• Korean Journal of Microbiology
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• v.38 no.4
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• pp.318-321
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• 2002

To induce recombinant protein under phosphate restricted conditions such as soil, we have constructed the expression vector (pEAAP) with phoA gene promoter of Enterobacter aerogenes. To construct the pEAAP, deletion of the T7 promoter and lac operator from pET-22b(+) by BglII-XhoI digestion and addition of the phoA gene promoter (containing the pho box) were performed. To test pEAAP as an expression vector controled by phosphate limitation, pEAPHY1 was constructed with the phytate gene (Bsa-phy1) of Bacillus subtillis var. amyloliquefaciens (KCTC 8913P). Under the phosphate-limitation condition, CK-PHY1 ( Escherichia coli JM109 was transformed with pEAPHY1) expressed the 41 kD Bsa-Phy1 . Also CK-PHY1 formed the clear zone in solid medium containing phytate as a sole phosphate source.

• Korean Journal of Microbiology
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• v.46 no.2
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• pp.113-121
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• 2010

Bacillus subtilis PhoP-PhoR two-component system (TCS) senses phosphate deficiency conditions, and then controls expression of the Pho regulon to prolong survival. The sensor histidine kinase, PhoR, is autophosphorylated and transfers the phosphate to the response regulator, PhoP. Phosphorylated PhoP (PhoP~P) binds to repeated 6-bp consensus PhoP binding sequences of Pho regulon promoters and activates or represses gene expression. Pho signal transduction systems are part of interconnected signal transduction network involving at least three TCSs (PhoP-PhoR, ResD-ResE TCS, SpoOA phosphorelay), a global carbon metabolism regulator (CcpA), and transition state regulators (AbrB, ScoC). In addition, PhoP-PhoR TCS is cross related with YycF-YycG TCS by cross-regulation. While indescribable progress has been made in understanding phosphate deficiency stress response through refined expression of the Pho regulon in the recent past years, many important questions still remain. Solving these questions may provide important information for application study using B. subtilis.

• Biotechnology and Bioprocess Engineering:BBE
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• v.10 no.2
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• pp.149-154
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• 2005

Secretion of the expressed heterologous proteins can reduce the stress to the host cells and is beneficial to their recovery and purification. In this study, fed-batch cultures of Escherichia coli YK537 (pAET-8) were conducted in a 5-L fermentor for the secretory production of human epidermal growth factor (hEGF) whose expression was under the control of alkaline phosphatase promoter. The effects of feeding of glucose and complex nitrogen sources on hEGF production were investigated. When the fed-batch culture was conducted in a chemically de-fined medium, the cell density was 9.68 g/L and the secreted hEGF was 44.7 mg/L in a period of 60 h. When a complex medium was used and glucose was added in pH-stat mode, the secreted hEGF was improved to 345 mg/L. When the culture was fed with glucose at a constant specific rate of $0.25\;gg^{-1}h^{-1}$, hEGF reached 514 mg/L. The effects of adding a solution containing yeast extract and tryptone were further studied. Different rate of the nitrogen source feeding resulted in different levels of phosphate and acetic acid formation, thus affected hEGF expression. At the optimal feeding rate, hEGF production achieved 686 mg/L.

• Microbiology and Biotechnology Letters
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• v.21 no.3
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• pp.214-220
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• 1993

The mating type a of yeast, Saccharomyces cerevisiae mutant with hmla2-102 and sir3-8ts was changed to type alpha by changing the growth temperature from 25C to 35C. A temperature-sensitive expression vector system was constructed using mating factor alpha1 (Mfalpha1) gene encoding alpha factor which is expressed in the type alpha cells. Vectors with different copy numbers were constructed by joining the promoter and pre or prepro-secretion single sequence of Mfalpha1 to promoterless PHO5' gene as a reporter gene.

• 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.

