DOE PAGES title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: Toward industrial production of isoprenoids in Escherichia coli: Lessons learned from CRISPR-Cas9 based optimization of a chromosomally integrated mevalonate pathway

Abstract

Abstract Escherichia coli has been the organism of choice for the production of different chemicals by engineering native and heterologous pathways. In the present study, we simultaneously address some of the main issues associated with E. coli as an industrial platform for isoprenoids, including an inability to grow on sucrose, a lack of endogenous control over toxic mevalonate (MVA) pathway intermediates, and the limited pathway engineering into the chromosome. As a proof of concept, we generated an E. coli DH1 strain able to produce the isoprenoid bisabolene from sucrose by integrating the cscAKB operon into the chromosome and by expressing a heterologous MVA pathway under stress‐responsive control. Production levels dropped dramatically relative to plasmid‐mediated expression when the entire pathway was integrated into the chromosome. In order to optimize the chromosomally integrated MVA pathway, we established a CRISPR‐Cas9 system to rapidly and systematically replace promoter sequences. This strategy led to higher pathway expression and a fivefold improvement in bisabolene production. More interestingly, we analyzed proteomics data sets to understand and address some of the challenges associated with metabolic engineering of the chromosomally integrated pathway. This report shows that integrating plasmid‐optimized operons into the genome and making them work optimally is notmore » a straightforward task and any poor engineering choices on the chromosome may lead to cell death rather than just resulting in low titers. Based on these results, we also propose directions for chromosomal metabolic engineering.« less

Authors:
 [1];  [2];  [1];  [1];  [1];  [1];  [1]; ORCiD logo [3]; ORCiD logo [3];  [4]; ORCiD logo [1]
  1. Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
  2. Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Osaka Municipal Technical Research Inst. (Japan)
  3. Univ. of Queensland, St. Lucia, QLD (Australia)
  4. Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Technical Univ. of Denmark, Horsholm (Denmark); Univ. of California, Berkeley, CA (United States)
Publication Date:
Research Org.:
Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Biological and Environmental Research (BER)
OSTI Identifier:
1530349
Alternate Identifier(s):
OSTI ID: 1419808
Grant/Contract Number:  
AC02-05CH11231; DE‐AC02‐05CH11231
Resource Type:
Accepted Manuscript
Journal Name:
Biotechnology and Bioengineering
Additional Journal Information:
Journal Volume: 115; Journal Issue: 4; Journal ID: ISSN 0006-3592
Publisher:
Wiley
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES

