Directed combinatorial mutagenesis of Escherichia coli for complex phenotype engineering
Abstract
Strain engineering for industrial production requires a targeted improvement of multiple complex traits, which range from pathway flux to tolerance to mixed sugar utilization. Here, we report the use of an iterative CRISPR EnAbled Trackable genome Engineering (iCREATE) method to engineer rapid glucose and xylose co-consumption and tolerance to hydrolysate inhibitors in E. coli. Deep mutagenesis libraries were rationally designed, constructed, and screened to target ~40,000 mutations across 30 genes. These libraries included global and high-level regulators that regulate global gene expression, transcription factors that play important roles in genome-level transcription, enzymes that function in the sugar transport system, NAD(P)H metabolism, and the aldehyde reduction system. Specific mutants that conferred increased growth in mixed sugars and hydrolysate tolerance conditions were isolated, confirmed, and evaluated for changes in genome-wide expression levels. As a result, we tested the strain with positive combinatorial mutations for 3-hydroxypropionic acid (3HP) production under high furfural and high acetate hydrolysate fermentation, which demonstrated a 7- and 8-fold increase in 3HP productivity relative to the parent strain, respectively.
- Authors:
-
- Univ. of Colorado, Boulder, CO (United States)
- National Renewable Energy Lab. (NREL), Golden, CO (United States)
- Publication Date:
- Research Org.:
- National Renewable Energy Laboratory (NREL), Golden, CO (United States)
- Sponsoring Org.:
- USDOE Office of Energy Efficiency and Renewable Energy (EERE), Sustainable Transportation Office. Bioenergy Technologies Office
- OSTI Identifier:
- 1432610
- Alternate Identifier(s):
- OSTI ID: 1632227
- Report Number(s):
- NREL/JA-5100-71281
Journal ID: ISSN 1096-7176; MainId:16871;UUID:66cfdc6a-5538-e811-9c15-2c44fd93e385;MainAdminID:5349
- Grant/Contract Number:
- AC36-08GO28308; FOA-0000996
- Resource Type:
- Accepted Manuscript
- Journal Name:
- Metabolic Engineering
- Additional Journal Information:
- Journal Volume: 47; Journal Issue: C; Journal ID: ISSN 1096-7176
- Publisher:
- Elsevier
- Country of Publication:
- United States
- Language:
- English
- Subject:
- 09 BIOMASS FUELS; Iterative CRISPR EnAbled Trackable genome; engineering; genome engineering; combinatorial mutagenesis; lignocellulosic biomass
Citation Formats
Liu, Rongming, Liang, Liya, Garst, Andrew D., Choudhury, Alaksh, Nogue, Violeta Sanchez i., Beckham, Gregg T., and Gill, Ryan T. Directed combinatorial mutagenesis of Escherichia coli for complex phenotype engineering. United States: N. p., 2018.
Web. doi:10.1016/j.ymben.2018.02.007.
Liu, Rongming, Liang, Liya, Garst, Andrew D., Choudhury, Alaksh, Nogue, Violeta Sanchez i., Beckham, Gregg T., & Gill, Ryan T. Directed combinatorial mutagenesis of Escherichia coli for complex phenotype engineering. United States. https://doi.org/10.1016/j.ymben.2018.02.007
Liu, Rongming, Liang, Liya, Garst, Andrew D., Choudhury, Alaksh, Nogue, Violeta Sanchez i., Beckham, Gregg T., and Gill, Ryan T. Thu .
"Directed combinatorial mutagenesis of Escherichia coli for complex phenotype engineering". United States. https://doi.org/10.1016/j.ymben.2018.02.007. https://www.osti.gov/servlets/purl/1432610.
