NDLI: Modulating heterologous protein production in yeast: the applicability of truncated auxotrophic markers

Content Provider	Springer Nature Link
Author	Kazemi Seresht, Ali Nørgaard, Per Palmqvist, Eva Akke Andersen, Asser Sloth Olsson, Lisbeth
Copyright Year	2012
Abstract	The use of auxotrophic Saccharomyces cerevisiae strains for improved production of a heterologous protein was examined. Two different marker genes were investigated, encoding key enzymes in the metabolic pathways for amino acid (LEU2) and pyrimidine (URA3) biosynthesis, respectively. Expression plasmids, carrying the partly defective selection markers LEU2d and URA3d, were constructed. Two CEN.PK-derived strains were chosen and insulin analogue precursor was selected as a model protein. Different truncations of the LEU2 and URA3 promoters were used as the mean to titrate the plasmid copy number and thus the recombinant gene dosage in order to improve insulin productivity. Experiments were initially carried out in batch mode to examine the stability of yeast transformants and to select high yielding mutants. Next, chemostat cultivations were run at high cell density to address industrial applicability and long-term expression stability of the transformants. We found that the choice of auxotrophic marker is crucial for developing a yeast expression system with stable heterologous protein production. The incremental truncation of the URA3 promoter led to higher plasmid copy numbers and IAP yields, whereas the truncation of the LEU2 promoter caused low plasmid stability. We show that the modification of the level of the recombinant gene dosage by varying the degree of promoter truncation can be a strong tool for optimization of productivity. The application of the URA3d-based expression systems showed a high potential for industrial protein production and for further academic studies.
Starting Page	3939
Ending Page	3948
Page Count	10
File Format	PDF
ISSN	01757598
Journal	Applied Microbiology and Biotechnology
Volume Number	97
Issue Number	9
e-ISSN	14320614
Language	English
Publisher	Springer-Verlag
Publisher Date	2012-07-11
Publisher Place	Berlin, Heidelberg
Access Restriction	Subscribed
Subject Keyword	Promoter truncation URA3d LEU2d Human insulin Plasmid copy number High cell density cultivation 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

Correlation of cell growth and heterologous protein production by Saccharomyces cerevisiae

The first auxotrophic mutant of Zygosaccharomyces bailii for recombinant productions: a road to practical applications

A novel yeast expression system based on a hormone-induced transcriptional cascade

Stable overproducer of hepatitis B surface antigen in the methylotrophic yeast Hansenula polymorpha due to multiple integration of heterologous auxotrophic selective markers and defect in peroxisome biogenesis

Substrate feeding strategies in Pichia pastoris fed-batch cultivation processes: Analysis of key parameters influencing recombinant protein production

The impact of phosphate scarcity on pharmaceutical protein production in S. cerevisiae: linking transcriptomic insights to phenotypic responses

Use of a "universal" yeast vector (CoMed™) system for the production of proteins in Hansenula polymorpha and Arxula adeninivorans

Gene copy number and polyploidy on products formation in yeast

Controlling heterologous gene expression in yeast cell factories on different carbon substrates and across the diauxic shift: a comparison of yeast promoter activities

Modulating heterologous protein production in yeast: the applicability of truncated auxotrophic markers

Similar Documents

Correlation of cell growth and heterologous protein production by Saccharomyces cerevisiae

The first auxotrophic mutant of Zygosaccharomyces bailii for recombinant productions: a road to practical applications

A novel yeast expression system based on a hormone-induced transcriptional cascade

Stable overproducer of hepatitis B surface antigen in the methylotrophic yeast Hansenula polymorpha due to multiple integration of heterologous auxotrophic selective markers and defect in peroxisome biogenesis

Substrate feeding strategies in Pichia pastoris fed-batch cultivation processes: Analysis of key parameters influencing recombinant protein production

The impact of phosphate scarcity on pharmaceutical protein production in S. cerevisiae: linking transcriptomic insights to phenotypic responses

Use of a "universal" yeast vector (CoMed™) system for the production of proteins in Hansenula polymorpha and Arxula adeninivorans

Gene copy number and polyploidy on products formation in yeast

Controlling heterologous gene expression in yeast cell factories on different carbon substrates and across the diauxic shift: a comparison of yeast promoter activities

Modulating heterologous protein production in yeast: the applicability of truncated auxotrophic markers