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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.

147 2012    Construction and characterization of recombinant Bacillus subtilis JY123 able to transport xylose efficiently
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Journal of biotechnology 2012 161(4):402-406
2012/08/10  2821
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  3270
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2011/11/28  2859
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