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Enhancing isoprenol production by systematically tuning metabolic pathways using CRISPR interference in E. coli

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, respect...

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Autores principales: Kim, Jinho, Lee, Taek Soon
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10659101/
https://www.ncbi.nlm.nih.gov/pubmed/38026852
http://dx.doi.org/10.3389/fbioe.2023.1296132
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author Kim, Jinho
Lee, Taek Soon
author_facet Kim, Jinho
Lee, Taek Soon
author_sort Kim, Jinho
collection PubMed
description 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.
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spelling pubmed-106591012023-01-01 Enhancing isoprenol production by systematically tuning metabolic pathways using CRISPR interference in E. coli Kim, Jinho Lee, Taek Soon Front Bioeng Biotechnol Bioengineering and Biotechnology 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. Frontiers Media S.A. 2023-11-06 /pmc/articles/PMC10659101/ /pubmed/38026852 http://dx.doi.org/10.3389/fbioe.2023.1296132 Text en This work is authored by Jinho Kim and Taek Soon Lee on behalf of the U.S. Government and as regards Dr. Kim, Dr. Lee and the U.S. Government, is not subject to copyright protection in the United States. Foreign and other copyrights may apply. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Bioengineering and Biotechnology
Kim, Jinho
Lee, Taek Soon
Enhancing isoprenol production by systematically tuning metabolic pathways using CRISPR interference in E. coli
title Enhancing isoprenol production by systematically tuning metabolic pathways using CRISPR interference in E. coli
title_full Enhancing isoprenol production by systematically tuning metabolic pathways using CRISPR interference in E. coli
title_fullStr Enhancing isoprenol production by systematically tuning metabolic pathways using CRISPR interference in E. coli
title_full_unstemmed Enhancing isoprenol production by systematically tuning metabolic pathways using CRISPR interference in E. coli
title_short Enhancing isoprenol production by systematically tuning metabolic pathways using CRISPR interference in E. coli
title_sort enhancing isoprenol production by systematically tuning metabolic pathways using crispr interference in e. coli
topic Bioengineering and Biotechnology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10659101/
https://www.ncbi.nlm.nih.gov/pubmed/38026852
http://dx.doi.org/10.3389/fbioe.2023.1296132
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