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Title: Redirecting Metabolic Flux via Combinatorial Multiplex CRISPRi-Mediated Repression for Isopentenol Production in Escherichia coli

Abstract

CRISPR interference (CRISPRi) via target guide RNA (gRNA) arrays and a deactivated Cas9 (dCas9) protein has been shown to simultaneously repress expression of multiple genomic DNA loci. By knocking down endogenous genes in competing pathways, CRISPRi technology can be utilized to redirect metabolic flux toward target metabolite. In this study, we constructed a CRISPRi-mediated multiplex repression system to silence transcription of several endogenous genes to increase precursor availability in a heterologous isopentenol biosynthesis pathway. To identify genomic knockdown targets in competing pathways, we first designed a single-gRNA library with 15 individual targets, where 3 gRNA cassettes targeting gene asnA, prpE, and gldA increased isopentenol titer by 18-24%. We then combined the 3 single-gRNA cassettes into a two- or three-gRNA array and observed up to 98% enhancement in production by fine-tuning the repression level through titrating dCas9 expression. Our strategy shows that multiplex combinatorial knockdown of competing genes using CRISPRi can increase production of the target metabolite, while the repression level needs to be adjusted to balance the metabolic network and achieve the maximum titer improvement.

Authors:
 [1];  [2];  [1]; ORCiD logo [1]
  1. Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
  2. Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Univ. of California, Berkeley, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
Publication Date:
Research Org.:
Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Basic Energy Sciences (BES) (SC-22)
OSTI Identifier:
1580375
Grant/Contract Number:  
[AC02-05CH11231]
Resource Type:
Accepted Manuscript
Journal Name:
ACS Synthetic Biology
Additional Journal Information:
[ Journal Volume: 8; Journal Issue: 2]; Journal ID: ISSN 2161-5063
Publisher:
American Chemical Society (ACS)
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES

Citation Formats

Tian, Tian, Kang, Jing Wei, Kang, Aram, and Lee, Taek Soon. Redirecting Metabolic Flux via Combinatorial Multiplex CRISPRi-Mediated Repression for Isopentenol Production in Escherichia coli. United States: N. p., 2019. Web. doi:10.1021/acssynbio.8b00429.
Tian, Tian, Kang, Jing Wei, Kang, Aram, & Lee, Taek Soon. Redirecting Metabolic Flux via Combinatorial Multiplex CRISPRi-Mediated Repression for Isopentenol Production in Escherichia coli. United States. doi:10.1021/acssynbio.8b00429.
Tian, Tian, Kang, Jing Wei, Kang, Aram, and Lee, Taek Soon. Fri . "Redirecting Metabolic Flux via Combinatorial Multiplex CRISPRi-Mediated Repression for Isopentenol Production in Escherichia coli". United States. doi:10.1021/acssynbio.8b00429. https://www.osti.gov/servlets/purl/1580375.
@article{osti_1580375,
title = {Redirecting Metabolic Flux via Combinatorial Multiplex CRISPRi-Mediated Repression for Isopentenol Production in Escherichia coli},
author = {Tian, Tian and Kang, Jing Wei and Kang, Aram and Lee, Taek Soon},
abstractNote = {CRISPR interference (CRISPRi) via target guide RNA (gRNA) arrays and a deactivated Cas9 (dCas9) protein has been shown to simultaneously repress expression of multiple genomic DNA loci. By knocking down endogenous genes in competing pathways, CRISPRi technology can be utilized to redirect metabolic flux toward target metabolite. In this study, we constructed a CRISPRi-mediated multiplex repression system to silence transcription of several endogenous genes to increase precursor availability in a heterologous isopentenol biosynthesis pathway. To identify genomic knockdown targets in competing pathways, we first designed a single-gRNA library with 15 individual targets, where 3 gRNA cassettes targeting gene asnA, prpE, and gldA increased isopentenol titer by 18-24%. We then combined the 3 single-gRNA cassettes into a two- or three-gRNA array and observed up to 98% enhancement in production by fine-tuning the repression level through titrating dCas9 expression. Our strategy shows that multiplex combinatorial knockdown of competing genes using CRISPRi can increase production of the target metabolite, while the repression level needs to be adjusted to balance the metabolic network and achieve the maximum titer improvement.},
doi = {10.1021/acssynbio.8b00429},
journal = {ACS Synthetic Biology},
number = [2],
volume = [8],
place = {United States},
year = {2019},
month = {1}
}

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Works referencing / citing this record:

Establishment and application of CRISPR interference to affect sporulation, hydrogen peroxide detoxification, and mannitol catabolism in the methylotrophic thermophile Bacillus methanolicus
journal, May 2019

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