• Microbiology and Biotechnology Letters
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• v.26 no.1
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• pp.34-39
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• 1998

We have already prepared a human lysozyme (HLY) structural gene from chemically synthesized 38 oligomers with high codon usage in Saccharomyces cerevisiae. For directing the synthesis and secretion of HLY in S. cerevisiae, two types of expression vectors, a YCp centromere-based vector, pHK101 and a YEp 2-$\mu\textrm{m}$ circle-based vector, pHK501 were constructed. With the resulting plasmids, we have confirmed that yeast transformant harboring pHK501 has more secreted HLY than pHK101-transformant by using a lysoplate and a turbidimetric assay. In flask cultivation, pHK501-transformant produced active HLY about 8 times (55 units/$m\ell$) higher than pHK101-transformant. From batch cultivation, the HLY productivity was obtained with 1.12 units/$m\ell$/h, corresponding to a 1.8-fold increase compared with flask fermentation. These results indicate that yeast transformant with pHK501 vector overexpressed and secreted HLY than that of YCp type vector.

• Biotechnology and Bioprocess Engineering:BBE
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• v.8 no.2
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• pp.162-165
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• 2003

In order to study the secretion of the human urokinase-type plasminogen activator, u-PA, from the yeast yarrowia lipolytica, three kinds of integrative expression vector were constructed. These vectors differed only in their secretion control legions, pre-, pre-dip-(dipeptide Stretch) or pre-dip-pro sequences of the alkaline extracellular protease, which were joined inflame to the human u-PA cDNA. The recombinant Y. lipolytica Strains, transformed with the expression vectors, secreted the hyperglycosylated u-PA. A fibrin plate assay of the culture supernatants showed that the hyperglycosylated u-PA proteins could catalyze fibrinolysis, and that the pre-dip sequence was the most efficient secretory signal for the secretion of the u-PA from Y. lipolyica. This result suggests that Y. lipolytica can be developed as a potential host for the production of recombinant human u-PA.

• Journal of Microbiology and Biotechnology
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• v.20 no.6
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• pp.974-977
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• 2010

Cyclophilins contain the conserved activity of cis-trans peptidyl-prolyl isomerase, which is implicated in protein folding, and function as molecular chaperones. When the yeast cyclophilin A gene (cpr1) was subcloned into the prokaryotic expression vector pKM260, it was found that the expression of Cpr1 drastically increased the cell viability of E. coli BL21 when under abiotic stress conditions, as in the presence of cadmium, copper, hydrogen peroxide, heat, and SDS. Therefore, this study illustrates the importance of Cpr1 as a molecular chaperone that can improve the cellular stress responses when E. coli cells are exposed to adverse conditions, while also demonstrating its potential to increase the stability of E. coli strains utilized for the production of recombinant proteins.

• Biotechnology and Bioprocess Engineering:BBE
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• v.5 no.1
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• pp.1-6
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• 2000

For the rapid selection of higher recombinant hirudin producing strain in a methylotrophic yeast Hansenula polymorpha, a multiple gene integration and dose-dependent selection vector, based on a telomere-associated ARS and a bacterial aminoglycoside 3-phosphotransferase (aph) gene, was adopted. Two hirudin expression cassettes (HV1 and HV2) were constructed using the MOX promoter of H. polymorpha and the mating factor $\alpha$ secretion signal of S. cerevisiae. Multiple integrants of a transforming vector containing hirudin expression cassettes were easily selected by using an antibiotic, G418. Hirudin expression level and integrated plasmid copy number of the tested transformants increased with increasing the concentration of G418 used for selection. The expression level of HV1 was consistently higher than that of HV2 under the similar conditions, suggesting that the gene context might be quite important for the high-level gene expression in H. polymorpha. The highest hirudin producing strain selected in this study produced over 96 mg/L of biologically active hirudin in a 500-mL flask and 165 mg/L in a 5-L fermentor.

• KSBB Journal
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• v.19 no.5
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• pp.352-357
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• 2004

Human ferritin H- and L-chain genes (hfH and hfL) were cloned into the yeast shuttle vector YEp352 containing the GAL1 (galactokinase) and GAL10 (epimerase) divergent promoters and the vectors constructed were used to transform Saccharomyces cerevisiae 2805. SDS-PAGE displayed expression of the introduced hfH and hfL in both recombinant strains of Y1H10L and Y1L10H. The ferritin subunits, that represented ca. $22\%$ and $15\%$ of the soluble proteins in Y1H10L and Y1L10H, were spontaneously assembled into active ferritin heteropolymers. The H subunit content of the purified recombinant human ferritin heteropolymers was proven to reflect the relative expression yield of the subunits. When the cells of 2d culture were incubated with 14.3 mM Fe(2), the cellular iron concentration of Y1H10L and Y1L10H was 1.7 and 2.0 times, respectively, that of the control strain. It is assumed that increase in the iron uptake of the recombinant yeasts is closely related to ferritin expression and H subunit content.

