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

Tian, Tian; Kang, Jing Wei; Kang, Aram; Lee, Taek Soon

doi:10.1021/acssynbio.8b00429

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:

Tian, Tian ^[1]; Kang, Jing Wei ^[2]; Kang, Aram ^[1];

^[1]

Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
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:: 2019-01-04

Research Org.:: Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)

Sponsoring Org.:: USDOE Office of Science (SC), Basic Energy Sciences (BES)

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.

Copy to clipboard


                    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:https://doi.org/10.1021/acssynbio.8b00429

Copy to clipboard


                    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:https://doi.org/10.1021/acssynbio.8b00429.  https://www.osti.gov/servlets/purl/1580375.

Copy to clipboard


                    
@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}

}

Copy to clipboard

Journal Article:

Free Publicly Available Full Text

Accepted Manuscript (DOE)

Publisher's Version of Record

DOI: https://doi.org/10.1021/acssynbio.8b00429

Other availability

Search WorldCat to find libraries that may hold this journal

Citation Metrics:

Cited by: 16 works

Citation information provided by
Web of Science

Save / Share:

Export Metadata

Save to My Library

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

Schultenkämper, Kerstin; Brito, Luciana F.; López, Marina Gil
Applied Microbiology and Biotechnology, Vol. 103, Issue 14
DOI: 10.1007/s00253-019-09907-8

Impact of CRISPR interference on strain development in biotechnology
journal, January 2020

Schultenkämper, Kerstin; Brito, Luciana F.; Wendisch, Volker F.
Biotechnology and Applied Biochemistry, Vol. 67, Issue 1
DOI: 10.1002/bab.1901

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

Schultenkämper, Kerstin; Brito, Luciana F.; López, Marina Gil
Applied Microbiology and Biotechnology, Vol. 103, Issue 14
DOI: 10.1007/s00253-019-09907-8

Establishment and application of multiplexed CRISPR interference system in Bacillus licheniformis
journal, November 2019

Zhan, Yangyang; Xu, Yong; Zheng, Pengling
Applied Microbiology and Biotechnology, Vol. 104, Issue 1
DOI: 10.1007/s00253-019-10230-5

Increased Production of Dicinnamoylmethane Via Improving Cellular Malonyl-CoA Level by Using a CRISPRi in Escherichia coli
journal, December 2019

Chu, Luan Luong; Pandey, Ramesh Prasad; Dhakal, Dipesh
Applied Biochemistry and Biotechnology, Vol. 190, Issue 1
DOI: 10.1007/s12010-019-03206-8

Multiplex genome editing of microorganisms using CRISPR-Cas
journal, April 2019

Adiego-Pérez, Belén; Randazzo, Paola; Daran, Jean Marc
FEMS Microbiology Letters, Vol. 366, Issue 8
DOI: 10.1093/femsle/fnz086

Obtaining a series of native gradient promoter-5′-UTR sequences in Corynebacterium glutamicum ATCC 13032
journal, June 2020

Li, Ning; Zeng, Weizhu; Xu, Sha
Microbial Cell Factories, Vol. 19, Issue 1
DOI: 10.1186/s12934-020-01376-3

Similar Records in DOE PAGES and OSTI.GOV collections:

A High-efficacy CRISPRi System for Gene Function Discovery in Zymomonas mobilis

Journal Article Banta, Amy B. ; Enright, Amy L. ; Siletti, Cheta ; ... - Applied and Environmental Microbiology

Zymomonas mobilis is a promising biofuel producer due to its high alcohol tolerance and streamlined metabolism that efficiently converts sugar to ethanol. Z. mobilis genes are poorly characterized relative to those of model bacteria, hampering our ability to rationally engineer the genome with pathways capable of converting sugars from plant hydrolysates into valuable biofuels and bioproducts. Many of the unique properties that make Z. mobilis an attractive biofuel producer are controlled by essential genes; however, these genes cannot be manipulated using traditional genetic approaches (e.g., deletion or transposon insertion) because they are required for viability. CRISPR interference (CRISPRi) is amore »« less
DOI: https://doi.org/10.1128/AEM.01621-20
Transcriptional reprogramming in yeast using dCas9 and combinatorial gRNA strategies

Journal Article Jensen, Emil D. ; Ferreira, Raphael ; Jakočiūnas, Tadas ; ... - Microbial Cell Factories

