Enhancing isoprenol production by systematically tuning metabolic pathways using CRISPR interference in E. coli
Abstract
Regulation of metabolic gene expression is crucial for maximizing bioproduction titers. Recent engineering tools including CRISPR/Cas9, CRISPR interference (CRISPRi), and CRISPR activation (CRISPRa) have enabled effective knock-out, knock-down, and overexpression of endogenous pathway genes, respectively, for advanced strain engineering. CRISPRi in particular has emerged as a powerful tool for gene repression through the use of a deactivated Cas9 (dCas9) protein and target guide RNA (gRNA). By constructing gRNA arrays, CRISPRi has the capacity for multiplexed gene downregulation across multiple orthogonal pathways for enhanced bioproduction titers. In this study, we harnessed CRISPRi to downregulate 32 essential and non-essential genes in E. coli strains heterologously expressing either the original mevalonate pathway or isopentenyl diphosphate (IPP) bypass pathway for isoprenol biosynthesis. Isoprenol remains a candidate bioproduct both as a drop-in blend additive and as a precursor for the high-performance sustainable aviation fuel, 1,4-dimethylcyclooctane (DMCO). Of the 32 gRNAs targeting genes associated with isoprenol biosynthesis, a subset was found to vastly improve product titers. Construction of a multiplexed gRNA library based on single guide RNA (sgRNA) performance enabled simultaneous gene repression, yielding a 3 to 4.5-fold increase in isoprenol titer (1.82 ± 0.19 g/L) on M9-MOPS minimal medium. We then scaled the best performing CRISPRimore »
- Authors:
- Publication Date:
- Sponsoring Org.:
- USDOE
- OSTI Identifier:
- 2204892
- Resource Type:
- Published Article
- Journal Name:
- Frontiers in Bioengineering and Biotechnology
- Additional Journal Information:
- Journal Name: Frontiers in Bioengineering and Biotechnology Journal Volume: 11; Journal ID: ISSN 2296-4185
- Publisher:
- Frontiers Media SA
- Country of Publication:
- Switzerland
- Language:
- English
Citation Formats
Kim, Jinho, and Lee, Taek Soon. Enhancing isoprenol production by systematically tuning metabolic pathways using CRISPR interference in E. coli. Switzerland: N. p., 2023.
Web. doi:10.3389/fbioe.2023.1296132.
Kim, Jinho, & Lee, Taek Soon. Enhancing isoprenol production by systematically tuning metabolic pathways using CRISPR interference in E. coli. Switzerland. https://doi.org/10.3389/fbioe.2023.1296132
Kim, Jinho, and Lee, Taek Soon. Mon .
"Enhancing isoprenol production by systematically tuning metabolic pathways using CRISPR interference in E. coli". Switzerland. https://doi.org/10.3389/fbioe.2023.1296132.
@article{osti_2204892,
title = {Enhancing isoprenol production by systematically tuning metabolic pathways using CRISPR interference in E. coli},
author = {Kim, Jinho and Lee, Taek Soon},
abstractNote = {Regulation of metabolic gene expression is crucial for maximizing bioproduction titers. Recent engineering tools including CRISPR/Cas9, CRISPR interference (CRISPRi), and CRISPR activation (CRISPRa) have enabled effective knock-out, knock-down, and overexpression of endogenous pathway genes, respectively, for advanced strain engineering. CRISPRi in particular has emerged as a powerful tool for gene repression through the use of a deactivated Cas9 (dCas9) protein and target guide RNA (gRNA). By constructing gRNA arrays, CRISPRi has the capacity for multiplexed gene downregulation across multiple orthogonal pathways for enhanced bioproduction titers. In this study, we harnessed CRISPRi to downregulate 32 essential and non-essential genes in E. coli strains heterologously expressing either the original mevalonate pathway or isopentenyl diphosphate (IPP) bypass pathway for isoprenol biosynthesis. Isoprenol remains a candidate bioproduct both as a drop-in blend additive and as a precursor for the high-performance sustainable aviation fuel, 1,4-dimethylcyclooctane (DMCO). Of the 32 gRNAs targeting genes associated with isoprenol biosynthesis, a subset was found to vastly improve product titers. Construction of a multiplexed gRNA library based on single guide RNA (sgRNA) performance enabled simultaneous gene repression, yielding a 3 to 4.5-fold increase in isoprenol titer (1.82 ± 0.19 g/L) on M9-MOPS minimal medium. We then scaled the best performing CRISPRi strain to 2-L fed-batch cultivation and demonstrated translatable titer improvements, ultimately obtaining 12.4 ± 1.3 g/L isoprenol. Our strategy further establishes CRISPRi as a powerful tool for tuning metabolic flux in production hosts and that titer improvements are readily scalable with potential for applications in industrial bioproduction.},
