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Application of targeted proteomics to metabolically engineered Escherichia coli

Authors

  • Pragya Singh,

    1. Joint BioEnergy Institute, Emeryville, CA, USA
    2. Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA
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  • Tanveer S. Batth,

    1. Joint BioEnergy Institute, Emeryville, CA, USA
    2. Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA
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  • Darmawi Juminaga,

    1. California Institute for Quantitative Biosciences, University of California, Berkeley, CA, USA
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  • Robert H. Dahl,

    1. Joint BioEnergy Institute, Emeryville, CA, USA
    2. Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA
    3. Department of Chemical & Biomolecular Engineering, University of California, Berkeley, CA, USA
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  • Jay D. Keasling,

    1. Joint BioEnergy Institute, Emeryville, CA, USA
    2. Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA
    3. California Institute for Quantitative Biosciences, University of California, Berkeley, CA, USA
    4. Department of Chemical & Biomolecular Engineering, University of California, Berkeley, CA, USA
    5. Department of Bioengineering, University of California, Berkeley, CA, USA
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  • Paul D. Adams,

    1. Joint BioEnergy Institute, Emeryville, CA, USA
    2. Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA
    3. Department of Bioengineering, University of California, Berkeley, CA, USA
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  • Christopher J. Petzold

    Corresponding author
    1. Joint BioEnergy Institute, Emeryville, CA, USA
    2. Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA
    • Correspondence: Dr. Christopher J. Petzold, Joint BioEnergy Institute, Lawrence Berkeley National Laboratory, 5885 Hollis St., 4th floor, Emeryville, CA 94608, USA E-mail: cjpetzold@lbl.gov

      Fax: +1-510-486-5225

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Abstract

As synthetic biology matures to compete with chemical transformation of commodity and high-value compounds, a wide variety of well-characterized biological parts are needed to facilitate system design. Protein quantification based on selected-reaction monitoring (SRM) mass spectrometry compliments metabolite and transcript analysis for system characterization and optimizing flux through engineered pathways. By using SRM quantification, we assayed red fluorescent protein (RFP) expressed from plasmids containing several inducible and constitutive promoters and subsequently assessed protein production from the same promoters driving expression of eight mevalonate pathway proteins in Escherichia coli. For each of the promoter systems, the protein level for the first gene in the operon followed that of RFP, however, the levels of proteins produced from genes farther from the promoter were much less consistent. Second, we used targeted proteomics to characterize tyrosine biosynthesis pathway proteins after removal of native regulation. The changes were not expected to cause significant impact on protein levels, yet significant variation in protein abundance was observed and tyrosine production for these strains spanned a range from less than 1 mg/L to greater than 250 mg/L. Overall, our results underscore the importance of targeted proteomics for determining accurate protein levels in engineered systems and fine-tuning metabolic pathways.

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