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Title: A Cas9-based toolkit to program gene expression in Saccharomyces cerevisiae

Despite the extensive use of Saccharomyces cerevisiae as a platform for synthetic biology, strain engineering remains slow and laborious. Here, we employ CRISPR/Cas9 technology to build a cloning-free toolkit that addresses commonly encountered obstacles in metabolic engineering, including chromosomal integration locus and promoter selection, as well as protein localization and solubility. The toolkit includes 23 Cas9-sgRNA plasmids, 37 promoters of various strengths and temporal expression profiles, and 10 protein-localization, degradation and solubility tags. We facilitated the use of these parts via a web-based tool, that automates the generation of DNA fragments for integration. Our system builds upon existing gene editing methods in the thoroughness with which the parts are standardized and characterized, the types and number of parts available and the ease with which our methodology can be used to perform genetic edits in yeast. We demonstrated the applicability of this toolkit by optimizing the expression of a challenging but industrially important enzyme, taxadiene synthase (TXS). This approach enabled us to diagnose an issue with TXS solubility, the resolution of which yielded a 25-fold improvement in taxadiene production.
Authors:
 [1] ;  [1] ;  [1] ;  [1] ; ;  [1] ;  [1] ;  [1] ;  [2] ;  [3] ;  [4] ;  [1]
  1. Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Biological Systems and Engineering Division
  2. Univ. of California, Berkeley, CA (United States). Dept. of Chemical and Biomolecular Engineering
  3. Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Biological Systems and Engineering Division; USDOE Joint Genome Institute (JGI), Walnut Creek, CA (United States)
  4. Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Biological Systems and Engineering Division; Univ. of California, Berkeley, CA (United States). Dept. of Chemical and Biomolecular Engineering, Dept. of Bioengineering; Technical Univ. of Denmark, Lyngby (Denmark). Novo Nordisk Foundation Center for Sustainability
Publication Date:
Grant/Contract Number:
AC02-05CH11231
Type:
Accepted Manuscript
Journal Name:
Nucleic Acids Research
Additional Journal Information:
Journal Volume: 45; Journal Issue: 1; Journal ID: ISSN 0305-1048
Publisher:
Oxford University Press
Research Org:
Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
Sponsoring Org:
USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23)
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; 60 APPLIED LIFE SCIENCES; gene expression; plasmids; cloning; chromosomes; DNA; engineering; fluorescence; internet; oncogenes; saccharomyces cerevisiae; solubility; yeasts; enzymes; genetics; intracellular protein transport; catabolism; DNA fragments; crispr; donors; gene editing
OSTI Identifier:
1379656

Reider Apel, Amanda, d'Espaux, Leo, Wehrs, Maren, Sachs, Daniel, Li, Rachel A., Tong, Gary J., Garber, Megan, Nnadi, Oge, Zhuang, William, Hillson, Nathan J., Keasling, Jay D., and Mukhopadhyay, Aindrila. A Cas9-based toolkit to program gene expression in Saccharomyces cerevisiae. United States: N. p., Web. doi:10.1093/nar/gkw1023.
Reider Apel, Amanda, d'Espaux, Leo, Wehrs, Maren, Sachs, Daniel, Li, Rachel A., Tong, Gary J., Garber, Megan, Nnadi, Oge, Zhuang, William, Hillson, Nathan J., Keasling, Jay D., & Mukhopadhyay, Aindrila. A Cas9-based toolkit to program gene expression in Saccharomyces cerevisiae. United States. doi:10.1093/nar/gkw1023.
Reider Apel, Amanda, d'Espaux, Leo, Wehrs, Maren, Sachs, Daniel, Li, Rachel A., Tong, Gary J., Garber, Megan, Nnadi, Oge, Zhuang, William, Hillson, Nathan J., Keasling, Jay D., and Mukhopadhyay, Aindrila. 2016. "A Cas9-based toolkit to program gene expression in Saccharomyces cerevisiae". United States. doi:10.1093/nar/gkw1023. https://www.osti.gov/servlets/purl/1379656.
@article{osti_1379656,
title = {A Cas9-based toolkit to program gene expression in Saccharomyces cerevisiae},
author = {Reider Apel, Amanda and d'Espaux, Leo and Wehrs, Maren and Sachs, Daniel and Li, Rachel A. and Tong, Gary J. and Garber, Megan and Nnadi, Oge and Zhuang, William and Hillson, Nathan J. and Keasling, Jay D. and Mukhopadhyay, Aindrila},
abstractNote = {Despite the extensive use of Saccharomyces cerevisiae as a platform for synthetic biology, strain engineering remains slow and laborious. Here, we employ CRISPR/Cas9 technology to build a cloning-free toolkit that addresses commonly encountered obstacles in metabolic engineering, including chromosomal integration locus and promoter selection, as well as protein localization and solubility. The toolkit includes 23 Cas9-sgRNA plasmids, 37 promoters of various strengths and temporal expression profiles, and 10 protein-localization, degradation and solubility tags. We facilitated the use of these parts via a web-based tool, that automates the generation of DNA fragments for integration. Our system builds upon existing gene editing methods in the thoroughness with which the parts are standardized and characterized, the types and number of parts available and the ease with which our methodology can be used to perform genetic edits in yeast. We demonstrated the applicability of this toolkit by optimizing the expression of a challenging but industrially important enzyme, taxadiene synthase (TXS). This approach enabled us to diagnose an issue with TXS solubility, the resolution of which yielded a 25-fold improvement in taxadiene production.},
doi = {10.1093/nar/gkw1023},
journal = {Nucleic Acids Research},
number = 1,
volume = 45,
place = {United States},
year = {2016},
month = {11}
}