Search Menu
DOE PAGES title logo U.S. Department of Energy
Office of Scientific and Technical Information
Search Scholarly Publications

Title: Iterative genome editing of Escherichia coli for 3-hydroxypropionic acid production

Abstract

Synthetic biology requires strategies for the targeted, efficient, and combinatorial engineering of biological sub-systems at the molecular level. Here, we report the use of the iterative CRISPR EnAbled Trackable genome Engineering (iCREATE) method for the rapid construction of combinatorially modified genomes. We coupled this genome engineering strategy with high-throughput phenotypic screening and selections to recursively engineer multiple traits in Escherichia coli for improved production of the platform chemical 3- hydroxypropionic acid (3HP). Particularly, we engineered i) central carbon metabolism, ii) 3HP synthesis, and (iii) 3HP tolerance through design, construction and testing of ~162,000 mutations across 115 genes spanning global regulators, transcription factors, and enzymes involved in 3HP synthesis and tolerance. The iCREATE process required ~1 month to perform 13 rounds of combinatorial genome modifications with targeted gene knockouts, expression modification by ribosomal binding site (RBS) engineering, and genome-level site-saturation mutagenesis. Specific mutants conferring increased 3HP titer, yield, and productivity were identified and then combined to produce 3HP at a yield and concentration ~60-fold higher than the wild-type strain.

Authors:
 [1];  [1];  [1];  [1];  [1];  [1];  [1]
+ Show Author Affiliations
  1. Univ. of Colorado, Boulder, CO (United States)
Publication Date:
Research Org.:
Univ. of Colorado, Boulder, CO (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Biological and Environmental Research (BER)
OSTI Identifier:
1548398
Alternate Identifier(s):
OSTI ID: 1582780
Grant/Contract Number:  
SC0018368
Resource Type:
Accepted Manuscript
Journal Name:
Metabolic Engineering
Additional Journal Information:
Journal Volume: 47; Journal Issue: C; Journal ID: ISSN 1096-7176
Publisher:
Elsevier
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; Genome engineering; Combinatorial engineering; 3-hydroxypropionic acid; High-throughput

Citation Formats

Liu, Rongming, Liang, Liya, Choudhury, Alaksh, Bassalo, Marcelo C., Garst, Andrew D., Tarasava, Katia, and Gill, Ryan T. Iterative genome editing of Escherichia coli for 3-hydroxypropionic acid production. United States: N. p., 2018. Web. doi:10.1016/j.ymben.2018.04.007.
Copy to clipboard
Liu, Rongming, Liang, Liya, Choudhury, Alaksh, Bassalo, Marcelo C., Garst, Andrew D., Tarasava, Katia, & Gill, Ryan T. Iterative genome editing of Escherichia coli for 3-hydroxypropionic acid production. United States. https://doi.org/10.1016/j.ymben.2018.04.007
Copy to clipboard
Liu, Rongming, Liang, Liya, Choudhury, Alaksh, Bassalo, Marcelo C., Garst, Andrew D., Tarasava, Katia, and Gill, Ryan T. Sat . "Iterative genome editing of Escherichia coli for 3-hydroxypropionic acid production". United States. https://doi.org/10.1016/j.ymben.2018.04.007. https://www.osti.gov/servlets/purl/1548398.
Copy to clipboard
@article{osti_1548398,
title = {Iterative genome editing of Escherichia coli for 3-hydroxypropionic acid production},
author = {Liu, Rongming and Liang, Liya and Choudhury, Alaksh and Bassalo, Marcelo C. and Garst, Andrew D. and Tarasava, Katia and Gill, Ryan T.},
abstractNote = {Synthetic biology requires strategies for the targeted, efficient, and combinatorial engineering of biological sub-systems at the molecular level. Here, we report the use of the iterative CRISPR EnAbled Trackable genome Engineering (iCREATE) method for the rapid construction of combinatorially modified genomes. We coupled this genome engineering strategy with high-throughput phenotypic screening and selections to recursively engineer multiple traits in Escherichia coli for improved production of the platform chemical 3- hydroxypropionic acid (3HP). Particularly, we engineered i) central carbon metabolism, ii) 3HP synthesis, and (iii) 3HP tolerance through design, construction and testing of ~162,000 mutations across 115 genes spanning global regulators, transcription factors, and enzymes involved in 3HP synthesis and tolerance. The iCREATE process required ~1 month to perform 13 rounds of combinatorial genome modifications with targeted gene knockouts, expression modification by ribosomal binding site (RBS) engineering, and genome-level site-saturation mutagenesis. Specific mutants conferring increased 3HP titer, yield, and productivity were identified and then combined to produce 3HP at a yield and concentration ~60-fold higher than the wild-type strain.},
doi = {10.1016/j.ymben.2018.04.007},
journal = {Metabolic Engineering},
number = C,
volume = 47,
place = {United States},
year = {Sat Apr 14 00:00:00 EDT 2018},
month = {Sat Apr 14 00:00:00 EDT 2018}
}
Copy to clipboard
Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record
https://doi.org/10.1016/j.ymben.2018.04.007
Copyright Statement
Other availability
Search WorldCat Search WorldCat to find libraries that may hold this journal
Citation Metrics:
Cited by: 25 works
Citation information provided by
Web of Science
Save / Share:
Export Metadata
Save to My Library
You must Sign In or Create an Account in order to save documents to your library.

