• KSBB Journal
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• v.11 no.4
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• pp.445-452
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• 1996

To investigate the promoter effect on heterologous gene expression in S. cerevisiae, the recombinant plasmids pYI11, pYI12, pYI10-2, and pYIGP were constructed to contain the inulinase gene (INUI) as a reporter under the control of GAL10, GAL7, GAL1, and GAP promoters, respectively. When the yeasts transformants were cultivated on galactose-containing rich media, the cell growth reached to 36-39 OD600 at 72 hours of cultivation. The specific growth rates of the cells harboring the four different plasmids decreased similarly : they dropped from $0.24 h^{-1}$ during the glucose-consuming period to 0.04 -$0.10 h^{-1}$ during the galactose-consuming period (gene expression phase for GAL promoter system). After the depletion of glucose, the expression of inulinase gene was started and reached to maximal levels of 4.3(GAL1 promoter), 4.0(GAL10 promoter), 3.8(GAL7 promoter), and 1.6(GAP promoter) unit/mL at 72 hours of cultivation. Based on the maximal expression level and activity staining on the plate, the promoter strength was in the order of GAL1, GAL10, GAL7 and GAP promoter. While the GAL-promoter systems showed a high plasmid stabilities of more than 78%, the GAP-promoter plasmid revealed a lower plasmid stability of 55%. Most of inulinase activity (98%) was found in the extracellular medium, indicating that the secretion efficiency of inulinase is independent on the type of promoter.

• Journal of Sericultural and Entomological Science
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• v.41 no.2
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• pp.102-107
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• 1999

To analysis a promoter strength of Atographa californica nucler polyhedrosis virus (AcNPV) IE1 gene, an immediate viral gene, ${\beta}$-glactosidase gene as a reporter gene was introduced under the control of the IE1 promoter. The restriction fragment containing IE1 promoter and ${\beta}$-galctosidase gene from pAcIE1-gal were inserter into pBacPAK9 to yield transfer vector pAcNPV-IE1-gal. The pAcNPV-IE1-gal was cotransfected with AcNPV genomic DNA BacPAK6 into Sf9 cells to produce recombinant baculovirus AcNPV-IE1-gal. In addition, recombinant bacvulovirus AcNPV-gal, which express ${\beta}$-galac-tosidase under the control of the polyhedrin promoter, was constrer, was constructed to compared with AcNPV-IE1-gal. The recombinant viruses were respectively infected into Sf9 cells and characterized by the virus titer and expression of ${\beta}$-galactoxidase in Sf9 cells. The promoter strength of IE1 and polyhedrin promoters was determined by the amount of ${\beta}$-galactosidase secreted into medium by viral infection. The titer of AcNPV-IE1-Gal determined by plaque assays in Sf9 cells was similar to that of AcNPV-gal. However, expression level of ${\beta}$-galactosidase by AcNPV-IE1-gal was significantly lower than that by AcNPV-gal. In conclusion, promoter strength of IE1 was approximately 25-fold lower than that of polyhedrin.

• Microbiology and Biotechnology Letters
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• v.47 no.4
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• pp.662-666
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• 2019

In this study, the NABH558 gene expression system was constructed to efficiently produce neoagarobiose hydrolase (NABH) in Saccharomyces cerevisiae strain. The ADH1 and GAL10 promoters of the pAMFα-NABH and pGMFα-NABH plasmids were examined to determine the suitable promoter for the NABH558 gene expression, respectively. The effect of promoter and carbon sources on NABH558 gene expression was investigated by transforming each plasmid into the S. cerevisiae 2805 strain. The NABH activity in the 2805/pAMFα-NABH strain was 0.069 unit/ml/DCW in YPD medium, whereas that in the 2805/pGMFα-NABH strain was similar (0.02-0.027 unit/ml/DCW) irrespective of the medium composition. The higher NABH activity in the YPD medium was due to the increased NABH558 gene transcription. NABH produced in the recombinant strains could degrade agarose to galactose and AHG. This indicated that ADH1 promoter was a more optimal promoter for the expression of NABH558 gene than the GAL10 promoter. The NABH activity induced by the ADH1 promoter was about 3-fold higher than that induced by the GAL10 promoter.

• KSBB Journal
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• v.14 no.4
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• pp.476-481
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• 1999

S. cerevisiae mutant(reg1-501, gal1), which cannot use galactose and has alleviated glucose repression level, is used as host for optimizing induction of GAL promoter. The optimum concentration of galactose as inducer for recombinant protein production and the galactose consumption rate have been tested with S. cerevisiae mutant and compared with conventional S. cerevisiae. The extent of glucose repression were investigated for both strain and the degradation pattern of produced foreign protein have been compared in both cases. The effect of pH on foreign protein degradation pattern were studied for both strains. The secetion efficiency of both strains were carried out. Through these experiments, optimum condition of recombinant protein production by GAL promoter using S. cerevisiae mutant (reg1-501, gal1) were found.

