CRISPR EnAbled Trackable Genome Engineering for Isopropanol Production in Escherichia coli

Violeta Sanchez i Nogue, Gregg Beckham, Liya Liang, Rongming Liu, Andrew Garst, Thomas Lee, Ryan Gill

Research output: Contribution to journalArticlepeer-review

79 Scopus Citations


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 parent strain PA07). We demonstrate the ability to rapidly construct and test close to ~1000 designer strains and identify superior performers.

Original languageAmerican English
Pages (from-to)1-10
Number of pages10
JournalMetabolic Engineering
StatePublished - 2017

Bibliographical note

Publisher Copyright:
© 2017

NREL Publication Number

  • NREL/JA-5100-68055


  • CRISPR EnAbled Trackable genome Engineering
  • Engineered Escherichia coli
  • Isopropanol
  • Proteomic analysis
  • RBS library


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