Works referenced in this record:

Sequencing and overexpression of the Escherichia coli aroE gene encoding shikimate dehydrogenase
journal, January 1988

  • Anton, I. A.; Coggins, J. R.
  • Biochemical Journal, Vol. 249, Issue 2
  • DOI: 10.1042/bj2490319

Rapid and Efficient One-Step Metabolic Pathway Integration in E. coli
journal, April 2016

  • Bassalo, Marcelo C.; Garst, Andrew D.; Halweg-Edwards, Andrea L.
  • ACS Synthetic Biology, Vol. 5, Issue 7
  • DOI: 10.1021/acssynbio.5b00187

Establishing a synthetic pathway for high-level production of 3-hydroxypropionic acid in Saccharomyces cerevisiae via β-alanine
journal, January 2015

Enhanced production of 3-hydroxypropionic acid from glucose via malonyl-CoA pathway by engineered Escherichia coli
journal, January 2016

Biorefineries for the production of top building block chemicals and their derivatives
journal, March 2015

A set of recombineering plasmids for gram-negative bacteria
journal, September 2006

Efficient search, mapping, and optimization of multi-protein genetic systems in diverse bacteria
journal, June 2014

  • Farasat, I.; Kushwaha, M.; Collens, J.
  • Molecular Systems Biology, Vol. 10, Issue 6, p. 731-731
  • DOI: 10.15252/msb.20134955

Saturation editing of genomic regions by multiplex homology-directed repair
journal, September 2014

  • Findlay, Gregory M.; Boyle, Evan A.; Hause, Ronald J.
  • Nature, Vol. 513, Issue 7516, p. 120-123
  • DOI: 10.1038/nature13695

A new pathway for polyketide synthesis in microorganisms
journal, August 1999

  • Funa, Nobutaka; Ohnishi, Yasuo; Fujii, Isao
  • Nature, Vol. 400, Issue 6747
  • DOI: 10.1038/23748

Enzymatic assembly of DNA molecules up to several hundred kilobases
journal, April 2009

  • Gibson, Daniel G.; Young, Lei; Chuang, Ray-Yuan
  • Nature Methods, Vol. 6, Issue 5, p. 343-345
  • DOI: 10.1038/nmeth.1318

RNA-guided editing of bacterial genomes using CRISPR-Cas systems
journal, January 2013

  • Jiang, Wenyan; Bikard, David; Cox, David
  • Nature Biotechnology, Vol. 31, Issue 3, p. 233-239
  • DOI: 10.1038/nbt.2508

Multigene Editing in the Escherichia coli Genome via the CRISPR-Cas9 System
journal, January 2015

  • Jiang, Yu; Chen, Biao; Duan, Chunlan
  • Applied and Environmental Microbiology, Vol. 81, Issue 7, p. 2506-2514
  • DOI: 10.1128/AEM.04023-14

Production of polyhydroxyalkanoates by Escherichia coli mutants with defected mixed acid fermentation pathways
journal, June 2010

  • Jian, Jia; Zhang, Shao-Qin; Shi, Zhen-Yu
  • Applied Microbiology and Biotechnology, Vol. 87, Issue 6
  • DOI: 10.1007/s00253-010-2706-0

Evolution reveals a glutathione-dependent mechanism of 3-hydroxypropionic acid tolerance
journal, November 2014

Engineering and systems-level analysis of Saccharomyces cerevisiae for production of 3-hydroxypropionic acid via malonyl-CoA reductase-dependent pathway
journal, March 2016

