CRISPR interference-guided multiplex repression of endogenous competing pathway genes for redirecting metabolic flux in Escherichia coli

Microbial Cell Factories
Seong Keun KimSeung-Goo Lee

Abstract

Multiplex control of metabolic pathway genes is essential for maximizing product titers and conversion yields of fuels, chemicals, and pharmaceuticals in metabolic engineering. To achieve this goal, artificial transcriptional regulators, such as clustered regularly interspaced short palindromic repeats (CRISPR) interference (CRISPRi), have been developed to specifically repress genes of interest. In this study, we deployed a tunable CRISPRi system for multiplex repression of competing pathway genes and, thus, directed carbon flux toward production of molecules of interest in Escherichia coli. The tunable CRISPRi system with an array of sgRNAs successfully repressed four endogenous genes (pta, frdA, ldhA, and adhE) individually and in double, triple, or quadruple combination that are involved in the formation of byproducts (acetate, succinate, lactate, and ethanol) and the consumption of NADH in E. coli. Single-target CRISPRi effectively reduced the amount of each byproduct and, interestingly, pta repression also decreased ethanol production (41%), whereas ldhA repression increased ethanol production (197%). CRISPRi-mediated multiplex repression of competing pathway genes also resulted in simultaneous reductions of acetate, succ...Continue Reading

References

Jun 1, 2000·Proceedings of the National Academy of Sciences of the United States of America·K A Datsenko, B L Wanner
Feb 16, 2002·Methods : a Companion to Methods in Enzymology·K J Livak, T D Schmittgen
Aug 6, 2005·Biotechnology Progress·Cheryl R DittrichKa-Yiu San
Apr 18, 2007·Nature Chemical Biology·Michelle C Y ChangJay D Keasling
Oct 19, 2007·Metabolic Engineering·Shota AtsumiJames C Liao
Dec 25, 2007·Applied and Environmental Microbiology·Abhishek MurarkaRamon Gonzalez
Apr 14, 2009·Nature Methods·Daniel G GibsonHamilton O Smith
Oct 27, 2009·Microbial Cell Factories·Sara Castaño-CerezoManuel Cánovas
Mar 15, 2011·Applied and Environmental Microbiology·Claire R ShenJames C Liao
Oct 3, 2012·Metabolic Engineering·Kevin V SolomonKristala L J Prather
Jan 22, 2013·Nature Biotechnology·Dokyun NaSang Yup Lee
Jan 31, 2013·Nature Communications·Peng XuMattheos A G Koffas
Mar 9, 2013·Nucleic Acids Research·Lior ZelcbuchRon Milo
Jan 20, 2012·ACS Synthetic Biology·Vandana SharmaYohei Yokobayashi
May 11, 2013·Current Opinion in Chemical Biology·Miao WenMichelle C Y Chang
Jun 4, 2013·Metabolic Engineering·Jorge Alonso-GutierrezTaek Soon Lee
Jun 14, 2013·Nucleic Acids Research·David BikardLuciano A Marraffini
Mar 13, 2014·Nature Chemical Biology·Gabriel M RodriguezShota Atsumi
Sep 5, 2014·Nature Protocols·Ryan R GallagherFarren J Isaacs
Nov 14, 2014·Nucleic Acids Research·Esteban Martínez-GarcíaVíctor de Lorenzo
Dec 3, 2014·Current Opinion in Biotechnology·J Andrew JonesMattheos A G Koffas
Jul 5, 2015·Metabolic Engineering·Yifan LiXueming Zhao
Oct 9, 2015·Nature Biotechnology·Sang Yup Lee, Hyun Uk Kim
Jan 23, 2016·Scientific Reports·Carlotta RondaAlex Toftgaard Nielsen
Feb 3, 2016·FEMS Microbiology Letters·Chang-Ting Chen, James C Liao
Apr 14, 2016·ACS Synthetic Biology·Marcelo C BassaloRyan T Gill
Jul 28, 2016·Biotechnology and Bioengineering·Mu-En ChungYu-Chen Hu
Oct 4, 2016·Metabolic Engineering·Dina ElhadiGuo-Qiang Chen
Jan 18, 2017·Microbial Cell Factories·Brady F CressMattheos A G Koffas

❮ Previous
Next ❯

Citations

Apr 6, 2018·International Journal of Molecular Sciences·Suhyung ChoByung-Kwan Cho
Jun 5, 2018·Biotechnology Journal·Yawei ZhaoYinhua Lu
Jun 20, 2018·Biotechnology Journal·Katia TarasavaRyan T Gill
Jun 5, 2018·Biotechnology Journal·Takayuki ArazoeKeiji Nishida
Feb 18, 2020·Biotechnology and Applied Biochemistry·Kerstin SchultenkämperVolker F Wendisch
Apr 28, 2020·Applied Microbiology and Biotechnology·Xiangyu JiChunbo Lou
Aug 30, 2020·Biotechnology and Bioengineering·Dragan MiscevicC Perry Chou
Nov 21, 2019·Applied Microbiology and Biotechnology·Yangyang ZhanShouwen Chen
Jun 26, 2020·World Journal of Microbiology & Biotechnology·Atieh Hashemi
Dec 6, 2019·Biochemical Society Transactions·Tabinda ShakeelSyed Shams Yazdani
Apr 11, 2020·Applied Microbiology and Biotechnology·Luciana F BritoVolker F Wendisch
Mar 11, 2020·Nature Communications·Nicholas S McCartyRodrigo Ledesma-Amaro
Mar 19, 2020·Frontiers in Microbiology·Bumjoon KimSang Jun Lee
Sep 5, 2020·Microbial Cell Factories·Zhenquan LiuDawei Zhang
Feb 2, 2021·Trends in Biotechnology·Justin PanichSteven W Singer
Jun 3, 2020·Current Opinion in Biotechnology·Yoshihiro Toya, Hiroshi Shimizu
Jun 23, 2020·Metabolic Engineering·D Brisbane Tovilla-CoutiñoMark A Eiteman
Jun 15, 2021·EcoSal Plus·Nicholas Backes, Gregory J Phillips
Mar 16, 2018·ACS Synthetic Biology·Suhyung ChoByung-Kwan Cho
Jan 26, 2018·ACS Synthetic Biology·Drew M DeLorenzoTae Seok Moon

❮ Previous
Next ❯

Methods Mentioned

BETA
PCR
fluorescence assay
sgRNA array
acetylation
restriction digest

Software Mentioned

Agilent ChemStation
MAGE

Related Concepts

Related Feeds

CRISPR Ribonucleases Deactivation

CRISPR-Cas system enables the editing of genes to create or correct mutations. This feed focuses on mechanisms that underlie deactivation of CRISPR ribonucleases. Here is the latest research.

CRISPR (general)

Clustered regularly interspaced short palindromic repeats (CRISPR) are DNA sequences in the genome that are recognized and cleaved by CRISPR-associated proteins (Cas). CRISPR-Cas system enables the editing of genes to create or correct mutations. Discover the latest research on CRISPR here.

CRISPR for Genome Editing

Genome editing technologies enable the editing of genes to create or correct mutations. Clustered regularly interspaced short palindromic repeats (CRISPR) are DNA sequences in the genome that are recognized and cleaved by CRISPR-associated proteins (Cas). Here is the latest research on the use of CRISPR-Cas system in gene editing.