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Title: Directed combinatorial mutagenesis of Escherichia coli for complex phenotype engineering

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. As a result, 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.
Authors:
 [1] ;  [1] ;  [1] ;  [1] ;  [2] ;  [2] ;  [1]
  1. Univ. of Colorado, Boulder, CO (United States)
  2. National Renewable Energy Lab. (NREL), Golden, CO (United States)
Publication Date:
Report Number(s):
NREL/JA-5100-71271
Journal ID: ISSN 1096-7176
Grant/Contract Number:
AC36-08GO28308
Type:
Accepted Manuscript
Journal Name:
Metabolic Engineering
Additional Journal Information:
Journal Volume: 47; Journal Issue: C; Journal ID: ISSN 1096-7176
Publisher:
Elsevier
Research Org:
National Renewable Energy Lab. (NREL), Golden, CO (United States)
Sponsoring Org:
USDOE Office of Energy Efficiency and Renewable Energy (EERE), Bioenergy Technologies Office (EE-3B)
Country of Publication:
United States
Language:
English
Subject:
09 BIOMASS FUELS; Iterative CRISPR EnAbled Trackable genome; engineering; genome engineering; combinatorial mutagenesis; lignocellulosic biomass
OSTI Identifier:
1432610

Liu, Rongming, Liang, Liya, Garst, Andrew D., Choudhury, Alaksh, Nogue, Violeta Sanchez i., Beckham, Gregg T., and Gill, Ryan T.. Directed combinatorial mutagenesis of Escherichia coli for complex phenotype engineering. United States: N. p., Web. doi:10.1016/j.ymben.2018.02.007.
Liu, Rongming, Liang, Liya, Garst, Andrew D., Choudhury, Alaksh, Nogue, Violeta Sanchez i., Beckham, Gregg T., & Gill, Ryan T.. Directed combinatorial mutagenesis of Escherichia coli for complex phenotype engineering. United States. doi:10.1016/j.ymben.2018.02.007.
Liu, Rongming, Liang, Liya, Garst, Andrew D., Choudhury, Alaksh, Nogue, Violeta Sanchez i., Beckham, Gregg T., and Gill, Ryan T.. 2018. "Directed combinatorial mutagenesis of Escherichia coli for complex phenotype engineering". United States. doi:10.1016/j.ymben.2018.02.007.
@article{osti_1432610,
title = {Directed combinatorial mutagenesis of Escherichia coli for complex phenotype engineering},
author = {Liu, Rongming and Liang, Liya and Garst, Andrew D. and Choudhury, Alaksh and Nogue, Violeta Sanchez i. and Beckham, Gregg T. and Gill, Ryan T.},
abstractNote = {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. As a result, 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.},
doi = {10.1016/j.ymben.2018.02.007},
journal = {Metabolic Engineering},
number = C,
volume = 47,
place = {United States},
year = {2018},
month = {3}
}