Application of targeted proteomics to metabolically engineered Escherichia coli

Research output: Contribution to journalJournal articleResearch

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Application of targeted proteomics to metabolically engineered Escherichia coli. / Singh, Pragya; Batth, Tanveer S; Juminaga, Darmawi; Dahl, Robert H; Keasling, Jay D; Adams, Paul D; Petzold, Christopher J.

In: Proteomics, Vol. 12, No. 8, 04.2012, p. 1289-99.

Research output: Contribution to journalJournal articleResearch

Harvard

Singh, P, Batth, TS, Juminaga, D, Dahl, RH, Keasling, JD, Adams, PD & Petzold, CJ 2012, 'Application of targeted proteomics to metabolically engineered Escherichia coli', Proteomics, vol. 12, no. 8, pp. 1289-99. https://doi.org/10.1002/pmic.201100482

APA

Singh, P., Batth, T. S., Juminaga, D., Dahl, R. H., Keasling, J. D., Adams, P. D., & Petzold, C. J. (2012). Application of targeted proteomics to metabolically engineered Escherichia coli. Proteomics, 12(8), 1289-99. https://doi.org/10.1002/pmic.201100482

Vancouver

Singh P, Batth TS, Juminaga D, Dahl RH, Keasling JD, Adams PD et al. Application of targeted proteomics to metabolically engineered Escherichia coli. Proteomics. 2012 Apr;12(8):1289-99. https://doi.org/10.1002/pmic.201100482

Author

Singh, Pragya ; Batth, Tanveer S ; Juminaga, Darmawi ; Dahl, Robert H ; Keasling, Jay D ; Adams, Paul D ; Petzold, Christopher J. / Application of targeted proteomics to metabolically engineered Escherichia coli. In: Proteomics. 2012 ; Vol. 12, No. 8. pp. 1289-99.

Bibtex

@article{b048dfefd3b74cb280f4075d0f3076d7,
title = "Application of targeted proteomics to metabolically engineered Escherichia coli",
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.",
keywords = "Bacterial Proteins, Escherichia coli, Fungal Proteins, Genetic Variation, Luminescent Proteins, Mass Spectrometry, Metabolic Engineering, Mevalonic Acid, Operon, Plasmids, Promoter Regions, Genetic, Proteomics, Saccharomyces cerevisiae, Transformation, Bacterial, Tyrosine",
author = "Pragya Singh and Batth, {Tanveer S} and Darmawi Juminaga and Dahl, {Robert H} and Keasling, {Jay D} and Adams, {Paul D} and Petzold, {Christopher J}",
note = "{\circledC} 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.",
year = "2012",
month = "4",
doi = "10.1002/pmic.201100482",
language = "English",
volume = "12",
pages = "1289--99",
journal = "Proteomics",
issn = "1615-9853",
publisher = "Wiley - V C H Verlag GmbH & Co. KGaA",
number = "8",

}

RIS

TY - JOUR

T1 - Application of targeted proteomics to metabolically engineered Escherichia coli

AU - Singh, Pragya

AU - Batth, Tanveer S

AU - Juminaga, Darmawi

AU - Dahl, Robert H

AU - Keasling, Jay D

AU - Adams, Paul D

AU - Petzold, Christopher J

N1 - © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

PY - 2012/4

Y1 - 2012/4

N2 - 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.

AB - 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.

KW - Bacterial Proteins

KW - Escherichia coli

KW - Fungal Proteins

KW - Genetic Variation

KW - Luminescent Proteins

KW - Mass Spectrometry

KW - Metabolic Engineering

KW - Mevalonic Acid

KW - Operon

KW - Plasmids

KW - Promoter Regions, Genetic

KW - Proteomics

KW - Saccharomyces cerevisiae

KW - Transformation, Bacterial

KW - Tyrosine

U2 - 10.1002/pmic.201100482

DO - 10.1002/pmic.201100482

M3 - Journal article

C2 - 22577029

VL - 12

SP - 1289

EP - 1299

JO - Proteomics

JF - Proteomics

SN - 1615-9853

IS - 8

ER -

ID: 68159296