Citation Formats

Alonso‐Gutierrez, Jorge, Koma, Daisuke, Hu, Qijun, Yang, Yuchen, Chan, Leanne J. G., Petzold, Christopher J., Adams, Paul D., Vickers, Claudia E., Nielsen, Lars K., Keasling, Jay D., and Lee, Taek S. Toward industrial production of isoprenoids in Escherichia coli: Lessons learned from CRISPR-Cas9 based optimization of a chromosomally integrated mevalonate pathway. United States: N. p., 2017. Web. doi:10.1002/bit.26530.
Alonso‐Gutierrez, Jorge, Koma, Daisuke, Hu, Qijun, Yang, Yuchen, Chan, Leanne J. G., Petzold, Christopher J., Adams, Paul D., Vickers, Claudia E., Nielsen, Lars K., Keasling, Jay D., & Lee, Taek S. Toward industrial production of isoprenoids in Escherichia coli: Lessons learned from CRISPR-Cas9 based optimization of a chromosomally integrated mevalonate pathway. United States. https://doi.org/10.1002/bit.26530
Alonso‐Gutierrez, Jorge, Koma, Daisuke, Hu, Qijun, Yang, Yuchen, Chan, Leanne J. G., Petzold, Christopher J., Adams, Paul D., Vickers, Claudia E., Nielsen, Lars K., Keasling, Jay D., and Lee, Taek S. Tue . "Toward industrial production of isoprenoids in Escherichia coli: Lessons learned from CRISPR-Cas9 based optimization of a chromosomally integrated mevalonate pathway". United States. https://doi.org/10.1002/bit.26530. https://www.osti.gov/servlets/purl/1530349.
@article{osti_1530349,
title = {Toward industrial production of isoprenoids in Escherichia coli: Lessons learned from CRISPR-Cas9 based optimization of a chromosomally integrated mevalonate pathway},
author = {Alonso‐Gutierrez, Jorge and Koma, Daisuke and Hu, Qijun and Yang, Yuchen and Chan, Leanne J. G. and Petzold, Christopher J. and Adams, Paul D. and Vickers, Claudia E. and Nielsen, Lars K. and Keasling, Jay D. and Lee, Taek S.},
abstractNote = {Abstract Escherichia coli has been the organism of choice for the production of different chemicals by engineering native and heterologous pathways. In the present study, we simultaneously address some of the main issues associated with E. coli as an industrial platform for isoprenoids, including an inability to grow on sucrose, a lack of endogenous control over toxic mevalonate (MVA) pathway intermediates, and the limited pathway engineering into the chromosome. As a proof of concept, we generated an E. coli DH1 strain able to produce the isoprenoid bisabolene from sucrose by integrating the cscAKB operon into the chromosome and by expressing a heterologous MVA pathway under stress‐responsive control. Production levels dropped dramatically relative to plasmid‐mediated expression when the entire pathway was integrated into the chromosome. In order to optimize the chromosomally integrated MVA pathway, we established a CRISPR‐Cas9 system to rapidly and systematically replace promoter sequences. This strategy led to higher pathway expression and a fivefold improvement in bisabolene production. More interestingly, we analyzed proteomics data sets to understand and address some of the challenges associated with metabolic engineering of the chromosomally integrated pathway. This report shows that integrating plasmid‐optimized operons into the genome and making them work optimally is not a straightforward task and any poor engineering choices on the chromosome may lead to cell death rather than just resulting in low titers. Based on these results, we also propose directions for chromosomal metabolic engineering.},
doi = {10.1002/bit.26530},
journal = {Biotechnology and Bioengineering},
number = 4,
volume = 115,
place = {United States},
year = {Tue Dec 26 00:00:00 EST 2017},
month = {Tue Dec 26 00:00:00 EST 2017}
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record

Citation Metrics:
Cited by: 27 works
Citation information provided by
Web of Science

Save / Share:

Works referenced in this record:

Engineered reversal of the β-oxidation cycle for the synthesis of fuels and chemicals
journal, August 2011

  • Dellomonaco, Clementina; Clomburg, James M.; Miller, Elliot N.
  • Nature, Vol. 476, Issue 7360, p. 355-359
  • DOI: 10.1038/nature10333

Sustainable reduction of bioreactor contamination in an industrial fermentation pilot plant
journal, October 2006

  • Junker, Beth; Lester, Michael; Leporati, James
  • Journal of Bioscience and Bioengineering, Vol. 102, Issue 4
  • DOI: 10.1263/jbb.102.251

Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes
journal, May 1986


Principal component analysis of proteomics (PCAP) as a tool to direct metabolic engineering
journal, March 2015


The genome sequence of E. coli W (ATCC 9637): comparative genome analysis and an improved genome-scale reconstruction of E. coli
journal, January 2011

  • Archer, Colin T.; Kim, Jihyun F.; Jeong, Haeyoung
  • BMC Genomics, Vol. 12, Issue 1
  • DOI: 10.1186/1471-2164-12-9

Substantial improvements in methyl ketone production in E. coli and insights on the pathway from in vitro studies
journal, November 2014


Refactoring the nitrogen fixation gene cluster from Klebsiella oxytoca
journal, April 2012

  • Temme, K.; Zhao, D.; Voigt, C. A.
  • Proceedings of the National Academy of Sciences, Vol. 109, Issue 18
  • DOI: 10.1073/pnas.1120788109

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
  • DOI: 10.1038/nature08187

BglBrick vectors and datasheets: A synthetic biology platform for gene expression
journal, January 2011

  • Lee, Taek; Krupa, Rachel A.; Zhang, Fuzhong
  • Journal of Biological Engineering, Vol. 5, Issue 1
  • DOI: 10.1186/1754-1611-5-12

Antibiotic resistance is the quintessential One Health issue
journal, July 2016

  • Robinson, T. P.; Bu, D. P.; Carrique-Mas, J.
  • Transactions of The Royal Society of Tropical Medicine and Hygiene, Vol. 110, Issue 7
  • DOI: 10.1093/trstmh/trw048