doi = {10.3389/fbioe.2023.1296132},
journal = {Frontiers in Bioengineering and Biotechnology},
number = ,
volume = 11,
place = {Switzerland},
year = {Mon Nov 06 00:00:00 EST 2023},
month = {Mon Nov 06 00:00:00 EST 2023}
}
https://doi.org/10.3389/fbioe.2023.1296132
Works referenced in this record:
Application of different types of CRISPR/Cas-based systems in bacteria
journal, September 2020
- Liu, Zhenquan; Dong, Huina; Cui, Yali
- Microbial Cell Factories, Vol. 19, Issue 1
Uncovering the gene knockout landscape for improved lycopene production in E. coli
journal, April 2008
- Alper, Hal; Stephanopoulos, Gregory
- Applied Microbiology and Biotechnology, Vol. 78, Issue 5
Recent advances in systems metabolic engineering tools and strategies
journal, October 2017
- Chae, Tong Un; Choi, So Young; Kim, Je Woong
- Current Opinion in Biotechnology, Vol. 47
Microbial production of high octane and high sensitivity olefinic ester biofuels
journal, April 2023
- Carruthers, David N.; Kim, Jinho; Mendez-Perez, Daniel
- Biotechnology for Biofuels and Bioproducts, Vol. 16, Issue 1
Fatty acid biosynthesis revisited: structure elucidation and metabolic engineering
journal, January 2015
- Beld, Joris; Lee, D. John; Burkart, Michael D.
- Molecular BioSystems, Vol. 11, Issue 1
Enhancing flavonoid production by systematically tuning the central metabolic pathways based on a CRISPR interference system in Escherichia coli
journal, September 2015
- Wu, Junjun; Du, Guocheng; Chen, Jian
- Scientific Reports, Vol. 5, Issue 1
Simple fed-batch technique for high cell density cultivation of Escherichia coli
journal, February 1995
- Korz, D. J.; Rinas, U.; Hellmuth, K.
- Journal of Biotechnology, Vol. 39, Issue 1
Production Cost and Carbon Footprint of Biomass-Derived Dimethylcyclooctane as a High-Performance Jet Fuel Blendstock
journal, August 2021
- Baral, Nawa Raj; Yang, Minliang; Harvey, Benjamin G.
- ACS Sustainable Chemistry & Engineering, Vol. 9, Issue 35
Metabolic engineering for the high-yield production of isoprenoid-based C5 alcohols in E. coli
journal, June 2015
- George, Kevin W.; Thompson, Mitchell G.; Kang, Aram
- Scientific Reports, Vol. 5, Issue 1
Regulation of ATP levels in Escherichia coli using CRISPR interference for enhanced pinocembrin production
journal, September 2018
- Tao, Sha; Qian, Ying; Wang, Xin
- Microbial Cell Factories, Vol. 17, Issue 1
Targeted and high-throughput gene knockdown in diverse bacteria using synthetic sRNAs
journal, April 2023
- Cho, Jae Sung; Yang, Dongsoo; Prabowo, Cindy Pricilia Surya
- Nature Communications, Vol. 14, Issue 1
Optimization of the IPP-bypass mevalonate pathway and fed-batch fermentation for the production of isoprenol in Escherichia coli
journal, December 2019
- Kang, Aram; Mendez-Perez, Daniel; Goh, Ee-Been
- Metabolic Engineering, Vol. 56
Dynamic pathway regulation: recent advances and methods of construction
journal, December 2017
- Tan, Sue Zanne; Prather, Kristala LJ
- Current Opinion in Chemical Biology, Vol. 41
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
High-throughput enzyme screening platform for the IPP-bypass mevalonate pathway for isopentenol production
journal, May 2017
- Kang, Aram; Meadows, Corey W.; Canu, Nicolas
- Metabolic Engineering, Vol. 41
Metabolic engineering of menaquinone‐8 pathway of Escherichia coli as a microbial platform for vitamin K production
journal, April 2011
- Kong, Min Kyung; Lee, Pyung Cheon
- Biotechnology and Bioengineering, Vol. 108, Issue 8
Microbial production of advanced biofuels
journal, June 2021
- Keasling, Jay; Garcia Martin, Hector; Lee, Taek Soon
- Nature Reviews Microbiology, Vol. 19, Issue 11
Applications of CRISPR/Cas System to Bacterial Metabolic Engineering
journal, April 2018
- Cho, Suhyung; Shin, Jongoh; Cho, Byung-Kwan
- International Journal of Molecular Sciences, Vol. 19, Issue 4
Rapid editing and evolution of bacterial genomes using libraries of synthetic DNA
journal, September 2014
- Gallagher, Ryan R.; Li, Zhe; Lewis, Aaron O.
