• Microbiology and Biotechnology Letters
• /
• v.22 no.2
• /
• pp.152-157
• /
• 1994

Inulinase of Kluyveromyces marxianus origin was produced by recombinant yeast Saccharomyces cerevisiae under the control of GAL1 promoter, to examine the expression and localization of inulinase in a repressed(galactose-free) or derepressed(galactose-containinga) medium. The inulinase gene(INU1A) was constitutively expressed at 6.7 units/ml in a repressed medium. When the cell started to utilize galactose in a derepressed medium, the INU1A gene began to be expressed, and the final expression level reached about 45 units/ml. According to be the nondenaturingPAGE analysis, inulinase produced by S. cerevisiae was found to be less glycosylated than the bakers yeast invertase. In addition, its glycosylation pattern was less heterogeneous than the K. marxianus inulinase. The supplementation of inulin or raffinose into the derepressed medium increased the cell growth rate, while the expression of INU1A was repressed. Regardless of the carbon sources examined, most of inulinase activity (more than 98%) was found in the extracellular medium, indicating excellent secretion efficiency.

• Proceedings of the Korean Society of Life Science Conference
• /
• 2007.10a
• /
• pp.59.2-59.2
• /
• 2007

See Full Text

• Proceedings of the Korean Society of Life Science Conference
• /
• 2001.02a
• /
• pp.82-82
• /
• 2001

• Proceedings of the Korean Society of Applied Pharmacology
• /
• 2005.04a
• /
• pp.5-14
• /
• 2005

Recently, data from several groups have raised the concept of 'checkpoint' in transcription. As capping of nascent RNA transcript is tightly coupled to RNA polymerase II transcription, we seek to obtain direct evidence that transcripiton checkpoint via capping enzyme functions in this early regulatory step. One of temperature sensitive (ts) alleles of ceg1, a guanylyltransferase subunit of the Saccharomyces cerevisiaecapping enzyme, showed 6-azauracil (6AU) sensitivity at the permissive growth temperature, which is a phenotype that is correlated with a transcription elongational defect. This ts allele, ceg1-63 also has an impaired ability to induce PUR5 in response to a 6AU treatment. However, this cellular and molecular defect is not due to the preferential degradation of the transcript attributed from a lack of guanylyltransferase activity. On the contrary, the data suggests that the guanylyltransferase subunit of the capping enzyme plays a role in transcription elongation. First, in addition to the 6AU sensitivity, ceg1-63is synthetically lethal with elongation defective mutations of the largest subunit of RNA polymerase II. Secondly, it exhibited a lower GAL1 mRNA turn-over after glucoseshut off. Third, it decreased the transcription read through a tandem array of promoter proximal pause sites in an orientation dependent manner. Interestingly, this mutant also showed lower pass through a pause site located further downstream of the promoter. Taken together, these results suggest that the capping enzyme plays the role of an early transcription checkpoint possibly in the step of the reversion of repression by stimulating polymerase to escape from the promoter proximal arrest once RNA becomes appropriately capped.

• Journal of Life Science
• /
• v.26 no.12
• /
• pp.1376-1382
• /
• 2016

Xylitol is widely used in the food and medical industry. It is produced by the reduction of xylose (lignocellulosic biomass) in the Saccharomyces cerevisiae strain, which is considered genetically safe. In this study, the expression system of the GRE3 (YHR104W) gene that encodes xylose reductase was constructed to efficiently produce xylitol in the S. cerevisiae strain, and the secretory production of xylose reductase was investigated. To select a suitable promoter for the expression of the GRE3 gene, pGMF-GRE3 and pAMF-GRE3 plasmid with GAL10 promoter and ADH1 promoter, respectively, were constructed. The mating factor ${\alpha}$ ($MF{\alpha}$) signal sequence was also connected to each promoter for secretory production. Each plasmid was transformed into S. cerevisiae $SEY2102{\Delta}trp1$, and $SEY2102{\Delta}trp1$/pGMF- GRE3 and $SEY2102{\Delta}trp1$/pAMF-GRE3 transformants were selected. In the $SEY2102{\Delta}trp1$/pGMF-GRE3 strain, the total activity of xylose reductase reached 0.34 unit/mg-protein when NADPH was used as a cofactor; this activity was 1.5 fold higher than that in $SEY2102{\Delta}trp1$/pAMF-GRE3 with ADH1 as the promoter. The secretion efficiency was 91% in both strains, indicating that most of the recombinant xylose reductase was efficiently secreted in the extracellular fraction. In a baffled flask culture of the $SEY2102{\Delta}trp1$/pGMF-GRE3 strain, 12.1 g/l of xylitol was produced from 20 g/l of xylose, and ~83% of the consumed xylose was reduced to xylitol.