Industrial fermentation of renewable diesel fuels
journal, June 2011


Cloning in Escherichia coli and molecular analysis of the sucrose system of the Salmonella plasmid SCR-53
journal, January 1985

  • Garc�a, Jos� L.
  • MGG Molecular & General Genetics, Vol. 201, Issue 3
  • DOI: 10.1007/BF00331358

Microbial Biosynthesis of Alkanes
journal, July 2010


Evolution in Regulatory Regions Rapidly Compensates the Cost of Nonoptimal Codon Usage
journal, April 2010

  • Amoros-Moya, D.; Bedhomme, S.; Hermann, M.
  • Molecular Biology and Evolution, Vol. 27, Issue 9
  • DOI: 10.1093/molbev/msq103

Synthesis of three advanced biofuels from ionic liquid-pretreated switchgrass using engineered Escherichia coli
journal, November 2011

  • Bokinsky, G.; Peralta-Yahya, P. P.; George, A.
  • Proceedings of the National Academy of Sciences, Vol. 108, Issue 50, p. 19949-19954
  • DOI: 10.1073/pnas.1106958108

Modular control of multiple pathways using engineered orthogonal T7 polymerases
journal, June 2012

  • Temme, Karsten; Hill, Rena; Segall-Shapiro, Thomas H.
  • Nucleic Acids Research, Vol. 40, Issue 17
  • DOI: 10.1093/nar/gks597

Stabilized gene duplication enables long-term selection-free heterologous pathway expression
journal, July 2009

  • Tyo, Keith E. J.; Ajikumar, Parayil Kumaran; Stephanopoulos, Gregory
  • Nature Biotechnology, Vol. 27, Issue 8
  • DOI: 10.1038/nbt.1555

Engineering dynamic pathway regulation using stress-response promoters
journal, October 2013

  • Dahl, Robert H.; Zhang, Fuzhong; Alonso-Gutierrez, Jorge
  • Nature Biotechnology, Vol. 31, Issue 11
  • DOI: 10.1038/nbt.2689

High-level semi-synthetic production of the potent antimalarial artemisinin
journal, April 2013

  • Paddon, C. J.; Westfall, P. J.; Pitera, D. J.
  • Nature, Vol. 496, Issue 7446
  • DOI: 10.1038/nature12051

Enzymatic assembly of DNA molecules up to several hundred kilobases
journal, April 2009

  • Gibson, Daniel G.; Young, Lei; Chuang, Ray-Yuan
  • Nature Methods, Vol. 6, Issue 5, p. 343-345
  • DOI: 10.1038/nmeth.1318

Metabolic engineering of Escherichia coli for direct production of 1,4-butanediol
journal, May 2011

  • Yim, Harry; Haselbeck, Robert; Niu, Wei
  • Nature Chemical Biology, Vol. 7, Issue 7
  • DOI: 10.1038/nchembio.580

Targeted proteomics for metabolic pathway optimization: Application to terpene production
journal, March 2011

  • Redding-Johanson, Alyssa M.; Batth, Tanveer S.; Chan, Rossana
  • Metabolic Engineering, Vol. 13, Issue 2
  • DOI: 10.1016/j.ymben.2010.12.005

An environmental life cycle assessment comparing Australian sugarcane with US corn and UK sugar beet as producers of sugars for fermentation
journal, December 2008


Non-fermentative pathways for synthesis of branched-chain higher alcohols as biofuels
journal, January 2008

  • Atsumi, Shota; Hanai, Taizo; Liao, James C.
  • Nature, Vol. 451, Issue 7174, p. 86-89
  • DOI: 10.1038/nature06450

Circuitry linking the Csr and stringent response global regulatory systems: Csr and stringent response systems
journal, May 2011


Technoeconomic analysis of renewable aviation fuel from microalgae, Pongamia pinnata , and sugarcane
journal, April 2013

  • Klein-Marcuschamer, Daniel; Turner, Christopher; Allen, Mark
  • Biofuels, Bioproducts and Biorefining, Vol. 7, Issue 4
  • DOI: 10.1002/bbb.1404

The double-strand-break repair model for recombination
journal, May 1983


A convenient method for multiple insertions of desired genes into target loci on the Escherichia coli chromosome
journal, November 2011

