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Title: A synthetic biochemistry platform for cell free production of monoterpenes from glucose

Abstract

Cell-free systems designed to perform complex chemical conversions of biomass to biofuels or commodity chemicals are emerging as promising alternatives to the metabolic engineering of living cells. We design a system comprises 27 enzymes for the conversion of glucose into monoterpenes that generates both NAD(P)H and ATP in a modified glucose breakdown module and utilizes both cofactors for building terpenes. Different monoterpenes are produced in our system by changing the terpene synthase enzyme. The system is stable for the production of limonene, pinene and sabinene, and can operate continuously for at least 5 days from a single addition of glucose. We also obtain conversion yields 495% and titres 415 g l-1. The titres are an order of magnitude over cellular toxicity limits and thus difficult to achieve using cell-based systems. Overall, these results highlight the potential of synthetic biochemistry approaches for producing bio-based chemicals.

Authors:
 [1];  [1];  [1]
+ Show Author Affiliations
  1. Univ. of California, Los Angeles, CA (United States). Dept. of Chemistry and Biochemistry
Publication Date:
Research Org.:
Univ. of California, Los Angeles, CA (United States)
Sponsoring Org.:
USDOE
OSTI Identifier:
1389298
Grant/Contract Number:  
FC02-02ER63421; AR0000556
Resource Type:
Accepted Manuscript
Journal Name:
Nature Communications
Additional Journal Information:
Journal Volume: 8; Journal ID: ISSN 2041-1723
Publisher:
Nature Publishing Group
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; 60 APPLIED LIFE SCIENCES; enzymes; metabolic engineering; metabolic pathways; synthetic biology

Citation Formats

Korman, Tyler P., Opgenorth, Paul H., and Bowie, James U. A synthetic biochemistry platform for cell free production of monoterpenes from glucose. United States: N. p., 2017. Web. doi:10.1038/ncomms15526.
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Korman, Tyler P., Opgenorth, Paul H., & Bowie, James U. A synthetic biochemistry platform for cell free production of monoterpenes from glucose. United States. https://doi.org/10.1038/ncomms15526
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Korman, Tyler P., Opgenorth, Paul H., and Bowie, James U. Wed . "A synthetic biochemistry platform for cell free production of monoterpenes from glucose". United States. https://doi.org/10.1038/ncomms15526. https://www.osti.gov/servlets/purl/1389298.
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@article{osti_1389298,
title = {A synthetic biochemistry platform for cell free production of monoterpenes from glucose},
author = {Korman, Tyler P. and Opgenorth, Paul H. and Bowie, James U.},
abstractNote = {Cell-free systems designed to perform complex chemical conversions of biomass to biofuels or commodity chemicals are emerging as promising alternatives to the metabolic engineering of living cells. We design a system comprises 27 enzymes for the conversion of glucose into monoterpenes that generates both NAD(P)H and ATP in a modified glucose breakdown module and utilizes both cofactors for building terpenes. Different monoterpenes are produced in our system by changing the terpene synthase enzyme. The system is stable for the production of limonene, pinene and sabinene, and can operate continuously for at least 5 days from a single addition of glucose. We also obtain conversion yields 495% and titres 415 g l-1. The titres are an order of magnitude over cellular toxicity limits and thus difficult to achieve using cell-based systems. Overall, these results highlight the potential of synthetic biochemistry approaches for producing bio-based chemicals.},
doi = {10.1038/ncomms15526},
journal = {Nature Communications},
number = ,
volume = 8,
place = {United States},
year = {Wed May 24 00:00:00 EDT 2017},
month = {Wed May 24 00:00:00 EDT 2017}
}
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Cell-free metabolic engineering: Biomanufacturing beyond the cell
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Biotechnological production of limonene in microorganisms
journal, February 2016

  • Jongedijk, Esmer; Cankar, Katarina; Buchhaupt, Markus
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  • DOI: 10.1007/s00253-016-7337-7

Biotechnological production of flavours and fragrances
journal, January 1998

  • Krings, U.; Berger, R. G.
  • Applied Microbiology and Biotechnology, Vol. 49, Issue 1
  • DOI: 10.1007/s002530051129

Identifying gene targets for the metabolic engineering of lycopene biosynthesis in Escherichia coli
journal, May 2005

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

Lysate of engineered Escherichia coli supports high-level conversion of glucose to 2,3-butanediol
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ATP Recycling with Cell Lysate for Enzyme-Catalyzed Chemical Synthesis, Protein Expression and PCR
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Engineering a mevalonate pathway in Escherichia coli for production of terpenoids
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A synthetic biochemistry module for production of bio-based chemicals from glucose
journal, April 2016

  • Opgenorth, Paul H.; Korman, Tyler P.; Bowie, James U.
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Identification and microbial production of a terpene-based advanced biofuel
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A synthetic biochemistry molecular purge valve module that maintains redox balance
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  • Opgenorth, Paul H.; Korman, Tyler P.; Bowie, James U.
  • Nature Communications, Vol. 5, Issue 1
  • DOI: 10.1038/ncomms5113

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 microorganisms for isoprenoid production
journal, January 2008

  • Kirby, James; Keasling, Jay D.
  • Natural Product Reports, Vol. 25, Issue 4
  • DOI: 10.1039/b802939c

Production of amorphadiene in yeast, and its conversion to dihydroartemisinic acid, precursor to the antimalarial agent artemisinin
journal, January 2012

  • Westfall, P. J.; Pitera, D. J.; Lenihan, J. R.
  • Proceedings of the National Academy of Sciences, Vol. 109, Issue 3, p. E111-E118
  • DOI: 10.1073/pnas.1110740109

Functional analysis of (4 S )-limonene synthase mutants reveals determinants of catalytic outcome in a model monoterpene synthase
journal, March 2015

  • Srividya, Narayanan; Davis, Edward M.; Croteau, Rodney B.
  • Proceedings of the National Academy of Sciences, Vol. 112, Issue 11
  • DOI: 10.1073/pnas.1501203112

Plant terpenoid synthases: Molecular biology and phylogenetic analysis
journal, April 1998

  • Bohlmann, J.; Meyer-Gauen, G.; Croteau, R.
  • Proceedings of the National Academy of Sciences, Vol. 95, Issue 8
  • DOI: 10.1073/pnas.95.8.4126

COPASI--a COmplex PAthway SImulator
journal, October 2006

Insect Attack and Wounding Induce Traumatic Resin Duct Development and Gene Expression of (—)-Pinene Synthase in Sitka Spruce
journal, August 2003

  • McKay, S. Ashley Byun; Hunter, William L.; Godard, Kimberley-Ann
  • Plant Physiology, Vol. 133, Issue 1
  • DOI: 10.1104/pp.103.022723

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

Microbial production of sabinene—a new terpene-based precursor of advanced biofuel
journal, January 2014

Thermostable ATP regeneration system using polyphosphate kinase from Thermosynechococcus elongatus BP-1 for d-amino acid dipeptide synthesis
journal, February 2007

  • Sato, Masaru; Masuda, Yusuke; Kirimura, Kohtaro
  • Journal of Bioscience and Bioengineering, Vol. 103, Issue 2, p. 179-184
  • DOI: 10.1263/jbb.103.179
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