- Nature Protocols, Vol. 9, Issue 10
Identifying gene targets for the metabolic engineering of lycopene biosynthesis in Escherichia coli
journal, May 2005
- Alper, Hal; Jin, Yong-Su; Moxley, J. F.
- Metabolic Engineering, Vol. 7, Issue 3
CRISPR interference-guided balancing of a biosynthetic mevalonate pathway increases terpenoid production
journal, November 2016
- Kim, Seong Keun; Han, Gui Hwan; Seong, Wonjae
- Metabolic Engineering, Vol. 38
β-Ketoacyl-Acyl Carrier Protein Synthase III (FabH) Is Essential for Bacterial Fatty Acid Synthesis
journal, September 2003
- Lai, Chiou-Yan; Cronan, John E.
- Journal of Biological Chemistry, Vol. 278, Issue 51
Improving isoprenol production via systematic CRISPRi screening in engineered Escherichia coli
journal, January 2022
- Wang, Jian; Jiang, Tian; Milligan, Sierra
- Green Chemistry, Vol. 24, Issue 18
Efficient de novo synthesis of resveratrol by metabolically engineered Escherichia coli
journal, March 2017
- Wu, Junjun; Zhou, Peng; Zhang, Xia
- Journal of Industrial Microbiology & Biotechnology, Vol. 44, Issue 7
Tuning a high performing multiplexed-CRISPRi Pseudomonas putida strain to further enhance indigoidine production
journal, December 2022
- Czajka, Jeffrey J.; Banerjee, Deepanwita; Eng, Thomas
- Metabolic Engineering Communications, Vol. 15
One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products
journal, May 2000
- Datsenko, K. A.; Wanner, B. L.
- Proceedings of the National Academy of Sciences, Vol. 97, Issue 12, p. 6640-6645
Systems strategies for developing industrial microbial strains
journal, October 2015
- Lee, Sang Yup; Kim, Hyun Uk
- Nature Biotechnology, Vol. 33, Issue 10
Metabolic engineering of Escherichia coli for production of mixed isoprenoid alcohols and their derivatives
journal, July 2018
- Zada, Bakht; Wang, Chonglong; Park, Ji-Bin
- Biotechnology for Biofuels, Vol. 11, Issue 1
The Essential Genome of Escherichia coli K-12
journal, February 2018
- Goodall, Emily C. A.; Robinson, Ashley; Johnston, Iain G.
- mBio, Vol. 9, Issue 1
Redirecting Metabolic Flux via Combinatorial Multiplex CRISPRi-Mediated Repression for Isopentenol Production in Escherichia coli
journal, January 2019
- Tian, Tian; Kang, Jing Wei; Kang, Aram
- ACS Synthetic Biology, Vol. 8, Issue 2
Programmable repression and activation of bacterial gene expression using an engineered CRISPR-Cas system
journal, June 2013
- Bikard, David; Jiang, Wenyan; Samai, Poulami
- Nucleic Acids Research, Vol. 41, Issue 15
CRISPR interference-guided multiplex repression of endogenous competing pathway genes for redirecting metabolic flux in Escherichia coli
journal, November 2017
- Kim, Seong Keun; Seong, Wonjae; Han, Gui Hwan
- Microbial Cell Factories, Vol. 16, Issue 1
[4 + 4]-cycloaddition of isoprene for the production of high-performance bio-based jet fuel
journal, January 2019
- Rosenkoetter, Kyle E.; Kennedy, C. Rose; Chirik, Paul J.
- Green Chemistry, Vol. 21, Issue 20
Isopentenyl diphosphate (IPP)-bypass mevalonate pathways for isopentenol production
journal, March 2016
- Kang, Aram; George, Kevin W.; Wang, George
- Metabolic Engineering, Vol. 34
Characterization of MenA (isoprenyl diphosphate:1,4-dihydroxy-2-naphthoate isoprenyltransferase) from Mycobacterium tuberculosis
journal, April 2019
- Dhiman, Rakesh K.; Pujari, Venugopal; Kincaid, James M.
- PLOS ONE, Vol. 14, Issue 4
Integrated analysis of isopentenyl pyrophosphate (IPP) toxicity in isoprenoid-producing Escherichia coli
journal, May 2018
- George, Kevin W.; Thompson, Mitchell G.; Kim, Joonhoon
- Metabolic Engineering, Vol. 47
The impact of genetic diversity on gene essentiality within the Escherichia coli species
journal, January 2021
- Rousset, François; Cabezas-Caballero, Jose; Piastra-Facon, Florence
- Nature Microbiology, Vol. 6, Issue 3
Improving Microbial Biogasoline Production in Escherichia coli Using Tolerance Engineering
journal, November 2014
- Foo, Jee Loon; Jensen, Heather M.; Dahl, Robert H.
- mBio, Vol. 5, Issue 6