• Journal of Microbiology and Biotechnology
• /
• v.16 no.3
• /
• pp.360-367
• /
• 2006

The endoinulinase gene (inu1) from Pseudomonas mucidolens was expressed on the cell surface of Saccharomyces cerevisiae by fusing with Aga2p linked to the membrane anchored protein, Aga1p. The inu1 gene of P. mucidolens was subcloned into the surface display vector, pCTcon (GAL1 promoter). The constructed plasmid, pCTENIU (8.5kb), was then introduced to S. cerevisiae EBY100 cells and the yeast transformants selected on synthetic defined media lacking uracil and inulin-containing media. The inu1 gene under the control of the GAL1 promoter was successfully expressed in the yeast transformants, and the surface display of endoinulinase confirmed by immunofluorescence microscopy, along with its enzymatic ability to form inulooligosaccharides (IOSs) from inulin. The total endoinulinase activity reached about 2.31 units/ml when the yeast transform ants were cultivated on a YPDG medium. To efficiently hydrolyze the inulin, various reaction conditions were examined, including the pH, temperature, and inulin source. The optimized conditions were then determined as follows: pH, 7.0; temperature, $50^{\circ}C$; inulin source, Jerusalem artichoke. Under the optimized condition and 46 units of endoinulinase per g of inulin, IOSs started to be produced after 10 min of enzymatic reaction. The highest yield, 71.2% of IOSs, was achieved after 30 h of reaction without any significant loss of the initial enzyme activity. As a result of the reaction with inulin, IOSs consisting of inulobiose (F2), inulotriose (F3), inulotetraose (F4), and inulopentaose (F5) were produced, and F4 was the major product.

• Proceedings of the KSAR Conference
• /
• 2001.03a
• /
• pp.16-16
• /
• 2001

Apoptosis로 일컬어지는 예정된 세포사멸(programmed cell death)은 개별 세포의 입장에서는 곧바로 사멸을 의미하지만, 정상적인 고등 생물의 입장에서는 개체의 발생과 분화하는데 프로그램된 과정이다. 자발적 세포사멸은 다른 조직에 비해 생식 조직인 난소나 정소에서 복잡한 apoptosis 기작들을 가지리라 사료된다. 본 연구는 Bcl-2 family중 apoptotic protein인 Bax에 대해 suppression하는 유전자를 yeast system을 활용하여 돼지 정소와 난소로부터 각각 cDNA library를 구축한 후 탐색하였다. 탐색에 활용된 cDNA library는 돼지의 정소와 난소로부터 mRNA를 분리하여 yeast vector인 pAD-GAL4-2.1에 구축하였고, 마우스 bax 유전자는 gal 1 promoter의 조절 하에 glucose 배지에서는 유도되지 않고, galactose 배지에서만 선택적으로 Bax를 발현할 수 있는 효모 vector(pL19-bax)를 구축하였다. Bax에 의한 apoptosis suppressor를 탐색하기 위해 우선 효모 W303에 pL19-bax를 transform하여 glucose 배지에서 Bax의 발현을 억제하였다. pL19-bax를 가진 효모에 정소와 난소로부터 구축된 cDNA library를 transform 시키고, transform된 효모는 각각 Bax에 의한 toxicity를 저해하는 유전자를 찾기 위해 스크린되었다. 이러한 방법으로 정소 cDNA library 탐색에서는 5 $\times$ $10^{6}$ transformant중 39개, 난소cDNA library 탐색에서는 2 $\times$ $10^{6}$ transformant중 26개의 콜로니가 생존하였다. 이들 콜로니로부터 유전자를 분리하여 분석해 본 결과 여러 그룹으로 분류할 수 있었다. 각 그룹의 관련 유전자는 protein synthesis/degradation 12종, oxidation/reductation 5종, detoxin/ cell cycle promoter 3종, signal transduction/growth factor 5종, 그리고 알려지지 않은 유전자 9종이었다. 그 중, bax-toxicity inhibition에 강력한 survival phenotype을 가지는 유전자(pSEDL)를 동정하였다. 이것은 T3-4-1 콜로니로부터 분리하였는데 140개 아미노산으로 이루어진 인간 SEDL(GenBank, XM_013096) 유전자와 매우 유사한 homology를 가지며, bax와 관련된 기능은 밝혀져 있지 않다. 이외에도 분리된 유전자에는 NADH, thioreduction, 그리고 cytochrome oxidase와 같은 positive 유전자 군이 크로닝되어, Bax를 이용한 효모에서 apoptosis suppressor에 관련된 유전자를 손쉽게 스크린하는 것이 가능하고, 분리된 유전자의 기능을 예측할 수 있어 지금까지 보고된 유전자 크로닝법 보다는 강력한 수단으로 활용될 수 있다는 사실을 시사하였다. 그러나, ORF에 관계없이 Bax 발현에 저항하는 유전자군이 선발된다든지 하는 문제점은 금후 검토가 필요하리라 사료된다.