  • Kildegaard, Kanchana R.; Jensen, Niels B.; Schneider, Konstantin
  • Microbial Cell Factories, Vol. 15, Issue 1
  • DOI: 10.1186/s12934-016-0451-5

Production of 3-hydroxypropionic acid from glucose and xylose by metabolically engineered Saccharomyces cerevisiae
journal, December 2015

Acetate and Formate Stress: Opposite Responses in the Proteome of Escherichia coli
journal, November 2001

Escherichia coli Peptide Binding Protein OppA Has a Preference for Positively Charged Peptides
journal, November 2011

Enhanced poly(3-hydroxypropionate) production via β-alanine pathway in recombinant Escherichia coli
journal, March 2017

Systems metabolic engineering of microorganisms for natural and non-natural chemicals
journal, May 2012

  • Lee, Jeong Wook; Na, Dokyun; Park, Jong Myoung
  • Nature Chemical Biology, Vol. 8, Issue 6
  • DOI: 10.1038/nchembio.970

Development of a 3-hydroxypropionate resistant Escherichia coli strain
journal, January 2016

SCALEs: multiscale analysis of library enrichment
journal, November 2006

  • Lynch, Michael D.; Warnecke, Tanya; Gill, Ryan T.
  • Nature Methods, Vol. 4, Issue 1
  • DOI: 10.1038/nmeth946

Identifying global regulators in transcriptional regulatory networks in bacteria
journal, October 2003

  • Martı́nez-Antonio, Agustino; Collado-Vides, Julio
  • Current Opinion in Microbiology, Vol. 6, Issue 5
  • DOI: 10.1016/j.mib.2003.09.002

Genome-Wide Identification of Transcription Start Sites, Promoters and Transcription Factor Binding Sites in E. coli
journal, October 2009

Rational design of a synthetic Entner–Doudoroff pathway for improved and controllable NADPH regeneration
journal, May 2015

Synergies between synthetic biology and metabolic engineering
journal, August 2011

  • Nielsen, Jens; Keasling, Jay D.
  • Nature Biotechnology, Vol. 29, Issue 8
  • DOI: 10.1038/nbt.1937

Mutations in Escherichia coli aceE and ribB Genes Allow Survival of Strains Defective in the First Step of the Isoprenoid Biosynthesis Pathway
journal, August 2012

Production of 3-hydroxypropionic acid via malonyl-CoA pathway using recombinant Escherichia coli strains
journal, February 2012

  • Rathnasingh, Chelladurai; Raj, Subramanian Mohan; Lee, Youjin
  • Journal of Biotechnology, Vol. 157, Issue 4, p. 633-640
  • DOI: 10.1016/j.jbiotec.2011.06.008

The no-SCAR (Scarless Cas9 Assisted Recombineering) system for genome editing in Escherichia coli
journal, October 2015

  • Reisch, Chris R.; Prather, Kristala L. J.
  • Scientific Reports, Vol. 5, Issue 1
  • DOI: 10.1038/srep15096

Perturbation of Anion Balance during Inhibition of Growth of Escherichia coli by Weak Acids
journal, February 1998

CRMAGE: CRISPR Optimized MAGE Recombineering
journal, January 2016

  • Ronda, Carlotta; Pedersen, Lasse Ebdrup; Sommer, Morten O. A.
  • Scientific Reports, Vol. 6, Article No. 19452
  • DOI: 10.1038/srep19452

Strategy for directing combinatorial genome engineering in Escherichia coli
journal, June 2012

  • Sandoval, N. R.; Kim, J. Y. H.; Glebes, T. Y.
  • Proceedings of the National Academy of Sciences, Vol. 109, Issue 26, p. 10540-10545
  • DOI: 10.1073/pnas.1206299109

Elucidating acetate tolerance in E. coli using a genome-wide approach
journal, March 2011

Probing Cellular Processes with Oligo-Mediated Recombination and Using the Knowledge Gained to Optimize Recombineering
journal, March 2011

  • Sawitzke, James A.; Costantino, Nina; Li, Xin-tian
  • Journal of Molecular Biology, Vol. 407, Issue 1, p. 45-59
  • DOI: 10.1016/j.jmb.2011.01.030

Serine hydroxymethyltransferase from Escherichia coli: purification and properties.
journal, January 1985

Functional optimization of gene clusters by combinatorial design and assembly
journal, November 2014

  • Smanski, Michael J.; Bhatia, Swapnil; Zhao, Dehua
  • Nature Biotechnology, Vol. 32, Issue 12, p. 1241-1249
  • DOI: 10.1038/nbt.3063

