skip to main content
DOE PAGES title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: Cell-free biosynthesis of limonene using enzyme-enriched Escherichia coli lysates

Abstract

Isoprenoids are an attractive class of metabolites for enzymatic synthesis from renewable substrates. However, metabolic engineering of microorganisms for monoterpenoid production is limited by the need for time-consuming, and often non-intuitive, combinatorial tuning of biosynthetic pathway variations to meet design criteria. Towards alleviating this limitation, the goal of this work was to build a modular, cell-free platform for construction and testing of monoterpenoid pathways, using the fragrance and flavoring molecule limonene as a model. In this platform, multiple Escherichia coli lysates, each enriched with a single overexpressed pathway enzyme, are mixed to construct the full biosynthetic pathway. First, we show the ability to synthesize limonene from six enriched lysates with mevalonate substrate, an adenosine triphosphate (ATP) source, and cofactors. Next, we extend the pathway to use glucose as a substrate, which relies on native metabolism in the extract to convert glucose to acetyl-CoA along with three additional enzymes to convert acetyl-CoA to mevalonate. We find that the native E. coli farnesyl diphosphate synthase (IspA) is active in the lysate and diverts flux from the pathway intermediate geranyl pyrophospahte to farnesyl pyrophsophate and the byproduct farnesol. By adjusting the relative levels of cofactors NAD+, ATP and CoA, the system can synthesizemore » 0.66 mM (90.2 mg l-1) limonene over 24 h, a productivity of 3.8 mg l-1 h-1. Our results highlight the flexibility of crude lysates to sustain complex metabolism and, by activating a glucose-to-limonene pathway with 9 heterologous enzymes encompassing 20 biosynthetic steps, expands an approach of using enzyme-enriched lysates for constructing, characterizing and prototyping enzymatic pathways.« less

Authors:
 [1];  [1];  [2]
  1. Department of Chemical and Biological Engineering, Northwestern University, Evanston, IL, USA
  2. Department of Chemical and Biological Engineering, Northwestern University, Evanston, IL, USA, Chemistry of Life Processes Institute, Northwestern University, Evanston, IL, USA, Center for Synthetic Biology, Northwestern University, Evanston, IL, USA, Robert H. Lurie Comprehensive Cancer Center, Simpson Querrey Institute Northwestern University, Chicago, IL, USA, Simpson Querrey Institute Northwestern University, Chicago, IL, USA
Publication Date:
Research Org.:
Northwestern Univ., Evanston, IL (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Biological and Environmental Research (BER). Biological Systems Science Division; David and Lucile Packard Foundation; Dreyfus; Dreyfus Teacher-Scholar Program; National Institutes of Health (NIH)
OSTI Identifier:
1496316
Alternate Identifier(s):
OSTI ID: 1660452
Grant/Contract Number:  
SC0018249; 503280
Resource Type:
Published Article
Journal Name:
Synthetic Biology
Additional Journal Information:
Journal Name: Synthetic Biology Journal Volume: 4 Journal Issue: 1; Journal ID: ISSN 2397-7000
Publisher:
Oxford University Press
Country of Publication:
United Kingdom
Language:
English
Subject:
cell-free metabolic engineering; limonene; metabolic pathway prototyping; cell-free synthetic biology; E. coli crude lysate

