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Title: CRISPR EnAbled trackable genome engineering for isopropanol production in Escherichia coli

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 17.5 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 different RBS levels for each gene. The best producing variant, PA14, produced isopropanol at titers of 7.1 g/L at 24 h, with a yield of 0.75 (mol/mol), and maximum productivity of 0.62 g/L/h (which was 0.22 g/L/h above the parentmore » strain PA07). As a result, we demonstrate the ability to rapidly construct and test close to ~1000 designer strains and identify superior performers.« less
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  1. Univ. of Colorado, Boulder, CO (United States)
  2. Muse Biotechnology Inc., Boulder, CO (United States)
  3. National Renewable Energy Lab. (NREL), Golden, CO (United States)
Publication Date:
Report Number(s):
Journal ID: ISSN 1096-7176
Grant/Contract Number:
AC36-08GO28308; FOA-0000996
Accepted Manuscript
Journal Name:
Metabolic Engineering
Additional Journal Information:
Journal Volume: 41; Journal Issue: C; Journal ID: ISSN 1096-7176
Research Org:
National Renewable Energy Lab. (NREL), Golden, CO (United States)
Sponsoring Org:
USDOE Office of Energy Efficiency and Renewable Energy (EERE)
Country of Publication:
United States
09 BIOMASS FUELS; isopropanol; CRISPR EnAbled Trackable genome Engineering; RBS library; engineered Escherichia coli; proteomic analysis
OSTI Identifier:
Alternate Identifier(s):
OSTI ID: 1417503