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Proteomic Applications for Engineering Escherichia coli for Biofuel Production

  • Author(s): Rutherford, Becky Joanna Gail
  • Advisor(s): Keasling, Jay D
  • et al.
Abstract

Concerns over energy security, carbon emissions, and other limitations of petroleum-based chemicals have led to increased interest in using biological systems for the production of commodity chemicals such as fuels. It is vital that the microbes used for these processes be able to grow with minimal nutrients, produce large amounts of the target molecule, and be tolerant to the chemicals being produced. Naturally occurring strains are unable to meet all these criteria and need to be further optimized as production hosts. Proteomics is the study of cellular proteins. It has become a powerful tool for host engineering, and can give vital information about pathway performance and strain responses. Using LC-MS/MS mass spectrometry, proteomics strategies were utilized to study cellular responses to a potential biofuel, n-butanol, and to overexpression of the carbon storage regulator, which increased flux through three key biofuel production pathways.

For each study, proteomics provided valuable cues for understanding how the host was adapting to an engineered environment. In the case of product toxicity, n-butanol stressed cells were iTRAQ labeled, separated into 20 fractions using HPLC, and spectra collected on an ESI-Q-TOF instrument. Analysis of the data indicates that n-butanol stress has components common to other stress responses including perturbation of respiratory functions, oxidative stress, heat shock and cell envelope stress, and metabolite transport and biosynthesis. Results from these analyses allow identification of key genes, such as degP, nlpD, and phoU, which were used to alleviate stress. Proteomics work was also vital for studying the cellular effects of the carbon storage regulator and the increased flux through biofuel pathways. Extracted proteins were analyzed on a 5600 Triple-TOF mass spectrometer. Increased levels of proteins involved with energy metabolism, particularly TCA cycle, were noted, along with approximately 20 proteins that were strongly up regulated, indicating previously unknown regulation. This dataset is the first cell-wide proteomics study of this global regulator, and may provide further clues for studying its complex regulation.

Finally, a novel method for multiplexing targeted proteomics samples is described, along with an outline of key experiments to validate its accuracy and usefulness to synthetic biologists. A concluding chapter outlining future work on butanol responsive genes, a targeted proteomics stress diagnostic, and challenges in developing beneficial phenotypes for biofuel production is included with several previously unpublished results.

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