title: A Cas9-based toolkit to program gene expression in Saccharomyces cerevisiae.
abstract: 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.affiliation: DOE Joint BioEnergy Institute, Emeryville, California, CA 94608, USA.|Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, California, CA 94720, USA.
authors.forename: Amanda
authors.initials: A
authors.lastname: Reider Apel
authors.affiliation: DOE Joint BioEnergy Institute, Emeryville, California, CA 94608, USA.|Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, California, CA 94720, USA.
authors.forename: Leo
authors.initials: L
authors.lastname: d'Espaux
authors.affiliation: DOE Joint BioEnergy Institute, Emeryville, California, CA 94608, USA.|Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, California, CA 94720, USA.
authors.forename: Maren
authors.initials: M
authors.lastname: Wehrs
authors.affiliation: DOE Joint BioEnergy Institute, Emeryville, California, CA 94608, USA.|Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, California, CA 94720, USA.
authors.forename: Daniel
authors.initials: D
authors.lastname: Sachs
authors.affiliation: DOE Joint BioEnergy Institute, Emeryville, California, CA 94608, USA.|Department of Plant and Microbial Biology, University of California, Berkeley, California, CA 94720, USA.
authors.forename: Rachel A
authors.initials: RA
authors.lastname: Li
authors.affiliation: DOE Joint BioEnergy Institute, Emeryville, California, CA 94608, USA.|Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, California, CA 94720, USA.
authors.forename: Gary J
authors.initials: GJ
authors.lastname: Tong
authors.affiliation: DOE Joint BioEnergy Institute, Emeryville, California, CA 94608, USA.|Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, California, CA 94720, USA.
authors.forename: Megan
authors.initials: M
authors.lastname: Garber
authors.affiliation: DOE Joint BioEnergy Institute, Emeryville, California, CA 94608, USA.|Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, California, CA 94720, USA.
authors.forename: Oge
authors.initials: O
authors.lastname: Nnadi
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of California, Berkeley, California, CA 94720, USA.
authors.forename: William
authors.initials: W
authors.lastname: Zhuang
authors.affiliation: DOE Joint BioEnergy Institute, Emeryville, California, CA 94608, USA.|Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, California, CA 94720, USA.|DOE Joint Genome Institute, Walnut Creek, California, CA 94598, USA.
authors.forename: Nathan J
authors.initials: NJ
authors.lastname: Hillson
authors.affiliation: DOE Joint BioEnergy Institute, Emeryville, California, CA 94608, USA.|Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, California, CA 94720, USA.|Department of Chemical and Biomolecular Engineering, University of California, Berkeley, California, CA 94720, USA.|DOE Joint Genome Institute, Walnut Creek, California, CA 94598, USA.|The Novo Nordisk Foundation Center for Sustainability, Technical University of Denmark, 2800 Kgs. Lyngby, Denmark.
authors.forename: Jay D
authors.initials: JD
authors.lastname: Keasling
authors.affiliation: DOE Joint BioEnergy Institute, Emeryville, California, CA 94608, USA amukhopadhyay@lbl.gov.|Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, California, CA 94720, USA.
authors.forename: Aindrila
authors.initials: A
authors.lastname: Mukhopadhyay
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D000076987:CRISPR-Associated Protein 9; D064113:CRISPR-Cas Systems; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D004271:DNA, Fungal / Q000235:genetics* / Q000378:metabolism; D004720:Endonucleases / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D005818:Genetic Engineering / Q000379:methods*; D007535:Isomerases / Q000235:genetics / Q000378:metabolism; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism; D011401:Promoter Regions, Genetic; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D012984:Software
chemical_list: D001426:Bacterial Proteins; D004271:DNA, Fungal; D000094704:RNA, Guide, CRISPR-Cas Systems; D000076987:CRISPR-Associated Protein 9; C000606107:Cas9 endonuclease Streptococcus pyogenes; D004720:Endonucleases; D007535:Isomerases; C095926:taxa-4(5),11(12)-diene synthase