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Download Hi-Res ImageDownload to MS-PowerPointCite This:ACS Synth. Biol. 2018, 7, 11, 2566-2576
    Research Article

    Parallel Integration and Chromosomal Expansion of Metabolic Pathways
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    • 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
      More by Jorge Alonso-Gutierrez
    • 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
      More by Taek Soon Lee
    • 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
      More by Hector Garcia Martin
      Orcidhttp://orcid.org/0000-0002-4556-9685
    • 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]
      More by Nathan J. Hillson
      Orcidhttp://orcid.org/0000-0002-9169-3978
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    ACS Synthetic Biology

    Cite this: ACS Synth. Biol. 2018, 7, 11, 2566–2576
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    https://doi.org/10.1021/acssynbio.8b00243
    Published October 23, 2018
    Copyright © 2018 American Chemical Society
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    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.

    Copyright © 2018 American Chemical Society

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    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)

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    This article is cited by 5 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. Fei Du, Zijia Li, Xin Li, Duoduo Zhang, Feng Zhang, Zixu Zhang, Yingshuang Xu, Jin Tang, Yongqian Li, Xingxu Huang, Yang Gu, Xiaoman Sun, He Huang. Optimizing multicopy chromosomal integration for stable high-performing strains. Nature Chemical Biology 2024, 20 (12) , 1670-1679. https://doi.org/10.1038/s41589-024-01650-0
    3. Seong Keun Kim, Haseong Kim, Seung Gyun Woo, Tae Hyun Kim, Eugene Rha, Kil Koang Kwon, Hyewon Lee, Seung-Goo Lee, Dae-Hee Lee. CRISPRi-based programmable logic inverter cascade for antibiotic-free selection and maintenance of multiple plasmids. Nucleic Acids Research 2022, 50 (22) , 13155-13171. https://doi.org/10.1093/nar/gkac1104
    4. Peter B Otoupal, Brady F Cress, Jennifer A Doudna, Joseph S Schoeniger. CRISPR-RNAa: targeted activation of translation using dCas13 fusions to translation initiation factors. Nucleic Acids Research 2022, 50 (15) , 8986-8998. https://doi.org/10.1093/nar/gkac680
    5. 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
    6. 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. 2021https://doi.org/10.1101/2021.09.29.462453
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    ACS Synthetic Biology

    Cite this: ACS Synth. Biol. 2018, 7, 11, 2566–2576
    Click to copy citationCitation copied!
    https://doi.org/10.1021/acssynbio.8b00243
    Published October 23, 2018
    Copyright © 2018 American Chemical Society
    Request reuse permissions

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