Citation Formats

Dudley, Quentin M., Nash, Connor J., and Jewett, Michael C. Cell-free biosynthesis of limonene using enzyme-enriched Escherichia coli lysates. United Kingdom: N. p., 2019. Web. doi:10.1093/synbio/ysz003.
Dudley, Quentin M., Nash, Connor J., & Jewett, Michael C. Cell-free biosynthesis of limonene using enzyme-enriched Escherichia coli lysates. United Kingdom. doi:10.1093/synbio/ysz003.
Dudley, Quentin M., Nash, Connor J., and Jewett, Michael C. Mon . "Cell-free biosynthesis of limonene using enzyme-enriched Escherichia coli lysates". United Kingdom. doi:10.1093/synbio/ysz003.
@article{osti_1496316,
title = {Cell-free biosynthesis of limonene using enzyme-enriched Escherichia coli lysates},
author = {Dudley, Quentin M. and Nash, Connor J. and Jewett, Michael C.},
abstractNote = {Isoprenoids are an attractive class of metabolites for enzymatic synthesis from renewable substrates. However, metabolic engineering of microorganisms for monoterpenoid production is limited by the need for time-consuming, and often non-intuitive, combinatorial tuning of biosynthetic pathway variations to meet design criteria. Towards alleviating this limitation, the goal of this work was to build a modular, cell-free platform for construction and testing of monoterpenoid pathways, using the fragrance and flavoring molecule limonene as a model. In this platform, multiple Escherichia coli lysates, each enriched with a single overexpressed pathway enzyme, are mixed to construct the full biosynthetic pathway. First, we show the ability to synthesize limonene from six enriched lysates with mevalonate substrate, an adenosine triphosphate (ATP) source, and cofactors. Next, we extend the pathway to use glucose as a substrate, which relies on native metabolism in the extract to convert glucose to acetyl-CoA along with three additional enzymes to convert acetyl-CoA to mevalonate. We find that the native E. coli farnesyl diphosphate synthase (IspA) is active in the lysate and diverts flux from the pathway intermediate geranyl pyrophospahte to farnesyl pyrophsophate and the byproduct farnesol. By adjusting the relative levels of cofactors NAD+, ATP and CoA, the system can synthesize 0.66 mM (90.2 mg l-1) limonene over 24 h, a productivity of 3.8 mg l-1 h-1. Our results highlight the flexibility of crude lysates to sustain complex metabolism and, by activating a glucose-to-limonene pathway with 9 heterologous enzymes encompassing 20 biosynthetic steps, expands an approach of using enzyme-enriched lysates for constructing, characterizing and prototyping enzymatic pathways.},
doi = {10.1093/synbio/ysz003},
journal = {Synthetic Biology},
number = 1,
volume = 4,
place = {United Kingdom},
year = {2019},
month = {1}
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record
DOI: 10.1093/synbio/ysz003

Save / Share:

Works referenced in this record:

Expression optimization and synthetic gene networks in cell-free systems
journal, December 2011

  • Karig, David K.; Iyer, Sukanya; Simpson, Michael L.
  • Nucleic Acids Research, Vol. 40, Issue 8
  • DOI: 10.1093/nar/gkr1191

In Vitro Reconstitution and Analysis of the 6-Deoxyerythronolide B Synthase
journal, October 2013

  • Lowry, Brian; Robbins, Thomas; Weng, Chih-Hisang
  • Journal of the American Chemical Society, Vol. 135, Issue 45
  • DOI: 10.1021/ja409048k

Customized optimization of metabolic pathways by combinatorial transcriptional engineering
journal, June 2012

  • Du, Jing; Yuan, Yongbo; Si, Tong
  • Nucleic Acids Research, Vol. 40, Issue 18
  • DOI: 10.1093/nar/gks549

Cell-free prototyping strategies for enhancing the sustainable production of polyhydroxyalkanoates bioplastics
journal, January 2018

  • Kelwick, Richard; Ricci, Luca; Chee, Soo Mei
  • Synthetic Biology, Vol. 3, Issue 1
  • DOI: 10.1093/synbio/ysy016

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


Enhanced protein and biochemical production using CRISPRi-based growth switches
journal, November 2016


High-throughput preparation methods of crude extract for robust cell-free protein synthesis
journal, March 2015

  • Kwon, Yong-Chan; Jewett, Michael C.
  • Scientific Reports, Vol. 5, Issue 1
  • DOI: 10.1038/srep08663

Cell-free metabolic engineering: Biomanufacturing beyond the cell
journal, October 2014

  • Dudley, Quentin M.; Karim, Ashty S.; Jewett, Michael C.
  • Biotechnology Journal, Vol. 10, Issue 1
  • DOI: 10.1002/biot.201400330

Directed evolution of Escherichia coli farnesyl diphosphate synthase (IspA) reveals novel structural determinants of chain length specificity
journal, January 2005


New biotechnology paradigm: cell-free biosystems for biomanufacturing
journal, January 2013

  • Rollin, Joseph A.; Tam, Tsz Kin; Zhang, Y. -H. Percival
  • Green Chemistry, Vol. 15, Issue 7
  • DOI: 10.1039/c3gc40625c

Cell-free synthetic biology for in vitro biosynthesis of pharmaceutical natural products
journal, June 2018


Characterization of a Feedback-Resistant Mevalonate Kinase from the Archaeon Methanosarcina mazei
journal, September 2011

  • Primak, Yuliya A.; Du, Mai; Miller, Michael C.
  • Applied and Environmental Microbiology, Vol. 77, Issue 21
  • DOI: 10.1128/AEM.05761-11

In Vitro Reconstruction of Nonribosomal Peptide Biosynthesis Directly from DNA Using Cell-Free Protein Synthesis
journal, August 2016