@article{osti_1432610,
title = {Directed combinatorial mutagenesis of Escherichia coli for complex phenotype engineering},
author = {Liu, Rongming and Liang, Liya and Garst, Andrew D. and Choudhury, Alaksh and Nogue, Violeta Sanchez i. and Beckham, Gregg T. and Gill, Ryan T.},
abstractNote = {Strain engineering for industrial production requires a targeted improvement of multiple complex traits, which range from pathway flux to tolerance to mixed sugar utilization. Here, we report the use of an iterative CRISPR EnAbled Trackable genome Engineering (iCREATE) method to engineer rapid glucose and xylose co-consumption and tolerance to hydrolysate inhibitors in E. coli. Deep mutagenesis libraries were rationally designed, constructed, and screened to target ~40,000 mutations across 30 genes. These libraries included global and high-level regulators that regulate global gene expression, transcription factors that play important roles in genome-level transcription, enzymes that function in the sugar transport system, NAD(P)H metabolism, and the aldehyde reduction system. Specific mutants that conferred increased growth in mixed sugars and hydrolysate tolerance conditions were isolated, confirmed, and evaluated for changes in genome-wide expression levels. As a result, we tested the strain with positive combinatorial mutations for 3-hydroxypropionic acid (3HP) production under high furfural and high acetate hydrolysate fermentation, which demonstrated a 7- and 8-fold increase in 3HP productivity relative to the parent strain, respectively.},
doi = {10.1016/j.ymben.2018.02.007},
journal = {Metabolic Engineering},
number = C,
volume = 47,
place = {United States},
year = {Thu Mar 29 00:00:00 EDT 2018},
month = {Thu Mar 29 00:00:00 EDT 2018}
}
Free Publicly Available Full Text
Publisher's Version of Record
Other availability
Search WorldCat to find libraries that may hold this journal
Cited by: 22 works
Citation information provided by
Web of Science
Web of Science
Save to My Library
You must Sign In or Create an Account in order to save documents to your library.
Works referenced in this record:
Activation of multiple antibiotic resistance and binding of stress-inducible promoters by Escherichia coli Rob protein.
journal, January 1995
- Ariza, R. R.; Li, Z.; Ringstad, N.
- Journal of bacteriology, Vol. 177, Issue 7
Escherichia coli rpoB Mutants Have Increased Evolvability in Proportion to Their Fitness Defects
journal, January 2010
- Barrick, J. E.; Kauth, M. R.; Strelioff, C. C.
- Molecular Biology and Evolution, Vol. 27, Issue 6
Genome dynamics during experimental evolution
journal, October 2013
- Barrick, Jeffrey E.; Lenski, Richard E.
- Nature Reviews Genetics, Vol. 14, Issue 12
Error-prone PCR of global transcription factor cyclic AMP receptor protein for enhanced organic solvent (toluene) tolerance
journal, December 2012
- Basak, Souvik; Song, Hao; Jiang, Rongrong
- Process Biochemistry, Vol. 47, Issue 12
Rapid and Efficient One-Step Metabolic Pathway Integration in E. coli
journal, April 2016
- Bassalo, Marcelo C.; Garst, Andrew D.; Halweg-Edwards, Andrea L.
- ACS Synthetic Biology, Vol. 5, Issue 7
Interruption of the ferredoxin (flavodoxin) NADP+ oxidoreductase gene of Escherichia coli does not affect anaerobic growth but increases sensitivity to paraquat.
journal, January 1995
- Bianchi, V.; Haggård-Ljungquist, E.; Pontis, E.
- Journal of bacteriology, Vol. 177, Issue 15
Deconstruction of Lignocellulosic Biomass to Fuels and Chemicals
journal, July 2011
- Chundawat, Shishir P. S.; Beckham, Gregg T.; Himmel, Michael E.
- Annual Review of Chemical and Biomolecular Engineering, Vol. 2, Issue 1, p. 121-145
Genetic and functional analysis of the multiple antibiotic resistance (mar) locus in Escherichia coli.
journal, March 1993
- Cohen, S. P.; Hächler, H.; Levy, S. B.
