title: CRISPR EnAbled Trackable genome Engineering for isopropanol production in Escherichia coli.
abstract: Isopropanol is an important target molecule for sustainable production of fuels and chemicals. Increases in DNA synthesis and synthetic biology capabilities have resulted in the development of a range of new strategies for the more rapid design, construction, and testing of production strains. Here, we report on the use of such capabilities to construct and test 903 different variants of the isopropanol production pathway in Escherichia coli. We first constructed variants to explore the effect of codon optimization, copy number, and translation initiation rates on isopropanol production. The best strain (PA06) produced isopropanol at titers of 17.5g/L, with a yield of 0.62 (mol/mol), and maximum productivity of 0.40g/L/h. We next integrated the isopropanol synthetic pathway into the genome and then used the CRISPR EnAbled Trackable genome Engineering (CREATE) strategy to generate an additional 640 individual RBS library variants for further evaluation. After testing each of these variants, we constructed a combinatorial library containing 256 total variants from four different RBS levels for each gene. The best producing variant, PA14, produced isopropanol at titers of 7.1g/L at 24h, with a yield of 0.75 (mol/mol), and maximum productivity of 0.62g/L/h (which was 0.22g/L/h above the parent strain PA07). We demonstrate the ability to rapidly construct and test close to ~1000 designer strains and identify superior performers.
authors.affiliation: Renewable and Sustainable Energy Institute (RASEI), University of Colorado Boulder, Boulder, CO 80303, United States.
authors.forename: Liya
authors.initials: L
authors.lastname: Liang
authors.affiliation: Renewable and Sustainable Energy Institute (RASEI), University of Colorado Boulder, Boulder, CO 80303, United States.
authors.forename: Rongming
authors.initials: R
authors.lastname: Liu
authors.affiliation: Muse Biotechnology Inc., 4001 Discovery Dr., Boulder, CO 80303, United States.
authors.forename: Andrew D
authors.initials: AD
authors.lastname: Garst
authors.affiliation: Department of Chemistry and Biochemistry, University of Colorado Boulder, Boulder, CO 80309 United States.
authors.forename: Thomas
authors.initials: T
authors.lastname: Lee
authors.affiliation: National Bioenergy Center, National Renewable Energy Laboratory, Golden, CO 80401, United States.
authors.forename: Violeta Sànchez I
authors.initials: VSI
authors.lastname: Nogué
authors.affiliation: National Bioenergy Center, National Renewable Energy Laboratory, Golden, CO 80401, United States.
authors.forename: Gregg T
authors.initials: GT
authors.lastname: Beckham
authors.affiliation: Renewable and Sustainable Energy Institute (RASEI), University of Colorado Boulder, Boulder, CO 80303, United States. Electronic address: rtg@colorado.edu.
authors.forename: Ryan T
authors.initials: RT
authors.lastname: Gill
mesh_terms: D019840:2-Propanol / Q000378:metabolism*; D064113:CRISPR-Cas Systems; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D000072669:Gene Editing / Q000379:methods*; D060847:Metabolic Engineering / Q000379:methods*
chemical_list: D019840:2-Propanol
keywords: CRISPR EnAbled Trackable genome Engineering; Engineered Escherichia coli; Isopropanol; Proteomic analysis; RBS library