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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]
  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) (SC-23)
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.
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. doi:10.1016/j.ymben.2018.04.007.
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. doi:10.1016/j.ymben.2018.04.007. https://www.osti.gov/servlets/purl/1548398.
@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 = {2018},
month = {4}
}

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Works referencing / citing this record:

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