  • Koma, Daisuke; Yamanaka, Hayato; Moriyoshi, Kunihiko
  • Applied Microbiology and Biotechnology, Vol. 93, Issue 2
  • DOI: 10.1007/s00253-011-3735-z

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
  • DOI: 10.1128/AEM.04023-14

Studies on transformation of Escherichia coli with plasmids
journal, June 1983


CRMAGE: CRISPR Optimized MAGE Recombineering
journal, January 2016

  • Ronda, Carlotta; Pedersen, Lasse Ebdrup; Sommer, Morten O. A.
  • Scientific Reports, Vol. 6, Article No. 19452
  • DOI: 10.1038/srep19452

Metabolic engineering of Escherichia coli using CRISPR–Cas9 meditated genome editing
journal, September 2015


Chromosome Replication and Segregation in Bacteria
journal, December 2012


Rewriting yeast central carbon metabolism for industrial isoprenoid production
journal, September 2016

  • Meadows, Adam L.; Hawkins, Kristy M.; Tsegaye, Yoseph
  • Nature, Vol. 537, Issue 7622
  • DOI: 10.1038/nature19769

Automated design of synthetic ribosome binding sites to control protein expression
journal, October 2009

  • Salis, Howard M.; Mirsky, Ethan A.; Voigt, Christopher A.
  • Nature Biotechnology, Vol. 27, Issue 10, p. 946-950
  • DOI: 10.1038/nbt.1568

Engineering a mevalonate pathway in Escherichia coli for production of terpenoids
journal, June 2003

  • Martin, Vincent J. J.; Pitera, Douglas J.; Withers, Sydnor T.
  • Nature Biotechnology, Vol. 21, Issue 7
  • DOI: 10.1038/nbt833

Precise Manipulation of Chromosomes in Vivo Enables Genome-Wide Codon Replacement
journal, July 2011

  • Isaacs, Farren J.; Carr, Peter A.; Wang, Harris H.
  • Science, Vol. 333, Issue 6040, p. 348-353
  • DOI: 10.1126/science.1205822

A transferable sucrose utilization approach for non-sucrose-utilizing Escherichia coli strains
journal, September 2012


High-Level Production of Amorpha-4,11-Diene, a Precursor of the Antimalarial Agent Artemisinin, in Escherichia coli
journal, February 2009


Metabolic engineering of Escherichia coli for limonene and perillyl alcohol production
journal, September 2013


Correlation analysis of targeted proteins and metabolites to assess and engineer microbial isopentenol production: Targeted Proteomics-Based Correlation Analysis
journal, May 2014

  • George, Kevin W.; Chen, Amy; Jain, Aakriti
  • Biotechnology and Bioengineering, Vol. 111, Issue 8
  • DOI: 10.1002/bit.25226

Microbial production of fatty-acid-derived fuels and chemicals from plant biomass
journal, January 2010

  • Steen, Eric J.; Kang, Yisheng; Bokinsky, Gregory
  • Nature, Vol. 463, Issue 7280, p. 559-562
  • DOI: 10.1038/nature08721

A brief history of synthetic biology
journal, April 2014

  • Cameron, D. Ewen; Bashor, Caleb J.; Collins, James J.
  • Nature Reviews Microbiology, Vol. 12, Issue 5
  • DOI: 10.1038/nrmicro3239

Genome-scale genetic engineering in Escherichia coli
journal, November 2013


Site-specific chromosomal integration of large synthetic constructs
journal, January 2010

  • Kuhlman, Thomas E.; Cox, Edward C.
  • Nucleic Acids Research, Vol. 38, Issue 6
  • DOI: 10.1093/nar/gkp1193

BglBricks: A flexible standard for biological part assembly
journal, January 2010

  • Anderson, J. Christopher; Dueber, John E.; Leguia, Mariana
  • Journal of Biological Engineering, Vol. 4, Issue 1
  • DOI: 10.1186/1754-1611-4-1

A constructive debate
journal, December 2012

  • Keasling, Jay D.; Mendoza, Abraham; Baran, Phil S.
  • Nature, Vol. 492, Issue 7428
  • DOI: 10.1038/492188a

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
  • DOI: 10.1073/pnas.120163297

