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

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

doi:10.1093/nar/gkw1023

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

Journal Article · Mon Nov 28 00:00:00 EST 2016 · Nucleic Acids Research

DOI:https://doi.org/10.1093/nar/gkw1023· OSTI ID:1379656

Reider Apel, Amanda ^[1]; d'Espaux, Leo ^[1]; Wehrs, Maren ^[1]; Sachs, Daniel ^[1]; Li, Rachel A.; Tong, Gary J. ^[1]; Garber, Megan ^[1]; Nnadi, Oge ^[1]; Zhuang, William ^[2]; Hillson, Nathan J. ^[3]; Keasling, Jay D. ^[4]; Mukhopadhyay, Aindrila ^[1]

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
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)
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

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.

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Research Organization:: Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)

Sponsoring Organization:: USDOE Office of Science (SC), Biological and Environmental Research (BER)

Grant/Contract Number:: AC02-05CH11231

OSTI ID:: 1379656

Journal Information:: Nucleic Acids Research, Vol. 45, Issue 1; ISSN 0305-1048

Publisher:: Oxford University PressCopyright Statement

Country of Publication:: United States

Language:: English

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Cited by: 159 works

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59 BASIC BIOLOGICAL SCIENCES
60 APPLIED LIFE SCIENCES
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solubility
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Title: A Cas9-based toolkit to program gene expression in Saccharomyces cerevisiae

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