• Journal of Microbiology and Biotechnology
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• v.15 no.1
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• pp.202-205
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• 2005

Our previous study showed that the overexpression of carboxypeptidase Y (CPY) of Saccharomyces cerevisiae in Escherichia coli resulted in the formation of insoluble inclusion bodies. To produce soluble CPY, we designed a novel Pichia pastoris expression system, in which the following were inserted into expression vectors: three different signal sequences derived from the mating factor a1 of S. cerevisiae, an inulinase of Kluyveromyces marxianus, and the endogenous signal sequence of CPY. The expression vector pHIL-D2-SSinul-proCPY was the most effective in the production of proCPY among the vectors examined. The purified active CPY was obtained from proCPY by treating with proteinase K, followed by QExcellose ion-exchange column chromatography.

• Microbiology and Biotechnology Letters
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• v.19 no.6
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• pp.568-574
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• 1991

The cloned glyceraldehyde-3-phosphate dehydrogenase (GAP) gene of Saccharomyces cereviszae (Holland et al., 1983) has been characterized. Based on the communication, we have also cloned 2.1 kb CAP DNA fragment and modified this fragment as a portable promoter. Two yeast expression vectors, one is YCp type vector being maintained at low copy number (1 or 2) and the other is YEp type vector at high copy number, have been constructed with the GAP promoter and the PH05' gene as a reporter. Our plasrnids were introduc,ed into S. cerevisiae HY-1, which has been improved. The $Trp^+$ transformants expressed APase activity efficiently and showed high level of PH05' transcripts.

• Journal of Microbiology and Biotechnology
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• v.26 no.5
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• pp.846-853
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• 2016

We displayed four types of Solanum nigrum metallothionein (SMT) for the first time on the surface of Saccharomyces cerevisiae using an α-agglutinin-based display system. The SMT genes were amplified by RT-PCR. The plasmid pYES2 was used to construct the expression vector. Transformed yeast strains were confirmed by PCR amplification and custom sequencing. Surface-expressed metallothioneins were indirectly indicated by the enhanced cadmium sorption capacity. Flame atomic absorption spectrophotometry was used to examine the concentration of Cd²⁺ in this study. The transformed yeast strains showed much higher resistance ability to Cd²⁺ compared with the control. Strikingly, their Cd²⁺ accumulation was almost twice as much as that of the wild-type yeast cells. Furthermore, surface-engineered yeast strains could effectively adsorb ultra-trace cadmium and accumulate Cd²⁺ under a wide range of pH levels, from 3 to 7, without disturbing the Cu²⁺ and Hg²⁺. Four types of surfaceengineered Saccharomyces cerevisiae strains were constructed and they could be used to purify Cd²⁺-contaminated water and adsorb ultra-trace cadmium effectively. The surface-engineered Saccharomyces cerevisiae strains would be useful tools for the bioremediation and biosorption of environmental cadmium contaminants.

• Journal of Microbiology and Biotechnology
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• v.20 no.2
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• pp.356-362
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• 2010

Ceramide is important not only for the maintenance of the barrier function of the skin but also for the water-binding capacity of the stratum corneum. Although the exact role of ceramide in the human skin is not fully understood, ceramide has become a widely used ingredient in cosmetic and pharmaceutical industries. Compared with other microorganisms, yeast is more suitable for the production of ceramide because yeast grows fast and is non-toxic. However, production of ceramide from yeast has not been widely studied and most work in this area has been carried out using Saccharomyces cerevisiae. Regulating the genes that are involved in sphingolipid synthesis is necessary to increase ceramide production. In this study, we investigated the effect of the genes involved in the synthesis of ceramide, lcb1, lcb2, tsc10, lac1, lag1, and sur2, on ceramide production levels. The genes were cloned into pYES2 high copy number vectors. S. cerevisiae was cultivated on YPDG medium at $30^{\circ}C$. Ceramide was purified from the cell extracts by solvent extraction and the ceramide content was analyzed by HPLC using ELSD. The maximum production of ceramide (9.8 mg ceramide/g cell) was obtained when the tsc10 gene was amplified by the pYES2 vector. Real-time RT-PCR analysis showed that the increase in ceramide content was proportional to the increase in the tsc10 gene expression level, which was 4.56 times higher than that of the control strain.

• Journal of Life Science
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• v.17 no.1 s.81
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• pp.39-44
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• 2007

The Rqh1 gene is essential for vegetative growth in fission Yeast. The rqh1 mutant showed that sensitivity of DNA damaging agent, a wild range of phenotype including abnormal gene expression and cell elongation. This result showed that the rqhl-overexpression cell was sensitivity to DNA damaging agent like rqhl mutant. When Rqh1 have an over-expression by $nmt1^+$ promoter of pREP vector, rqh1 mutant DNA damaging agent sensitivity could be compensated. We isolated two strong mutant containing complementation gene, rqh156 and rqh172, respectively. This result observed that the DNA damaging agent sensitivity of rqhl mutant was complemented by the expression of rqh156 and rqh172. They induced mRNA expression in a dose-dependent manner HU, MMS and UV. The HU sensitivity of the rqhl was complemented by the expression of rqh156 and rqh172. The mRNA expression of rqh156 decreased on HU dose dependent but the mRNA expression of rqh172 did not decrease on HU dose dependent. The MMS and W sensitivity of the rqhl was complemented by the expression of rqh156 and rqh172. These results indicate that the isolated rqhl gene may play an important role in DNA metabolism.