Directed Combinatorial Mutagenesis of Escherichia coli for Complex Phenotype Engineering

Rongming Liu, Liya Liang, Andrew D. Garst, Alaksh Choudhury, Violeta Sànchez i. Nogué, Gregg T. Beckham, Ryan T. Gill

Research output: Contribution to journalArticlepeer-review

26 Scopus Citations


Strain engineering for industrial production requires a targeted improvement of multiple complex traits, which range from pathway flux to tolerance to mixed sugar utilization. Here, we report the use of an iterative CRISPR EnAbled Trackable genome Engineering (iCREATE) method to engineer rapid glucose and xylose co-consumption and tolerance to hydrolysate inhibitors in E. coli. Deep mutagenesis libraries were rationally designed, constructed, and screened to target ~40,000 mutations across 30 genes. These libraries included global and high-level regulators that regulate global gene expression, transcription factors that play important roles in genome-level transcription, enzymes that function in the sugar transport system, NAD(P)H metabolism, and the aldehyde reduction system. Specific mutants that conferred increased growth in mixed sugars and hydrolysate tolerance conditions were isolated, confirmed, and evaluated for changes in genome-wide expression levels. We tested the strain with positive combinatorial mutations for 3-hydroxypropionic acid (3HP) production under high furfural and high acetate hydrolysate fermentation, which demonstrated a 7- and 8-fold increase in 3HP productivity relative to the parent strain, respectively.

Original languageAmerican English
Pages (from-to)10-20
Number of pages11
JournalMetabolic Engineering
StatePublished - 2018

Bibliographical note

Publisher Copyright:
© 2018

NREL Publication Number

  • NREL/JA-5100-71281


  • Combinatorial mutagenesis
  • Genome engineering
  • Iterative CRISPR EnAbled Trackable genome Engineering
  • Lignocellulosic biomass


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