- Journal of Bacteriology, Vol. 175, Issue 5
Replacement of the glucose phosphotransferase transport system by galactose permease reduces acetate accumulation and improves process performance of Escherichia coli for recombinant protein production without impairment of growth rate
journal, May 2006
- De Anda, Ramón; Lara, Alvaro R.; Hernández, Vanessa
- Metabolic Engineering, Vol. 8, Issue 3, p. 281-290
Metabolic shutdown in Escherichia coli cells lacking the outer membrane channel TolC: Metabolic shutdown in TolC mutants
journal, June 2010
- Dhamdhere, Girija; Zgurskaya, Helen I.
- Molecular Microbiology, Vol. 77, Issue 3
Efficient search, mapping, and optimization of multi-protein genetic systems in diverse bacteria
journal, June 2014
- Farasat, I.; Kushwaha, M.; Collens, J.
- Molecular Systems Biology, Vol. 10, Issue 6, p. 731-731
Enhancing Tolerance to Short-Chain Alcohols by Engineering the Escherichia coli AcrB Efflux Pump to Secrete the Non-native Substrate n-Butanol
journal, September 2013
- Fisher, Michael A.; Boyarskiy, Sergey; Yamada, Masaki R.
- ACS Synthetic Biology, Vol. 3, Issue 1, p. 30-40
Use of the rpoB gene to determine the specificity of base substitution mutations on the Escherichia coli chromosome
journal, May 2003
- Garibyan, L.
- DNA Repair, Vol. 2, Issue 5
Glucose-6-phosphate dehydrogenase and ferredoxin-NADP(H) reductase contribute to damage repair during the soxRS response of Escherichia coli
journal, April 2006
- Giró, Mariana; Carrillo, Néstor; Krapp, Adriana R.
- Microbiology, Vol. 152, Issue 4
Genome-Wide Mapping of Furfural Tolerance Genes in Escherichia coli
journal, January 2014
- Glebes, Tirzah Y.; Sandoval, Nicholas R.; Reeder, Philippa J.
- PLoS ONE, Vol. 9, Issue 1
Proteolytic degradation of Escherichia coli transcription activators SoxS and MarA as the mechanism for reversing the induction of the superoxide (SoxRS) and multiple antibiotic resistance (Mar) regulons: Proteolytic degradation of SoxS and MarA
journal, February 2004
- Griffith, Kevin L.; Shah, Ishita M.; E. Wolf, Richard
- Molecular Microbiology, Vol. 51, Issue 6
Multiplex growth rate phenotyping of synthetic mutants in selection to engineer glucose and xylose co-utilization in Escherichia coli: Multiplex Growth Rate Phenotyping
journal, December 2016
- Groot, Joost; Cepress-Mclean, Sidney C.; Robbins-Pianka, Adam
- Biotechnology and Bioengineering, Vol. 114, Issue 4
Expression of galP and glk in a Escherichia coli PTS mutant restores glucose transport and increases glycolytic flux to fermentation products
journal, June 2003
- Hernández-Montalvo, Verónica; Martínez, Alfredo; Hernández-Chavez, Georgina
- Biotechnology and Bioengineering, Vol. 83, Issue 6
Biomass Recalcitrance: Engineering Plants and Enzymes for Biofuels Production
journal, February 2007
- Himmel, M. E.; Ding, S.-Y.; Johnson, D. K.
