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POT1-mediated δ-integration strategy for high-copy, stable expression of heterologous proteins in Saccharomyces cerevisiae.
- Source :
-
FEMS yeast research [FEMS Yeast Res] 2017 Sep 01; Vol. 17 (6). - Publication Year :
- 2017
-
Abstract
- In biotechnological industry, increased expression cassette stability and copy number serve as important means of maintaining consistently high production levels of heterologous proteins in Saccharomyces cerevisiae. In this study, we combined δ sequences for site-specific integration with TPI1 gene from Schizosaccharomyces pombe (POT1) as a selection marker to realize high-copy integration and stable expression of heterologous proteins in S. cerevisiae. With the newly developed POT1 platform, a 32-copy integration of enhanced green fluorescent protein (EGFP) expression cassette was obtained in a single round and was stably maintained after 100 generations of growth in a rich complex medium. Talaromyces emersonii cellobiohydrolase I gene was synthesized with S. cerevisiae codon bias and expressed under the control of TPI1 promoter and terminator via POT1-mediated δ-integration; the highest specific activity yielded 238 mU g-1 of dry cell weight when p-nitrophenyl-β-D-cellobioside was used as substrate, whereas the highest activity in cellulose hydrolysis reached 67% Avicel conversion. POT1-mediated δ-integration produces high protein levels over a wide dynamic range and enables broad applications in metabolic engineering and synthetic biology.<br /> (© FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)
- Subjects :
- Cellulose 1,4-beta-Cellobiosidase analysis
Cellulose 1,4-beta-Cellobiosidase genetics
Eurotiales enzymology
Eurotiales genetics
Genes, Reporter
Genomic Instability
Green Fluorescent Proteins analysis
Green Fluorescent Proteins genetics
Recombinant Proteins biosynthesis
Recombination, Genetic
Schizosaccharomyces enzymology
Schizosaccharomyces genetics
Gene Dosage
Gene Expression
Promoter Regions, Genetic
Recombinant Proteins genetics
Saccharomyces cerevisiae genetics
Saccharomyces cerevisiae metabolism
Subjects
Details
- Language :
- English
- ISSN :
- 1567-1364
- Volume :
- 17
- Issue :
- 6
- Database :
- MEDLINE
- Journal :
- FEMS yeast research
- Publication Type :
- Academic Journal
- Accession number :
- 28922845
- Full Text :
- https://doi.org/10.1093/femsyr/fox064