Development of sucrose-utilizing Escherichia coli K-12 strain by cloning β-fructofuranosidases and its application for l-threonine production
journal, August 2010

  • Lee, Jeong Wook; Choi, Sol; Park, Jin Hwan
  • Applied Microbiology and Biotechnology, Vol. 88, Issue 4
  • DOI: 10.1007/s00253-010-2825-7

Balancing a heterologous mevalonate pathway for improved isoprenoid production in Escherichia coli
journal, March 2007


Genome-scale promoter engineering by coselection MAGE
journal, April 2012

  • Wang, Harris H.; Kim, Hwangbeom; Cong, Le
  • Nature Methods, Vol. 9, Issue 6, p. 591-593
  • DOI: 10.1038/nmeth.1971

Manufacturing Molecules Through Metabolic Engineering
journal, December 2010


Regulatory consequences of gene translocation in bacteria
journal, July 2012

  • Block, Dena H. S.; Hussein, Razika; Liang, Lusha W.
  • Nucleic Acids Research, Vol. 40, Issue 18
  • DOI: 10.1093/nar/gks694

Lambda Red Recombineering in Escherichia coli Occurs Through a Fully Single-Stranded Intermediate
journal, September 2010


Chromosome position effects on gene expression in Escherichia coli K-12
journal, September 2014

  • Bryant, Jack A.; Sellars, Laura E.; Busby, Stephen J. W.
  • Nucleic Acids Research, Vol. 42, Issue 18
  • DOI: 10.1093/nar/gku828

Isoprenoid Pathway Optimization for Taxol Precursor Overproduction in Escherichia coli
journal, September 2010


RNA-guided editing of bacterial genomes using CRISPR-Cas systems
journal, January 2013

  • Jiang, Wenyan; Bikard, David; Cox, David
  • Nature Biotechnology, Vol. 31, Issue 3, p. 233-239
  • DOI: 10.1038/nbt.2508

Antibiotic resistance in the wild: an eco-evolutionary perspective
journal, January 2017

  • Hiltunen, Teppo; Virta, Marko; Laine, Anna-Liisa
  • Philosophical Transactions of the Royal Society B: Biological Sciences, Vol. 372, Issue 1712
  • DOI: 10.1098/rstb.2016.0039

Analysis of gene control signals by DNA fusion and cloning in Escherichia coli
journal, April 1980


Works referencing / citing this record:

Techniques for chromosomal integration and expression optimization in Escherichia coli
journal, July 2018

  • Ou, Bingming; Garcia, Carolina; Wang, Yejun
  • Biotechnology and Bioengineering, Vol. 115, Issue 10
  • DOI: 10.1002/bit.26790

Modular biomanufacturing for a sustainable production of terpenoid-based insect deterrents
journal, January 2018

  • Mischko, Wolfgang; Hirte, Max; Roehrer, Simon
  • Green Chemistry, Vol. 20, Issue 11
  • DOI: 10.1039/c8gc00434j

CRISPR Gene Perturbations Provide Insights for Improving Bacterial Biofuel Tolerance
journal, September 2018

  • Otoupal, Peter B.; Chatterjee, Anushree
  • Frontiers in Bioengineering and Biotechnology, Vol. 6
  • DOI: 10.3389/fbioe.2018.00122

Microbial Platform for Terpenoid Production: Escherichia coli and Yeast
journal, October 2018


Advancement of Metabolic Engineering Assisted by Synthetic Biology
journal, December 2018


Bacterial terpene biosynthesis: challenges and opportunities for pathway engineering
journal, January 2019

  • Helfrich, Eric J. N.; Lin, Geng-Min; Voigt, Christopher A.
  • Beilstein Journal of Organic Chemistry, Vol. 15
  • DOI: 10.3762/bjoc.15.283

Identification of parallel and divergent optimization solutions for homologous metabolic enzymes
journal, June 2018

  • Standaert, Robert F.; Giannone, Richard J.; Michener, Joshua K.
  • Metabolic Engineering Communications, Vol. 6
  • DOI: 10.1016/j.meteno.2018.04.002

Challenges and tackles in metabolic engineering for microbial production of carotenoids
journal, March 2019


Microbial Platform for Terpenoid Production: Escherichia coli and Yeast
journal, October 2018