- Science, Vol. 315, Issue 5813, p. 804-807
Sequence analysis of two temperature-sensitive mutations in the alpha subunit gene ( rpoA ) of Escherichia coli RNA polymerase
journal, January 1990
- Igarashi, Kazuhiko; Fujita, Nobuyuki; Ishihama, Akira
- Nucleic Acids Research, Vol. 18, Issue 20
Ambidextrous transcriptional activation by SoxS: requirement for the C-terminal domain of the RNA polymerase alpha subunit in a subset of Escherichia coli superoxide-inducible genes
journal, January 1996
- Jair, Kam-Wing; Fawcett, William P.; Fujita, Nobuyuki
- Molecular Microbiology, Vol. 19, Issue 2
Succinic acid production from corn stalk hydrolysate in an E. coli mutant generated by atmospheric and room-temperature plasmas and metabolic evolution strategies
journal, October 2013
- Jiang, Min; Wan, Qing; Liu, Rongming
- Journal of Industrial Microbiology & Biotechnology, Vol. 41, Issue 1
Multigene Editing in the Escherichia coli Genome via the CRISPR-Cas9 System
journal, January 2015
- Jiang, Yu; Chen, Biao; Duan, Chunlan
- Applied and Environmental Microbiology, Vol. 81, Issue 7, p. 2506-2514
Characterization of the pleiotropic phenotypes of rifampin-resistant rpoB mutants of Escherichia coli.
journal, January 1989
- Jin, D. J.; Gross, C. A.
- Journal of Bacteriology, Vol. 171, Issue 9
Mapping and sequencing of mutations in the Escherichia colirpoB gene that lead to rifampicin resistance
journal, July 1988
- Jin, Ding Jun; Gross, Carol A.
- Journal of Molecular Biology, Vol. 202, Issue 1
Dilute acid pretreatment of lignocellulose for whole slurry ethanol fermentation
journal, March 2013
- Jung, Young Hoon; Kim, In Jung; Kim, Hyun Kyung
- Bioresource Technology, Vol. 132
Enhanced tolerance of Saccharomyces cerevisiae to multiple lignocellulose-derived inhibitors through modulation of spermidine contents
journal, May 2015
- Kim, Sun-Ki; Jin, Yong-Su; Choi, In-Geol
- Metabolic Engineering, Vol. 29
Escherichia coli RNA polymerase mutants that enhance or diminish the SOS response constitutively expressed in the absence of RNase HI activity.
journal, January 1994
- Kogoma, T.
- Journal of Bacteriology, Vol. 176, Issue 5
Factors affecting redox potential and differential sensitivity of SoxR to redox-active compounds: SoxR activation by redox-active compounds
journal, June 2015
- Lee, Kang-Lok; Singh, Atul K.; Heo, Lim
- Molecular Microbiology, Vol. 97, Issue 5
Effects of overexpression of NAPRTase, NAMNAT, and NAD synthetase in the NAD(H) biosynthetic pathways on the NAD(H) pool, NADH/NAD+ ratio, and succinic acid production with different carbon sources by metabolically engineered Escherichia coli
journal, December 2013
- Liang, Liya; Liu, Rongming; Chen, Xu
- Biochemical Engineering Journal, Vol. 81
Comparison of individual component deletions in a glucose-specific phosphotransferase system revealed their different applications
journal, August 2015
- Liang, Quanfeng; Zhang, Fengyu; Li, Yikui
- Scientific Reports, Vol. 5, Issue 1
Mutation to rifampicin resistance at the beginning of the RNA polymerase β subunit gene in Escherichia coli
journal, August 1984
- Lisitsyn, N. A.; Sverdlov, E. D.; Moiseyeva, E. P.
- Molecular and General Genetics MGG, Vol. 196, Issue 1
Succinate production by metabolically engineered Escherichia coli using sugarcane bagasse hydrolysate as the carbon source
journal, May 2013
- Liu, Rongming; Liang, Liya; Cao, Weijia
- Bioresource Technology, Vol. 135
Efficient succinic acid production from lignocellulosic biomass by simultaneous utilization of glucose and xylose in engineered Escherichia coli
journal, December 2013
- Liu, Rongming; Liang, Liya; Li, Feng
- Bioresource Technology, Vol. 149
Combinatorial modulation of galP and glk gene expression for improved alternative glucose utilization
journal, December 2011
- Lu, Jiao; Tang, Jinlei; Liu, Yi
- Applied Microbiology and Biotechnology, Vol. 93, Issue 6
Competition among seven Escherichia coli sigma subunits: relative binding affinities to the core RNA polymerase
journal, September 2000
- Maeda, H.
