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CRISPR EnAbled Trackable genome Engineering for isopropanol production in Escherichia coli.pdf
Metabolic Engineering 41 (2017) 1–10
Contents lists available at ScienceDirect
Metabolic Engineering
journal homepage: /locate/ymben
CRISPR EnAbled Trackable genome Engineering for isopropanol production in Escherichia coli
Liya Lianga, Rongming Liua, Andrew D. Garstb, Thomas Leec, Violeta Sànchez i. Noguéd, Gregg T. Beckhamd, Ryan T. Gilla,?
a Renewable and Sustainable Energy Institute (RASEI), University of Colorado Boulder, Boulder, CO 80303, United States b Muse Biotechnology Inc., 4001 Discovery Dr., Boulder, CO 80303, United States c Department of Chemistry and Biochemistry, University of Colorado Boulder, Boulder, CO 80309 United States d National Bioenergy Center, National Renewable Energy Laboratory, Golden, CO 80401, United States
MARK
ARTICLE INFO
Keywords: Isopropanol CRISPR EnAbled Trackable genome Engineering RBS library Engineered Escherichia coli Proteomic analysis
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 di?erent variants of the isopropanol production pathway in Escherichia coli. We ?rst constructed variants to explore the e?ect of codon optimization, copy number, and translation initiation rates on isopropanol production. The best strain (PA06) produced isopropanol at titers of 17.5 g/L, with a yield of 0.62 (mol/mol), and maximum productivity of 0.40 g/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 di?erent RBS l
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