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

Liu, Rongming; Liang, Liya; Choudhury, Alaksh; Bassalo, Marcelo C.; Garst, Andrew D.; Tarasava, Katia; Gill, Ryan T.

doi:10.1016/j.ymben.2018.04.007

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

Journal Article · Sat Apr 14 00:00:00 UTC 2018 · Metabolic Engineering

DOI:https://doi.org/10.1016/j.ymben.2018.04.007· OSTI ID:1548398

Liu, Rongming ^[1]; Liang, Liya ^[2]; Choudhury, Alaksh ^[2]; Bassalo, Marcelo C. ^[2]; Garst, Andrew D. ^[2]; Tarasava, Katia ^[2]; Gill, Ryan T. ^[2]

Univ. of Colorado, Boulder, CO (United States); University of Colorado
Univ. of Colorado, Boulder, CO (United States)

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.

View Accepted Manuscript (DOE)

Research Organization:: Univ. of Colorado, Boulder, CO (United States)

Sponsoring Organization:: USDOE; USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23)

Grant/Contract Number:: SC0018368

OSTI ID:: 1548398

Alternate ID(s):: OSTI ID: 1582780

Journal Information:: Metabolic Engineering, Journal Name: Metabolic Engineering Journal Issue: C Vol. 47; ISSN 1096-7176

Publisher:: ElsevierCopyright Statement

Country of Publication:: United States

Language:: English

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59 BASIC BIOLOGICAL SCIENCES
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