Background Transcriptional reprogramming is a fundamental process of living cells in order to adapt to environmental and endogenous cues. In order to allow flexible and timely control over gene expression without the interference of native gene expression machinery, a large number of studies have focused on developing synthetic biology tools for orthogonal control of transcription. Most recently, the nuclease-deficient Cas9 (dCas9) has emerged as a flexible tool for controlling activation and repression of target genes, by the simple RNA-guided positioning of dCas9 in the vicinity of the target gene transcription start site. Results In this study we compared two differentmore »« less
DOI: https://doi.org/10.1186/s12934-017-0664-2

Full Text Available
Functional genomics of the rapidly replicating bacterium Vibrio natriegens by CRISPRi

Journal Article Lee, Henry H. ; Ostrov, Nili ; Wong, Brandon G. ; ... - Nature Microbiology

We present that the fast-growing Gram-negative bacterium Vibrio natriegens is an attractive microbial system for molecular biology and biotechnology due to its remarkably short generation time and metabolic prowess. However, efforts to uncover and utilize the mechanisms underlying its rapid growth are hampered by the scarcity of functional genomic data. Here, we develop a pooled genome-wide clustered regularly interspaced short palindromic repeats (CRISPR) interference (CRISPRi) screen to identify a minimal set of genes required for rapid wild-type growth. Targeting 4,565 (99.7%) of predicted protein-coding genes, our screen uncovered core genes comprising putative essential and growth-supporting genes that are enriched formore »« less
Cited by 19
DOI: https://doi.org/10.1038/s41564-019-0423-8

Full Text Available
Engineering Corynebacterium glutamicum to produce the biogasoline isopentenol from plant biomass hydrolysates

Journal Article Sasaki, Yusuke ; Eng, Thomas ; Herbert, Robin A. ; ... - Biotechnology for Biofuels

Background:Many microbes used for the rapid discovery and development of metabolic pathways have sensitivities to final products and process reagents. Isopentenol (3-methyl-3-buten-1-ol), a biogasoline candidate, has an established heterologous gene pathway but is toxic to several microbial hosts. Reagents used in the pretreatment of plant biomass, such as ionic liquids, also inhibit growth of many host strains. We explored the use of Corynebacterium glutamicum as an alternative host to address these constraints. Results:We found C. glutamicum ATCC 13032 to be tolerant to both the final product, isopentenol, as well to three classes of ionic liquids. A heterologous mevalonate-based isopentenol pathwaymore »« less
Cited by 6
DOI: https://doi.org/10.1186/s13068-019-1381-3
Genome-scale metabolic rewiring improves titers rates and yields of the non-native product indigoidine at scale

Journal Article Banerjee, Deepanwita ; Eng, Thomas ; Lau, Andrew K. ; ... - Nature Communications

Abstract High titer, rate, yield (TRY), and scalability are challenging metrics to achieve due to trade-offs between carbon use for growth and production. To achieve these metrics, we take the minimal cut set (MCS) approach that predicts metabolic reactions for elimination to couple metabolite production strongly with growth. We compute MCS solution-sets for a non-native product indigoidine, a sustainable pigment, in Pseudomonas putida KT2440, an emerging industrial microbe. From the 63 solution-sets, our omics guided process identifies one experimentally feasible solution requiring 14 simultaneous reaction interventions. We implement a total of 14 genes knockdowns using multiplex-CRISPRi. MCS-based solution shifts productionmore »« less
DOI: https://doi.org/10.1038/s41467-020-19171-4

Similar Records

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

Abstract

Citation Formats

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

Impact of CRISPR interference on strain development in biotechnology journal, January 2020

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

Establishment and application of multiplexed CRISPR interference system in Bacillus licheniformis journal, November 2019

Increased Production of Dicinnamoylmethane Via Improving Cellular Malonyl-CoA Level by Using a CRISPRi in Escherichia coli journal, December 2019

Multiplex genome editing of microorganisms using CRISPR-Cas journal, April 2019

Obtaining a series of native gradient promoter-5′-UTR sequences in Corynebacterium glutamicum ATCC 13032 journal, June 2020

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

Impact of CRISPR interference on strain development in biotechnology
journal, January 2020

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

Establishment and application of multiplexed CRISPR interference system in Bacillus licheniformis
journal, November 2019

Increased Production of Dicinnamoylmethane Via Improving Cellular Malonyl-CoA Level by Using a CRISPRi in Escherichia coli
journal, December 2019

Multiplex genome editing of microorganisms using CRISPR-Cas
journal, April 2019

Obtaining a series of native gradient promoter-5′-UTR sequences in Corynebacterium glutamicum ATCC 13032
journal, June 2020