Doctoral Dissertations
Date of Award
12-2020
Degree Type
Dissertation
Degree Name
Doctor of Philosophy
Major
Energy Science and Engineering
Major Professor
Mitchel Doktycz
Committee Members
Jennifer Morrell-Falvey, Dale Pelletier, Michael Simpson, Robert Hettich
Abstract
Next generation DNA sequencing has led to an accumulation of a putative biosynthetic gene clusters for many natural product classes of interest. In vivo extraction and heterologous expression do not have sufficient throughput to validate predicted enzyme functions and inform future annotations. Further, engineering the production of new natural products is laborious and limited by the trade-offs between cell growth and product synthesis. Conversely, cell-free platforms, particularly those capable of cell-free protein synthesis (CFPS), facilitate rapid screening of enzyme function and prototyping of metabolic pathways. However, the protein content and metabolic activity of many cell-free systems are poorly defined, increasing variability between lysates and impeding systematic engineering. Here, the strength of untargeted peptidomics as an enabling tool for the engineering of cell-free systems is established based upon its ability to measure both global protein abundances and newly synthesized peptides. Synthesis of peptide natural products was found to be more robust in purified enzyme CFPS systems compared to crude lysates; however, non-specific peptide degradation, detected through peptidomics, remains a concern. Crude cell-free systems were determined be better suited to small molecule production, due to the extensive metabolic networks they were found to possess. Perturbations of these networks, carried out through changes to growth media, were observed through shotgun proteomics and informed engineering of phenol biosynthesis in a crude Escherichia coli lysate. Implementing shotgun proteomics as an analytical tool for cell-free systems will increase reproducibility and further the development of a platform for high-throughput functional genomics and metabolic engineering.
Recommended Citation
Mohr, Benjamin, "Advancing a systems cell-free metabolic engineering approach to natural product synthesis and discovery. " PhD diss., University of Tennessee, 2020.
https://trace.tennessee.edu/utk_graddiss/6837