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

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

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Abstract

Despite the extensive use of Saccharomyces cere-visiae 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.
Original languageEnglish
JournalNucleic acids research
Volume45
Issue number1
Pages (from-to)496-508
ISSN0305-1048
DOIs
Publication statusPublished - 2017

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