• Proceedings of the Zoological Society Korea Conference
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• 1999.10b
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• pp.265.1-265
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• 1999

No Abstract, See Full Text

• Journal of Life Science
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• v.19 no.5
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• pp.566-572
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• 2009

A recombinant plasmid, pDH3, was obtained from the genomic library of Serattia marcescens KCTC 2172, and several recombinant subclones constructed from pDH3. The nucleotide sequence of a 5,137 bp segment, pPH4, was determined and three open reading frames were detected. The three ORFs encoded the phosphate specific transport (pst) operon, which was pstC, pstA, and pstB, with the same direction of transcription. Comparison of the pst operon of S. marcescens with that of other organisms revealed that the genes for pstS and phoU were missing. A potential CRP bonding site and pho box sequence was found in the upstream of the putative promoter at the regulatory region. Analysis of the nucleotide sequence showed that homology in amino acid sequences between the PstC protein and Yersinia sp., Vibrio sp., and Pseudomonas sp. were 49, 37 and 33%, respectively. The PstA protein and Yersinia sp., Vibrio sp., and Pseudomonas sp. showed homologies of 64, 51, and 47%, respectively. PstB protein and Methanocaldococcus sp., E. coli, and Mycoplasma sp. showed homologies of 60, 50, and 48%, respectively. The pst genes could be expressed in vivo and positively regulated by cAMP-CRP. The E. coli strain harboring plasmid pPH7, with pst genes, increased with the transport of phosphate.

• Microbiology and Biotechnology Letters
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• v.19 no.1
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• pp.37-44
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• 1991

- I aimed to isolate trans-acting factors involved in the basal expression level of eukaryotic genes. One of the yeast histidine biosynthetic gene, HIS5 was taken as a model for this study. HIS5 gene has a substantial basal level in amino acid rich medium and is derepressed if starved for any single amino acid. The derepression is mediated by cis-acting DNA sequences 5'-TGACTC-3' found in 5' non-transcribed region of the gene and trans-acting factors including GCN4 as positive factor and its negative factor GCDI 7, and GCNZ as a negative factor of GCD17. I first investigated the role of these trans-acting factors in HIS5 basal expression level by using HIS5-pH05 fusion in which expression of pH05 gene encoding inorganic phosphate-repressible acid phosphatase (APase) is regulated by HIS5 promoter. Strain with gcn2 or gcn4 mutation showed 3 to 4 fold lower APase activity than wild type. The level of APase activity was similar in gcn2 and gcn4 mutants. Trans-acting factors involved in basal level were identified by isolating 14 mutants showing increased expression of HISSPH05 fusion from gcn4 background. All the mutants carry a single nuclear recessive mutation and fall into four complementation groups, designated as bell (basal expression level), be12, be23 and be14.

• Journal of The Korean Society of Grassland and Forage Science
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• v.17 no.3
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• pp.285-292
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• 1997

This study was conducted to obtain the transgenic Brnssica napus plants with tobacco Apase gene using the binary vector system of Agrobacteriurn fumefociens. The results obtained were summarized as follows: A repressible acid phosphatase gene of Saccharon~yces cerevisiae, pho105 was used for screening of tobacco Apase cDNA. In order to identify Apase gene in tobacco genome, Southern blot analysis was pcrformed and the Apase gcnc may be present as a single copy, or at most two or three copies, in tobacco genome. To isolate the tobacco Apase gene, tobacco cDNA library was constructed using purifed mRNA from -Pi treated tobacco root and the plaque forming unit of the library was 2.8 x $10^5$ pfu/m${\ell}$, therefore the library might cover all expressed mRNAs. Using pho5 as a probe. tobacco Apase cDNA was cloned, and restriction mapping and Southern blot analysis of cDNA insert were revealed that the 3.6 kb cDNA contained tobacco acid phosphatase cDNA. Plasmid pGA695 -tcAPl was constructed by subcloning tobacco Apase cDNA into the Hind site of pGA695 with 35s promoter which can be expressed constitutively in plants. The Brassica napus cotyledonary petioles were cocultivated with the ,4 grobacteriunz and transferred to the selection medium. The transformed and regenerated plants were transplanted to soil medium. Southern blot analysis was done on the transformed plants, and it was confirmed that a foregin gene was stably integrated into the genonies of B. nnpus plants.