Actions for A Cas9-based toolkit to program gene expression in <i>Saccharomyces cerevisiae<

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A Cas9-based toolkit to program gene expression in <i>Saccharomyces cerevisiae</i> [electronic resource].

Published
Washington, D.C. : United States. Dept. of Energy. Office of Science, 2016.
Oak Ridge, Tenn. : Distributed by the Office of Scientific and Technical Information, U.S. Dept. of Energy
Physical Description
pages 496-508 : digital, PDF file
Additional Creators
Lawrence Berkeley National Laboratory, United States. Department of Energy. Office of Science, and United States. Department of Energy. Office of Scientific and Technical Information
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Summary
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.
Report Numbers
E 1.99:1379656
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Note
Published through SciTech Connect.
11/28/2016.
"ark:/13030/qt1t03r1tq"
Nucleic Acids Research 45 1 ISSN 0305-1048 AM
Amanda Reider Apel; Leo d'Espaux; Maren Wehrs; Daniel Sachs; Li, Rachel; Gary J. Tong; Megan Garber; Oge Nnadi; William Zhuang; Nathan J. Hillson; Jay D. Keasling; Aindrila Mukhopadhyay.
Funding Information
AC02-05CH11231
View MARC record | catkey: 23493853