- Nucleic Acids Research, Vol. 28, Issue 18
SoxRS Down-Regulation of rob Transcription
journal, September 2002
- Michán, Carmen; Manchado, Manuel; Pueyo, Carmen
- Journal of Bacteriology, Vol. 184, Issue 17
Cellulosic hydrolysate toxicity and tolerance mechanisms in Escherichia coli
journal, January 2009
- Mills, Tirzah Y.; Sandoval, Nicholas R.; Gill, Ryan T.
- Biotechnology for Biofuels, Vol. 2, Issue 1, Article No. 26
Rational design of a synthetic Entner–Doudoroff pathway for improved and controllable NADPH regeneration
journal, May 2015
- Ng, Chiam Yu; Farasat, Iman; Maranas, Costas D.
- Metabolic Engineering, Vol. 29
Two-stage control of an oxidative stress regulon: the Escherichia coli SoxR protein triggers redox-inducible expression of the soxS regulatory gene.
journal, January 1992
- Nunoshiba, T.; Hidalgo, E.; Amábile Cuevas, C. F.
- Journal of Bacteriology, Vol. 174, Issue 19
Impact of Global Transcriptional Regulation by ArcA, ArcB, Cra, Crp, Cya, Fnr, and Mlc on Glucose Catabolism in Escherichia coli
journal, April 2005
- Perrenoud, A.; Sauer, U.
- Journal of Bacteriology, Vol. 187, Issue 9
SoxRS-Regulated Expression and Genetic Analysis of the yggX Gene of Escherichia coli
journal, October 2003
- Pomposiello, P. J.; Koutsolioutsou, A.; Carrasco, D.
- Journal of Bacteriology, Vol. 185, Issue 22
The Path Forward for Biofuels and Biomaterials
journal, January 2006
- Ragauskas, Arthur J.; Williams, Charlotte K.; Davison, Brian H.
- Science, Vol. 311, Issue 5760, p. 484-489
Transcriptional analysis and adaptive evolution of Escherichia coli strains growing on acetate
journal, July 2016
- Rajaraman, Eashwar; Agarwal, Ankit; Crigler, Jacob
- Applied Microbiology and Biotechnology, Vol. 100, Issue 17
The no-SCAR (Scarless Cas9 Assisted Recombineering) system for genome editing in Escherichia coli
journal, October 2015
- Reisch, Chris R.; Prather, Kristala L. J.
- Scientific Reports, Vol. 5, Issue 1
CRMAGE: CRISPR Optimized MAGE Recombineering
journal, January 2016
- Ronda, Carlotta; Pedersen, Lasse Ebdrup; Sommer, Morten O. A.
- Scientific Reports, Vol. 6, Article No. 19452
Strategy for directing combinatorial genome engineering in Escherichia coli
journal, June 2012
- Sandoval, N. R.; Kim, J. Y. H.; Glebes, T. Y.
- Proceedings of the National Academy of Sciences, Vol. 109, Issue 26, p. 10540-10545
Elucidating acetate tolerance in E. coli using a genome-wide approach
journal, March 2011
- Sandoval, Nicholas R.; Mills, Tirzah Y.; Zhang, Min
- Metabolic Engineering, Vol. 13, Issue 2
Simultaneous generation of many RNA-seq libraries in a single reaction
journal, March 2015
- Shishkin, Alexander A.; Giannoukos, Georgia; Kucukural, Alper
- Nature Methods, Vol. 12, Issue 4
SoxR as a Single-Cell Biosensor for NADPH-Consuming Enzymes in Escherichia coli
journal, December 2013
- Siedler, Solvej; Schendzielorz, Georg; Binder, Stephan
- ACS Synthetic Biology, Vol. 3, Issue 1
Recruiting alternative glucose utilization pathways for improving succinate production
journal, August 2012
- Tang, Jinlei; Zhu, Xinna; Lu, Jiao
- Applied Microbiology and Biotechnology, Vol. 97, Issue 6
De novo assembly of bacterial transcriptomes from RNA-seq data
journal, January 2015
- Tjaden, Brian
- Genome Biology, Vol. 16, Issue 1
Valorization of Biomass: Deriving More Value from Waste
journal, August 2012
- Tuck, C. O.; Perez, E.; Horvath, I. T.