• Journal of Microbiology and Biotechnology
• /
• v.18 no.2
• /
• pp.219-227
• /
• 2008

The free-living photoheterotrophic Gram-negative bacterium Rhodobacter sphaeroides possesses a quorum-sensing (QS) regulatory system mediated by CerR-CerI, a member of the LuxR-LuxI family. To identify the genes affected by the regulatory system, random lacZ fusions were generated in the genome of R. sphaeroides strain 2.4.1 using a promoter-trapping vector, pSG2. About 20,000 clones were screened and 23 showed a significantly different level of ${\beta}$-gal activities upon the addition of synthetic 7,8-cis-N-tetradecenoyl-homoserine lactone (RAI). Among these 23 clones, the clone showing the highest level of induction was selected for further study, where about a ten-fold increase of ${\beta}$-gal activity was exhibited in the presence of RAI and induction was shown to be required for cerR. In this clone, the lacZ reporter was inserted in a putative gene that exhibited a low homology with catD. A genetic analysis showed that the expression of the catD homolog was initiated from a promoter of another gene present upstream of the catD. This upstream gene showed a strong homology with luxR and hence was named qsrR (quorum-sensing regulation regulator). A comparison of the total protein expression profiles for the wild-type cells and qsrR-null mutant cells using two-dimensional gel electrophoresis and a MALDI-TOF analysis allowed the identification of sets of genes modulated by the luxR homolog.

• KSBB Journal
• /
• v.10 no.3
• /
• pp.343-348
• /
• 1995

Human lipocorin-I(LCI) is a calcium ion-dependent and phospholipid-binding protein which exhibits an anti-inflammatory activity by inhibiting phospholipase A2 activity. In this study, the LCI gene containing its own terminator region was joined to GAL10 promoter-ppL (prepro-leader sequence of mating factor a). An ATG start codon of LCI gene was placed at downstream with KR endoprotease recognition site(Lys-Arg) of ppL. Recombinant S. cerevisiae harboring the LCI expression/secretion vector, pYGLPT5, was aerobicall grown on a liquid YPDG medium al $30^{\circ}C$ for 72hys. The whole cell and culture supernatant were separated after centrifugation, and the expressed LCI was analyzed by SDS-PAGE and western blotting methods. A majority fraction of the expressed LCI was found to be accumulated in the intracellular fraction, resulting in very low secretion efficiency of about 7.4%. About $500mg/\ell$ of LCI was extracellularly produced by the fed-batch culture employing the controlledfeeding of glucose and galactose. The secreted LCI was purified by ultrafiltration and hydroxylapatite column chromatography, and a purity of more than 99% was obtained.

• Korean Journal of Microbiology
• /
• v.36 no.3
• /
• pp.236-241
• /
• 2000

A defensin is an inducible antibacterial peptide from a fleshfly and contains 40 residues basic peptide with six cysteines. For the consiruction of recombinant S cerevisiae expressing defensin, the structural gene coding for active defensin was chemically synthesized and fused in fiam to GAP promoter, MFul preprosequence and the GAL7 transcription terminator, generating a recombinant plasnlid pGMD18. S. ce~evisine 2805 Gells were transror~ned to uracil prototroph by the pGMDl8 arid the transformed cells showing antibacterial activity against 111. luteus TAM1056 were selected by growth inhibition zone assay. The optimal culture conditions for the unprovement of the defensin production of a selected tmdonnant were investigated. The optirmzed medium containing 0.4% yeast extract, 2% corn steep liquor, 2.5% glucose and 0.05% $C_2CO_3$, could be determined and the optimum lemperature. and initial pH could be detennnied as $28^{\circ}C$ and pH 3, ~mpectively. The optimized conditioiis revealed the trvofold Increase in the cell growth and the fourfold in the antibaclerial activity. coinpar-ed with tllc Yl'D medium.