• Journal of Life Science
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• v.27 no.12
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• pp.1403-1409
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• 2017

In this study, the xylitol dehydrogenase (XYL2) gene was expressed in Saccharomyces cerevisiae as a host cell for ease of use in the degradation of lignocellulosic biomass (xylose). To select suitable expression systems for the S.XYL2 gene from S. cerevisiae and the P.XYL2 gene from Pichia stipitis, $pGMF{\alpha}-S.XYL2$, $pGMF{\alpha}-P.XYL2$, $pAMF{\alpha}-S.XYL2$ and $pAMF{\alpha}-P.XYL2$ plasmids with the GAL10 promoter and ADH1 promoter, respectively, were constructed. The mating factor ${\alpha}$ ($MF{\alpha}$) signal sequence was also connected to each promoter to allow secretion. Each plasmid was transformed into S. cerevisiae $SEY2102{\Delta}trp1$ strain and the xylitol dehydrogenase activity was investigated. The GAL10 promoter proved more suitable than the ADH1 promoter for expression of the XYL2 gene, and the xylitol dehydrogenase activity from P. stipitis was twice that from S. cerevisiae. The xylitol dehydrogenase showed $NAD^+$-dependent activity and about 77% of the recombinant xylitol dehydrogenase was secreted into the periplasmic space of the $SEY2102{\Delta}trp1/pGMF{\alpha}-P.XYL2$ strain. The xylitol dehydrogenase activity was increased by up to 41% when a glucose/xylose mixture was supplied as a carbon source, rather than glucose alone. The expression system and culture conditions optimized in this study resulted in large amounts of xylitol dehydrogenase using S. cerevisiae as the host strain, indicating the potential of this expression system for use in bioethanol production and industrial applications.

• Microbiology and Biotechnology Letters
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• v.40 no.4
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• pp.348-355
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• 2012

In the present study, we investigated the overexpression and characterization of bovine pancreatic (bp)- DNase I in Saccharomyces cerevisiae and Pichia pastoris. The bp-DNase I gene was fused in frame with the GAL10 promoter, $MF{\alpha}$, and GAL7 terminator sequences, resulting in the plasmid, pGAL-$MF{\alpha}$-DNaseI (6.4 kb). Also, the bp-DNase I gene was fused in frame with the AOX1 promoter, $MF{\alpha}$, and AOX1 terminator sequences, resulting in the plasmid, pPEXI (8.8 kb). The recombinant plasmids, pGAL-$MF{\alpha}$-DNaseI and pPEXI were introduced into S. cerevisiae and P. pastoris host cells, respectively. When the transformed yeast cells were cultured at $30^{\circ}C$ for 48 h in galactose or methanol medium, bp-DNase I was overexpressed and the most of activity was found in the extracellular fraction. P. pastoris transformant activity showed 45.5 unit/mL in the culture medium at 48 h cultivation, whereas S. cerevisiae transformant revealed 37.7 unit/mL in the extracellular fraction at 48 h cultivation. The enzymatic characteristics, such as DNA cleavage and half life were investigated. Treatment of the recombinant DNase I from P. pastoris induced degradation of the calf thymus DNA within 1 minute, and this DNA degradation rate was higher than that of commercial bp-DNase I (SIGMA) and the recombinant DNase I from S. cerevisiae.

• Microbiology and Biotechnology Letters
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• v.26 no.1
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• pp.68-75
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• 1998

The overproduction and high level secretion of Glucose Oxidase (GOD) from A. niger in S. cerevisiae was carried out by cloning GOD gene. For this purpose, using two different strong promoters (ADH1 promoter, GAL10 promoter) and signal sequences (${alpha}$-MF signal sequence of S. cerevisiae and ${alpha}$-amylase signal sequence of A. oryzae) and GAL7- and GOD terminator, four expression vectors were constructed. All the expression vectors were transformed in S. cerevisiae 2805 using auxotroph method. By the flask culture, transformants of pGAL expression vector series containing GAL 10 promotor showed much higher GOD productivity than transformants of pADH expression vector series containing ADH1 promoter Transformants of pGALGO2 containing GAL10 promotor and ${alpha}$-amylase signal sequence has shown the best productivity of GOD ($GOD_{total}$: 10.3 unit/mL, $GOD_{ex}$: 8.7 unit/mL) at 115 hr. This value was three fold higher than that of pGALGO1 containing GAL 10 promotor and ${alpha}$-MF signal sequence, even if the same promotor was involved. Through the ${alpha}$-amylase signal sequence of A. oryzae, GOD was secreted much more than the case of ${alpha}$-MF signal sequence from S. cerevisiae. These results suggest that signal sequence may play a important roles in not only the secretion but also the overproduction of foreign protein. Secretion rate of GOD in pGALGO1 and pGALGO2 was 89% and 84%, respectively, Because of the overglycosylation in S. cerevisiae the molecular weight of recombinant GOD in S. cerevisiae was much larger (250 kDa) than that of nature GOD in A. niger (170 kDa).