- Science, Vol. 337, Issue 6095, p. 695-699
The Orphan Gene ybjN Conveys Pleiotropic Effects on Multicellular Behavior and Survival of Escherichia coli
journal, September 2011
- Wang, Dongping; Calla, Bernarda; Vimolmangkang, Sornkanok
- PLoS ONE, Vol. 6, Issue 9
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
Engineering furfural tolerance in Escherichia coli improves the fermentation of lignocellulosic sugars into renewable chemicals
journal, February 2013
- Wang, X.; Yomano, L. P.; Lee, J. Y.
- Proceedings of the National Academy of Sciences, Vol. 110, Issue 10, p. 4021-4026
Paradigm for industrial strain improvement identifies sodium acetate tolerance loci in Zymomonas mobilis and Saccharomyces cerevisiae
journal, May 2010
- Yang, S.; Land, M. L.; Klingeman, D. M.
- Proceedings of the National Academy of Sciences, Vol. 107, Issue 23
Structure of the Escherichia coli RNA Polymerase Subunit Amino-Terminal Domain
journal, July 1998
- Zhang, G.
- Science, Vol. 281, Issue 5374
Random mutagenesis of global transcription factor cAMP receptor protein for improved osmotolerance
journal, December 2011
- Zhang, Hongfang; Chong, Huiqing; Ching, Chi Bun
- Biotechnology and Bioengineering, Vol. 109, Issue 5
Improving Escherichia coli FucO for Furfural Tolerance by Saturation Mutagenesis of Individual Amino Acid Positions
journal, March 2013
- Zheng, Huabao; Wang, Xuan; Yomano, Lorraine P.
- Applied and Environmental Microbiology, Vol. 79, Issue 10
The rpoB mutants destabilizing initiation complexes at stringently controlled promoters behave like "stringent" RNA polymerases in Escherichia coli
journal, March 1998
- Zhou, Y. N.; Jin, D. J.
- Proceedings of the National Academy of Sciences, Vol. 95, Issue 6
Isolation and Characterization of RNA Polymerase rpoB Mutations That Alter Transcription Slippage during Elongation in Escherichia coli
journal, December 2012
- Zhou, Yan Ning; Lubkowska, Lucyna; Hui, Monica
- Journal of Biological Chemistry, Vol. 288, Issue 4
Works referencing / citing this record:
Improved succinate production from galactose‐rich feedstocks by engineered Escherichia coli under anaerobic conditions
journal, January 2020
- Zhu, Fayin; San, Ka‐Yiu; Bennett, George N.
- Biotechnology and Bioengineering, Vol. 117, Issue 4
Retrons and their applications in genome engineering
journal, October 2019
- Simon, Anna J.; Ellington, Andrew D.; Finkelstein, Ilya J.
- Nucleic Acids Research, Vol. 47, Issue 21
Synthetic biology for evolutionary engineering: from perturbation of genotype to acquisition of desired phenotype
journal, May 2019
- Yang, Jina; Kim, Beomhee; Kim, Gi Yeon
- Biotechnology for Biofuels, Vol. 12, Issue 1
Retrons and their applications in genome engineering
journal, October 2019
- Simon, Anna J.; Ellington, Andrew D.; Finkelstein, Ilya J.
- Nucleic Acids Research, Vol. 47, Issue 21