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Subject   Characterization of Saccharomyces cerevisiae promoters for heterologous gene expression in Kluyveromyces marxianus
Dae-Hyuk Kweon et al.
Applied Microbiology and Biotechnology 2013 97 (5):2029–2041
Characterization of Saccharomyces cerevisiae promoters for heterologous gene expression in Kluyveromyces marxianus

Ki-Sung Lee; Jun-Seob Kim; Paul Heo; Tae-Jun Yang; Young-Je Sung; Yuna Cheon; Hyun Min Koo; Byung Jo Yu; Jin-Ho Seo; Yong-Su Jin; Jae Chan Park; Dae-Hyuk Kweon

Applied Microbiology and Biotechnology 2013 97 (5):2029–2041

ABSTRACT: Kluyveromyces marxianus is now considered one of the best choice of option for
industrial applications of yeast because the strain is able to grow at high temperature, utilizes
various carbon sources and grows fast. However, the use of K. marxianus as a host for
industrial applications is still limited. This limitation is largely due to a lack of knowledge on
the characteristics of the promoters since the time and amount of protein expression is
strongly dependent on the promoter employed. In this study, four well-known constitutive
promoters (PCYC, PTEF, PGPD and PADH) of Saccharomyces cerevisiae were characterized in
K. marxianus in terms of protein expression level and their stochastic behavior. After
constructing five URA3-auxotrophic K. marxianus strains and a plasmid vector, four cassettes
each comprising one of the promoters-the gene for the green fluorescence protein (GFP)-CYC1
terminator (TCYC) were inserted into the vector. GFP expression under the control of each
one of the promoters was analyzed by reverse transcription PCR, fluorescence microscopy
and flow cytometer. Using these combined methods, the promoter strength was
determined to be in the order of PGPD>PADH~PTEF>>PCYC. All promoters except for the PCYC
exhibited three distinctive populations, including non-expressing cells, weakly-expressing cells
and strongly-expressing cells. The relative ratios between populations were strongly
dependent on the promoter and culture time. Forward scattering was independent of GFP
fluorescence intensity, indicating that the different fluorescence intensities were not just
due to different cell sizes derived from budding. It was also excluded the possibility that
the non-expressing cells resulted from plasmid loss because plasmid stability was maintained
at almost 100% over the culture time. The same cassettes, cloned into a single copy plasmid
pRS416 and transformed into S. cerevisiae, showed only one population. When the cassettes
were integrated into the chromosome, the stochastic behavior was markedly reduced.
These combined results imply that the gene expression stochasticity should be overcome
in order to use this strain for delicate metabolic engineering, which would require the
co-expression of several genes.




No
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Subject
Name
Date
Hit
2012    Characterization of Saccharomyces cerevisiae promoters for heterologous gene expression in Kluyveromyces marxianus
Dae-Hyuk Kweon et al.
Applied Microbiology and Biotechnology 2013 97 (5):2029–2041
2012/07/16  3150
145 2012    Production of resveratrol from tyrosine in metabolically engineered Saccharomyces cerevisiae
So-Yeon Shin et al.
Enzyme and Microbial Technology 2012 51(4);211–216
2012/06/27  2674
144 2012    Effects of Trx2p and Sec23p expression on stable production of hepatitis B surface antigen S domain in recombinant Saccharomyces cerevisiae
Young-Kyoung Park et al.
Journal of Biotechnology 2012 160:151-160
2012/05/08  2607
143 2012    Aqueous ammonia pretreatment, saccharification, and fermentation evaluation of oil palm fronds for ethanol production
Kyoung Heon Kim et al.
Bioprocess and Biosystems Engineering 2012 35(9):1497-1503
2012/04/19  2879
142 2012    Whole cell biosynthesis of a functional oligosaccharide, 2-fucosyllactose, using engineered Escherichia coli
Won-Heong Lee et al.
Microbial Cell Factories 2012 11:48
2012/04/18  2783
141 2012    Electrostatic interaction-induced inclusion body formation of glucagon-like peptide-1 fused with ubiquitin and cationic tag
Sung-Gun Kim et al.
Protein Expression and Purification 2012 84(1):38-46
2012/04/17  2592
140 2012    pH-responsive high-density lipoprotein-like nanoparticles release paclitaxel at acidic pH in cancer chemotherapy
Sung-Gun Kim, Dae-Hyuk Kweon et al.
International Journal of Nanomedicine 2012:7 1–12
2012/04/16  2201
139 2012    Isobutanol production in engineered Saccharomyces scerevisiae by overexpression of 2-ketoisovalerate decarboxylase and valine biosynthetic enzymes
Won-Heong Lee et al.
Bioprocess and Biosystems Engineering 2012 35(9
2012/04/10  2135
138 2012    Modulation of guanosine nucleotides biosynthetic pathways enhanced GDP-L-fucose production in recombinant Escherichia coli
Won-Heong Lee et al.
Applied microbiology and biotechnology 2012 93:2327-2334
2011/11/28  2756
137 2011    Biotechnological production of human milk oligosaccharides
Nam Soo Han et al.
Biotechnology Advances 2012 30:1268-1278
2011/11/09  2975
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