Synthetic biology and the development of tools for metabolic engineering
journal, May 2012


In vitro reconstitution guide for targeted synthetic metabolism of chemicals, nutraceuticals and drug precursors
journal, March 2016


A kinetic-based approach to understanding heterologous mevalonate pathway function in E. coli : A Kinetic-Based Approach to Understanding Heterologous
journal, August 2014

  • Weaver, Lane J.; Sousa, Mirta M. L.; Wang, George
  • Biotechnology and Bioengineering, Vol. 112, Issue 1
  • DOI: 10.1002/bit.25323

CRISPR interference-guided balancing of a biosynthetic mevalonate pathway increases terpenoid production
journal, November 2016


Systems metabolic engineering of microorganisms for natural and non-natural chemicals
journal, May 2012

  • Lee, Jeong Wook; Na, Dokyun; Park, Jong Myoung
  • Nature Chemical Biology, Vol. 8, Issue 6
  • DOI: 10.1038/nchembio.970

Engineering Limonene and Bisabolene Production in Wild Type and a Glycogen-Deficient Mutant of Synechococcus sp. PCC 7002
journal, June 2014

  • Davies, Fiona K.; Work, Victoria H.; Beliaev, Alexander S.
  • Frontiers in Bioengineering and Biotechnology, Vol. 2
  • DOI: 10.3389/fbioe.2014.00021

Functional optimization of gene clusters by combinatorial design and assembly
journal, November 2014

  • Smanski, Michael J.; Bhatia, Swapnil; Zhao, Dehua
  • Nature Biotechnology, Vol. 32, Issue 12, p. 1241-1249
  • DOI: 10.1038/nbt.3063

Synthetic microbial consortia enable rapid assembly of pure translation machinery
journal, November 2017

  • Villarreal, Fernando; Contreras-Llano, Luis E.; Chavez, Michael
  • Nature Chemical Biology, Vol. 14, Issue 1
  • DOI: 10.1038/nchembio.2514

Establishing a High-Yielding Cell-Free Protein Synthesis Platform Derived from Vibrio natriegens
journal, August 2018

  • Des Soye, Benjamin J.; Davidson, Samuel R.; Weinstock, Matthew T.
  • ACS Synthetic Biology, Vol. 7, Issue 9
  • DOI: 10.1021/acssynbio.8b00252

Cell-Free Mixing of Escherichia coli Crude Extracts to Prototype and Rationally Engineer High-Titer Mevalonate Synthesis
journal, July 2016

  • Dudley, Quentin M.; Anderson, Kim C.; Jewett, Michael C.
  • ACS Synthetic Biology, Vol. 5, Issue 12
  • DOI: 10.1021/acssynbio.6b00154

Synthetic protein scaffolds provide modular control over metabolic flux
journal, August 2009

  • Dueber, John E.; Wu, Gabriel C.; Malmirchegini, G. Reza
  • Nature Biotechnology, Vol. 27, Issue 8, p. 753-759
  • DOI: 10.1038/nbt.1557

Lysate of engineered Escherichia coli supports high-level conversion of glucose to 2,3-butanediol
journal, November 2015


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

Mono and diterpene production in Escherichia coli
journal, January 2004

  • Reiling, K. Kinkead; Yoshikuni, Yasuo; Martin, Vincent J.J.
  • Biotechnology and Bioengineering, Vol. 87, Issue 2, p. 200-212
  • DOI: 10.1002/bit.20128

Multivariate modular metabolic engineering for pathway and strain optimization
journal, October 2014

  • Biggs, Bradley Walters; De Paepe, Brecht; Santos, Christine Nicole S.
  • Current Opinion in Biotechnology, Vol. 29
  • DOI: 10.1016/j.copbio.2014.05.005

Energizing cell-free protein synthesis with glucose metabolism
journal, January 2005

  • Calhoun, Kara A.; Swartz, James R.
  • Biotechnology and Bioengineering, Vol. 90, Issue 5
  • DOI: 10.1002/bit.20449

Enzymatic total synthesis of enterocin polyketides
journal, August 2007


Prolonging cell-free protein synthesis with a novel ATP regeneration system
journal, January 1999


Terpenoid biomaterials
journal, May 2008


Statistical Experimental Design Guided Optimization of a One-Pot Biphasic Multienzyme Total Synthesis of Amorpha-4,11-diene
journal, November 2013


Enhanced limonene production by optimizing the expression of limonene biosynthesis and MEP pathway genes in E. coli
journal, August 2014


