeding of yeast extract and glucose, both promoter systems gave the cell concentration of about 30 g-dry cell weight/1 byt the maximal exoinulinase activity of 3.70 unit/ml and plasmid stability of 96% in the ADH1 promoter were higher than those (2.70 unit/ml, 80%) of PGK sys- tem Taking into account the plasmid stability and extended culture time the ADH1 promoter systems would be the most feasible expression systems for the constitutive overproduction of exoinulinase through high cell-density fed- batch cultures using non-selective rich medium.

• BMB Reports
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• v.29 no.4
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• pp.359-364
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• 1996

By both in vitro hydroxylamine mutagenesis of the wild type 3-phosphoglycerate kinase gene (PGK) promoter DNA and insertion of the leu2-d gene, we have created yeast expression vectors potentially useful for production of eukaryotic genes in yeast. The guanine (G) to adenine (A) change at the -3 position from the ATG start codon of the PGK promoter-based vector rendered a 6~7 times elevated expression of the adjacent eukaryotic gene, and insertion of the leu2-d gene in the vector containing the mutated PGK promoter further enhanced the expression of the gene. When expression of the AIDS virus HIV1-gagP17 gene in a lacZ fusion form was examined with this new vector, a 15 times higher level of expression than that from the original PGK promoter was observed. Northern and Southern analysis showed that this elevated expression is due to the production of a high copy number of mRNA by leu2-d gene functioning and by efficient translation of the produced mRNA. Thus, the vector that contained the A at the -3 position from the ATG start codon in the promoter region and the leu2-d gene shows increased expression capability and will be potentially useful for production of eukaryotic genes in yeast.

• Korean Journal of Microbiology
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• v.28 no.1
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• pp.1-5
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• 1990

The coding sequence of hepatitis B viral core antigen (HBcAg) (subtype adr) contains two in-phase initiation codons, one for precore and the other for core antigen gene. To study the expression of core antigen and the role of precore region, the coding sequence of HBcAg with or without precore (pre-C) region were subcloned into yeast expression vector containing phosphoglycerate kinase (PGK) promoter. To study the role of upstream region in the expression of the core antigen, a series of 5' deletion mutants were also subcloned into the vector. After transformation into various host strains, the expression of HBcAg were analysed by radio-immunoassat. Under optimal condition of core antigen gene expression in yeast, the highest amount of antigen was detected in the cell line SHY4 containing pGKHBc plasmid composed of the yeast PGK gene promoter, terminator and C-gene. Regardless of the presence of precore region, core antigen was not detected in the medium but in cell extract. These results suggest that precore region cannot affect the secretion of core antigen in Saccharomyces cerevisiae.

• Journal of Microbiology and Biotechnology
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• v.20 no.4
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• pp.767-774
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• 2010

In this study, the problems of high caloric content, increased maturation time, and off-flavors in commercial beer manufacture arising from residual sugar, diacetyl, and acetaldehyde levels were addressed. A recombinant industrial brewing yeast strain (TQ1) was generated from T1 [Lipomyces starkeyi dextranase gene (LSD1) introduced, ${\alpha}$-acetohydroxyacid synthase gene (ILV2) disrupted] by introducing Saccharomyces cerevisiae glucoamylase (SGA1) and a strong promoter (PGK1), while disrupting the gene coding alcohol dehydrogenase (ADH2). The highest glucoamylase activity for TQ1 was 93.26 U/ml compared with host strain T1 (12.36 U/ml) and wild-type industrial yeast strain YSF5 (10.39 U/ml), respectively. European Brewery Convention (EBC) tube fermentation tests comparing the fermentation broths of TQ1 with T1 and YSF5 showed that the real extracts were reduced by 15.79% and 22.47%; the main residual maltotriose concentrations were reduced by 13.75% and 18.82%; the caloric contents were reduced by 27.18 and 35.39 calories per 12 oz. Owing to the disruption of the ADH2 gene in TQ1, the off-flavor acetaldehyde concentrations in the fermentation broth were 9.43% and 13.28%, respectively, lower than that of T1 and YSF5. No heterologous DNA sequences or drug resistance genes were introduced into TQ1. Hence, the gene manipulations in this work properly solved the addressed problems in commercial beer manufacture.