• Journal of Life Science
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• v.28 no.11
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• pp.1277-1284
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• 2018

In this study, a repeated yeast integrative plasmid (R-YIp) harboring Cre/loxP system was constructed to integrate various gene expression cassettes into the yeast chromosome. The R-YIp system contains a reusable selective marker (CgTRP1), loxP sequence, and target sequence for integration. Therefore, many gene expression cassettes can be integrated into the same position of the same yeast chromosome. In the present study, several model enzymes involving xylan/xylose metabolism were examined, including endoxylanase (XYLP), ${\beta}$-xylosidase (XYLB), xylose reductase (GRE3) and xylitol dehydrogenase (XYL2). Efficient expression of these genes was obtained using two promoters (GAL10p and ADH1p) and various plasmids (pGMF-GENE and pAMF-GENE plasmids) were constructed. The XYLP, XYLB, GRE3, and XYL2 genes were efficiently expressed under the control of the GAL10 promoter. Subsequently, R-YIps containing the GAL10p-GENE-GAL7t cassette were constructed, resulting in pRS-XylP, pRS-XylB, pRS-Gre3, and pRS-Xyl2 plasmids. These plasmids were sequentially integrated into chromosome VII of a Saccharomyces cerevisiae strain by repeated gene integration and selective marker rescue. These genes were integrated by the R-YIp system and were stably expressed in the yeast transformants to produce active recombinant enzymes. Therefore, we expect that the R-YIp system will be able to overcome current limitations of the host cells and allow selective marker selection for the integration of various genes into the yeast chromosome.

• Microbiology and Biotechnology Letters
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• v.32 no.3
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• pp.206-211
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• 2004

Levansucrase gene(lscA) from Pseudomonas aurantiaca was subcloned downstream of GAL1 promoter in pYES 2.0 and pYInu-AT [GAL10 promoter ＋ exoinulinase signal sequence of Kluyveromyces marxianus], resulting pYES-lscA and p YInu-lscA, respectively. The two expression plasmids were introduced into an invertase-deficient strain, Sacchromayces cerevisiae SEY2102, and transformants with high activity of levansucrase were selected. When each yeast transform ants was cultivated in medium containing galactose, the extracellular and intracellular activities of levansucrase reached about 8.62 U/ml with the strain harboring pYES-lscA and 5.43 U/ml with the strain harboring pYInu-lscA. The levansucrase activity of 80% was detected in the periplasmic space and cytoplasm. The levansucrase activity in the medium of SEY2102/pYInu-lscA was 0.87 U/ml whereas that of SEY2102/pYES-lscA was 0.47 U/ml, which implying the exoinulinase signal sequence didn't enhance the secretion efficiency of levansucrase. Furthermore, the recombinant levansucrase expressed in yeast seems to be produced as a hyper-glycosylated form.

• Molecules and Cells
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• v.27 no.5
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• pp.583-589
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• 2009

GTP cyclohydrolase I (GTPCH) is a key enzyme in the de novo synthesis of tetrahydrobiopterin. Previously, the Drosophila melanogaster GTPCH gene has been shown to be expressed from two different promoters (P1 and P2). In our study, the 5'-flanking DNA regions required for P1 and P2 promoter activities were characterized using transient expression assay. The DNA regions between -98 and +31, and between -73 and +35 are required for efficient P1 and P2 promoter activities, respectively. The regions between -98 and -56 and between -73 and -41 may contain critical elements required for the expression of GTPCH in Drosophila. By aligning the nucleotide sequences in the P1 and P2 promoter regions of the Drosophila melanogaster and Drosophila virilrs GTPCH genes, several conserved elements including palindromic sequences in the regions critical for P1 and P2 promoter activities were identified. Western blot analysis of transgenic flies transformed using P1 or P2 promoter-lacZ fusion plasmids further revealed that P1 promoter expression is restricted to the late pupae and adult developmental stages but that the P2 promoter driven expression of GTPCH is constitutive throughout fly development. In addition, X-gal staining of the embryos and imaginal discs of transgenic flies suggests that the P2 promoter is active from stage 13 of embryo and is generally active in most regions of the imaginal discs at the larval stages.