Transforming biochemical engineering with cell-free biology
journal, December 2011


In Vitro Reconstitution of Metabolic Pathways: Insights into Nature’s Chemical Logic
journal, March 2015


An integrated cell-free metabolic platform for protein production and synthetic biology
journal, October 2008

  • Jewett, Michael C.; Calhoun, Kara A.; Voloshin, Alexei
  • Molecular Systems Biology, Vol. 4, Issue 1, Article No. 220
  • DOI: 10.1038/msb.2008.57

Biotechnological production of limonene in microorganisms
journal, February 2016

  • Jongedijk, Esmer; Cankar, Katarina; Buchhaupt, Markus
  • Applied Microbiology and Biotechnology, Vol. 100, Issue 7
  • DOI: 10.1007/s00253-016-7337-7

A Pressure Test to Make 10 Molecules in 90 Days: External Evaluation of Methods to Engineer Biology
journal, February 2018

  • Casini, Arturo; Chang, Fang-Yuan; Eluere, Raissa
  • Journal of the American Chemical Society, Vol. 140, Issue 12
  • DOI: 10.1021/jacs.7b13292

Gene Circuit Performance Characterization and Resource Usage in a Cell-Free “Breadboard”
journal, April 2014

  • Siegal-Gaskins, Dan; Tuza, Zoltan A.; Kim, Jongmin
  • ACS Synthetic Biology, Vol. 3, Issue 6
  • DOI: 10.1021/sb400203p

Engineering the productivity of recombinant Escherichia coli for limonene formation from glycerol in minimal media
journal, June 2014

  • Willrodt, Christian; David, Christian; Cornelissen, Sjef
  • Biotechnology Journal, Vol. 9, Issue 8
  • DOI: 10.1002/biot.201400023

Synthetic non-oxidative glycolysis enables complete carbon conservation
journal, September 2013

  • Bogorad, Igor W.; Lin, Tzu-Shyang; Liao, James C.
  • Nature, Vol. 502, Issue 7473
  • DOI: 10.1038/nature12575

Cell-free protein synthesis: Applications come of age
journal, September 2012


Acute Limonene Toxicity in Escherichia coli Is Caused by Limonene Hydroperoxide and Alleviated by a Point Mutation in Alkyl Hydroperoxidase AhpC
journal, May 2015

  • Chubukov, Victor; Mingardon, Florence; Schackwitz, Wendy
  • Applied and Environmental Microbiology, Vol. 81, Issue 14
  • DOI: 10.1128/AEM.01102-15

Enzymatic Menthol Production: One-Pot Approach Using Engineered Escherichia coli
journal, June 2015

  • Toogood, Helen S.; Cheallaigh, Aisling Ní; Tait, Shirley
  • ACS Synthetic Biology, Vol. 4, Issue 10
  • DOI: 10.1021/acssynbio.5b00092

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

A synthetic biochemistry platform for cell free production of monoterpenes from glucose
journal, May 2017

  • Korman, Tyler P.; Opgenorth, Paul H.; Bowie, James U.
  • Nature Communications, Vol. 8, Issue 1
  • DOI: 10.1038/ncomms15526

Metabolic engineering of oleaginous yeast Yarrowia lipolytica for limonene overproduction
journal, October 2016


A synthetic biochemistry molecular purge valve module that maintains redox balance
journal, June 2014

  • Opgenorth, Paul H.; Korman, Tyler P.; Bowie, James U.
  • Nature Communications, Vol. 5, Issue 1
  • DOI: 10.1038/ncomms5113

Functional characterization of a geraniol synthase-encoding gene from Camptotheca acuminata and its application in production of geraniol in Escherichia coli
journal, June 2016

  • Chen, Fei; Li, Wei; Jiang, Liangzhen
  • Journal of Industrial Microbiology & Biotechnology, Vol. 43, Issue 9
  • DOI: 10.1007/s10295-016-1802-2

Tuning Gene Expression in Yarrowia lipolytica by a Hybrid Promoter Approach
journal, September 2011

  • Blazeck, John; Liu, Leqian; Redden, Heidi
  • Applied and Environmental Microbiology, Vol. 77, Issue 22
  • DOI: 10.1128/AEM.05763-11

In silico assessment of cell-free systems
journal, May 2012

  • Bujara, Matthias; Panke, Sven
  • Biotechnology and Bioengineering, Vol. 109, Issue 10
  • DOI: 10.1002/bit.24534

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


Bacterial Production of Pinene by a Laboratory-Evolved Pinene-Synthase
journal, June 2016


