NDLI: Characterization of Saccharomyces cerevisiae promoters for heterologous gene expression in Kluyveromyces marxianus

Content Provider	Springer Nature Link
Author	Lee, Ki Sung Kim, Jun Seob Heo, Paul Yang, Tae Jun Sung, Young Je Cheon, Yuna Koo, Hyun Min Yu, Byung Jo Seo, Jin Ho Jin, Yong Su Park, Jae Chan Kweon, Dae Hyuk
Copyright Year	2012
Abstract	Kluyveromyces marxianus is now considered one of the best choices 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 (P CYC , P TEF , P GPD , and P ADH ) 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 (T CYC ) 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 P GPD > P ADH ∼ P TEF >> P CYC . All promoters except for the P CYC 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 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.
Starting Page	2029
Ending Page	2041
Page Count	13
File Format	PDF
ISSN	01757598
Journal	Applied Microbiology and Biotechnology
Volume Number	97
Issue Number	5
e-ISSN	14320614
Language	English
Publisher	Springer-Verlag
Publisher Date	2012-08-22
Publisher Place	Berlin, Heidelberg
Access Restriction	Subscribed
Subject Keyword	Kluyveromyces marxianus Promoter Strength Stochasticity Flow cytometer Biotechnology Microbiology Microbial Genetics and Genomics
Content Type	Text
Resource Type	Article
Subject	Medicine Applied Microbiology and Biotechnology Biotechnology

Sl.	Authority	Responsibilities	Communication Details
1	Ministry of Education (GoI), Department of Higher Education	Sanctioning Authority	https://www.education.gov.in/ict-initiatives
2	Indian Institute of Technology Kharagpur	Host Institute of the Project: The host institute of the project is responsible for providing infrastructure support and hosting the project	https://www.iitkgp.ac.in
3	National Digital Library of India Office, Indian Institute of Technology Kharagpur	The administrative and infrastructural headquarters of the project	Dr. B. Sutradhar bsutra@ndl.gov.in
4	Project PI / Joint PI	Principal Investigator and Joint Principal Investigators of the project	Dr. B. Sutradhar bsutra@ndl.gov.in Prof. Saswat Chakrabarti will be added soon
5	Website/Portal (Helpdesk)	Queries regarding NDLI and its services	support@ndl.gov.in
6	Contents and Copyright Issues	Queries related to content curation and copyright issues	content@ndl.gov.in
7	National Digital Library of India Club (NDLI Club)	Queries related to NDLI Club formation, support, user awareness program, seminar/symposium, collaboration, social media, promotion, and outreach	clubsupport@ndl.gov.in
8	Digital Preservation Centre (DPC)	Assistance with digitizing and archiving copyright-free printed books	dpc@ndl.gov.in
9	IDR Setup or Support	Queries related to establishment and support of Institutional Digital Repository (IDR) and IDR workshops	idr@ndl.gov.in