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Parallel Integration and Chromosomal Expansion of Metabolic Pathways

  • Garima Goyal
    Garima Goyal
    Technologies, DOE Joint BioEnergy Institute, Emeryville, California 94608, United States
    DOE Agile BioFoundry, Emeryville, California 94608, United States
    Biological Systems & Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720, United States
    More by Garima Goyal
  • Zak Costello
    Zak Costello
    Biofuels and Bioproducts Divisions, DOE Joint BioEnergy Institute, Emeryville, California 94608, United States
    DOE Agile BioFoundry, Emeryville, California 94608, United States
    Biological Systems & Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720, United States
    More by Zak Costello
  • Jorge Alonso-Gutierrez
    Jorge Alonso-Gutierrez
    Biofuels and Bioproducts Divisions, DOE Joint BioEnergy Institute, Emeryville, California 94608, United States
    Biological Systems & Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720, United States
  • Aram Kang
    Aram Kang
    Biofuels and Bioproducts Divisions, DOE Joint BioEnergy Institute, Emeryville, California 94608, United States
    Biological Systems & Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720, United States
    More by Aram Kang
  • Taek Soon Lee
    Taek Soon Lee
    Biofuels and Bioproducts Divisions, DOE Joint BioEnergy Institute, Emeryville, California 94608, United States
    Biological Systems & Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720, United States
  • Hector Garcia Martin
    Hector Garcia Martin
    Biofuels and Bioproducts Divisions, DOE Joint BioEnergy Institute, Emeryville, California 94608, United States
    DOE Agile BioFoundry, Emeryville, California 94608, United States
    Biological Systems & Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720, United States
  • , and 
  • Nathan J. Hillson*
    Nathan J. Hillson
    Technologies, DOE Joint BioEnergy Institute, Emeryville, California 94608, United States
    DOE Agile BioFoundry, Emeryville, California 94608, United States
    Biological Systems & Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720, United States
    *E-mail: [email protected]
Cite this: ACS Synth. Biol. 2018, 7, 11, 2566–2576
Publication Date (Web):October 23, 2018
https://doi.org/10.1021/acssynbio.8b00243
Copyright © 2018 American Chemical Society
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Supporting Info (2)»

Abstract

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Robust fermentation of biomass-derived sugars into bioproducts demands the reliable microbial expression of metabolic pathways. Plasmid-based expression systems may suffer from instability and result in highly variable titers, rates, and yields. An established mitigation approach, chemical induced chromosomal expansion (CIChE), expands a singly integrated pathway to plasmid-like copy numbers while maintaining stability in the absence of antibiotic selection pressure. Here, we report parallel integration and chromosomal expansion (PIACE), extensions to CIChE that enable independent expansions of pathway components across multiple loci, use suicide vectors to achieve high-efficiency site-specific integration of sequence-validated multigene components, and introduce a heat-curable plasmid to obviate recA deletion post pathway expansion. We applied PIACE to stabilize an isopentenol pathway across three loci in E. coli DH1 and then generate libraries of pathway component copy number variants to screen for improved titers. Polynomial regressor statistical modeling of the production screening data suggests that increasing copy numbers of all isopentenol pathway components would further improve titers.

Supporting Information

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The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssynbio.8b00243.

  • Plasmid and chromosomal maps, fluorescence and qPCR measurements, support vector and random forest regressor modeling, table of plasmids and strains, PCR amplification and DNA assembly reactions, colony PCR DNA oligos, and qPCR DNA oligos (PDF)

  • Jupyter notebook (ZIP)

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Most electronic Supporting Information files are available without a subscription to ACS Web Editions. Such files may be downloaded by article for research use (if there is a public use license linked to the relevant article, that license may permit other uses). Permission may be obtained from ACS for other uses through requests via the RightsLink permission system: http://pubs.acs.org/page/copyright/permissions.html.

Cited By

This article is cited by 2 publications.

  1. Xin Xu, Zhi-Ming Rao, Jian-Zhong Xu, Wei-Guo Zhang. Enhancement of l-Pipecolic Acid Production by Dynamic Control of Substrates and Multiple Copies of the pipA Gene in the Escherichia coli Genome. ACS Synthetic Biology 2022, 11 (2) , 760-769. https://doi.org/10.1021/acssynbio.1c00467
  2. Deepanwita Banerjee, Thomas Eng, Yusuke Sasaki, Aparajitha Srinivasan, Asun Oka, Robin A. Herbert, Jessica Trinh, Vasanth R. Singan, Ning Sun, Dan Putnam, Corinne D. Scown, Blake Simmons, Aindrila Mukhopadhyay. Genomics Characterization of an Engineered Corynebacterium glutamicum in Bioreactor Cultivation Under Ionic Liquid Stress. Frontiers in Bioengineering and Biotechnology 2021, 9 https://doi.org/10.3389/fbioe.2021.766674

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