A cell-free framework for rapid biosynthetic pathway prototyping and enzyme discovery
journal, July 2016


How corrinoids are synthesized without oxygen: nature's first pathway to vitamin B12
journal, September 1997


Enhanced limonene production in cyanobacteria reveals photosynthesis limitations
journal, November 2016

  • Wang, Xin; Liu, Wei; Xin, Changpeng
  • Proceedings of the National Academy of Sciences, Vol. 113, Issue 50
  • DOI: 10.1073/pnas.1613340113

Controlling cell-free metabolism through physiochemical perturbations
journal, January 2018


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

Isoprenoid Synthesis in Escherichia coli. Separation and Partial Purification of Four Enzymes Involved in the Synthesis1
journal, April 1986


Production of jet fuel precursor monoterpenoids from engineered Escherichia coli : Production of Jet Fuel Precursor Monoterpenoids
journal, May 2017

  • Mendez-Perez, Daniel; Alonso-Gutierrez, Jorge; Hu, Qijun
  • Biotechnology and Bioengineering, Vol. 114, Issue 8
  • DOI: 10.1002/bit.26296

Cell-free synthetic biology: Thinking outside the cell
journal, May 2012


Synthesis of (R,R)-2,3-butanediol from starch in a hybrid cell-free reaction system
journal, November 2018


Biosynthesis “debugged”: Novel bioproduction strategies: Novel bioproduction strategies
journal, October 2012


High-Throughput Optimization Cycle of a Cell-Free Ribosome Assembly and Protein Synthesis System
journal, October 2018

  • Caschera, Filippo; Karim, Ashty S.; Gazzola, Gianluca
  • ACS Synthetic Biology, Vol. 7, Issue 12
  • DOI: 10.1021/acssynbio.8b00276

Mimicking theEscherichia coli cytoplasmic environment activates long-lived and efficient cell-free protein synthesis
journal, January 2004

  • Jewett, Michael C.; Swartz, James R.
  • Biotechnology and Bioengineering, Vol. 86, Issue 1
  • DOI: 10.1002/bit.20026

An Enzymatic Platform for the Synthesis of Isoprenoid Precursors
journal, August 2014


Engineering synergy in biotechnology
journal, April 2014

  • Nielsen, Jens; Fussenegger, Martin; Keasling, Jay
  • Nature Chemical Biology, Vol. 10, Issue 5
  • DOI: 10.1038/nchembio.1519

Developing fermentative terpenoid production for commercial usage
journal, February 2016


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


Engineering Cellular Metabolism
journal, March 2016


In vitro reconstitution of mevalonate pathway and targeted engineering of farnesene overproduction in Escherichia coli : In Vitro Reconstitution of Mevalonate Pathway
journal, February 2014

  • Zhu, Fayin; Zhong, Xiaofang; Hu, Mengzhu
  • Biotechnology and Bioengineering, Vol. 111, Issue 7
  • DOI: 10.1002/bit.25198

Rapidly Characterizing the Fast Dynamics of RNA Genetic Circuitry with Cell-Free Transcription–Translation (TX-TL) Systems
journal, March 2014

  • Takahashi, Melissa K.; Chappell, James; Hayes, Clarmyra A.
  • ACS Synthetic Biology, Vol. 4, Issue 5
  • DOI: 10.1021/sb400206c

Integrated analysis of isopentenyl pyrophosphate (IPP) toxicity in isoprenoid-producing Escherichia coli
journal, May 2018


Determination of key enzymes for threonine synthesis through in vitro metabolic pathway analysis
journal, June 2015


    Works referencing / citing this record:

    Cell‐free protein synthesis: The transition from batch reactions to minimal cells and microfluidic devices
    journal, January 2020

    • Ayoubi‐Joshaghani, Mohammad H.; Dianat‐Moghadam, Hassan; Seidi, Khaled
    • Biotechnology and Bioengineering, Vol. 117, Issue 4
    • DOI: 10.1002/bit.27248

    In vitro multi-enzymatic cascades using recombinant lysates of E. coli: an emerging biocatalysis platform
    journal, January 2020


    Cell-free gene expression: an expanded repertoire of applications
    journal, November 2019

    • Silverman, Adam D.; Karim, Ashty S.; Jewett, Michael C.
    • Nature Reviews Genetics, Vol. 21, Issue 3
    • DOI: 10.1038/s41576-019-0186-3

    Enzyme alchemy: cell-free synthetic biochemistry for natural products
    journal, September 2019