Microbial export of lactic and 3-hydroxypropanoic acid: implications for industrial fermentation processes
journal, October 2004

  • Maris, Antonius J. A. van; Konings, Wil N.; Dijken, Johannes P. van
  • Metabolic Engineering, Vol. 6, Issue 4
  • DOI: 10.1016/j.ymben.2004.05.001

Programming cells by multiplex genome engineering and accelerated evolution
journal, July 2009

  • Wang, Harris H.; Isaacs, Farren J.; Carr, Peter A.
  • Nature, Vol. 460, Issue 7257, p. 894-898
  • DOI: 10.1038/nature08187

Rapid dissection of a complex phenotype through genomic-scale mapping of fitness altering genes
journal, May 2010

  • Warnecke, T. E.; Lynch, M. D.; Karimpour-Fard, A.
  • Metabolic Engineering, Vol. 12, Issue 3, p. 241-250
  • DOI: 10.1016/j.ymben.2009.12.002

Identification of a 21 amino acid peptide conferring 3-hydroxypropionic acid stress-tolerance to Escherichia coli
journal, January 2012

  • Warnecke, T. E.; Lynch, M. D.; Lipscomb, M. L.
  • Biotechnology and Bioengineering, Vol. 109, Issue 5, p. 1347-1352
  • DOI: 10.1002/bit.24398

Rapid profiling of a microbial genome using mixtures of barcoded oligonucleotides
journal, July 2010

  • Warner, Joseph R.; Reeder, Philippa J.; Karimpour-Fard, Anis
  • Nature Biotechnology, Vol. 28, Issue 8, p. 856-862
  • DOI: 10.1038/nbt.1653

Modular optimization of multi-gene pathways for fatty acids production in E. coli
journal, January 2013

  • Xu, Peng; Gu, Qin; Wang, Wenya
  • Nature Communications, Vol. 4, Issue 1
  • DOI: 10.1038/ncomms2425

Metabolic engineering of Escherichia coli for direct production of 1,4-butanediol
journal, May 2011

  • Yim, Harry; Haselbeck, Robert; Niu, Wei
  • Nature Chemical Biology, Vol. 7, Issue 7
  • DOI: 10.1038/nchembio.580

Multiplexed tracking of combinatorial genomic mutations in engineered cell populations
journal, March 2015

  • Zeitoun, Ramsey I.; Garst, Andrew D.; Degen, George D.
  • Nature Biotechnology, Vol. 33, Issue 6, p. 631-637
  • DOI: 10.1038/nbt.3177

Quantitative Tracking of Combinatorially Engineered Populations with Multiplexed Binary Assemblies
journal, January 2017

An efficient one-step site-directed and site-saturation mutagenesis protocol
journal, August 2004

    Sort by:
    [ × clear filter / sort ]

    Works referencing / citing this record:

    Combining orthogonal CRISPR and CRISPRi systems for genome engineering and metabolic pathway modulation in Escherichia coli
    journal, February 2019

    • Sung, Li‐Yu; Wu, Meng‐Ying; Lin, Mei‐Wei
    • Biotechnology and Bioengineering, Vol. 116, Issue 5
    • DOI: 10.1002/bit.26915

    Production of 3-Hydroxypropanoic Acid From Glycerol by Metabolically Engineered Bacteria
    journal, May 2019

    • Jers, Carsten; Kalantari, Aida; Garg, Abhroop
    • Frontiers in Bioengineering and Biotechnology, Vol. 7
    • DOI: 10.3389/fbioe.2019.00124

    Synthetic chimeric nucleases function for efficient genome editing
    journal, December 2019

    Synthetic evolution
    journal, June 2019

    • Simon, Anna J.; d’Oelsnitz, Simon; Ellington, Andrew D.
    • Nature Biotechnology, Vol. 37, Issue 7
    • DOI: 10.1038/s41587-019-0157-4

    Synthetic chimeric nucleases function for efficient genome editing
    journal, December 2019

    Production of 3-Hydroxypropanoic Acid From Glycerol by Metabolically Engineered Bacteria
    journal, May 2019

    • Jers, Carsten; Kalantari, Aida; Garg, Abhroop
    • Frontiers in Bioengineering and Biotechnology, Vol. 7
    • DOI: 10.3389/fbioe.2019.00124
      Sort by:
      [ × clear filter / sort ]

      Similar Records in DOE PAGES and OSTI.GOV collections: