Utilizing a Cell-free Protein Synthesis System and Engineered Methyltransferases for Natural Product Synthesis

Simple item page
dc.contributor.advisorPhillips, George N
dc.contributor.advisorShamoo, Yousif
dc.creatorDitzel, Alex
dc.date.accessioned2023-08-09T18:46:58Z
dc.date.available2023-08-09T18:46:58Z
dc.date.created2023-05
dc.date.issued2023-04-20
dc.date.submittedMay 2023
dc.date.updated2023-08-09T18:46:58Z
dc.description.abstractNatural products are a rich source of pharmaceuticals, constituting the majority of small molecule drugs in use today. However, their production through organic synthesis or heterologous hosts can be difficult and time-consuming. To enable easier screening and production of natural products, a cell-free protein synthesis (CFPS) system was utilized to partially assemble natural products in vitro using SAM-dependent methyltransferase enzyme reactions. Caffeine synthesis by the tea caffeine synthase TCS1 was demonstrated within the CFPS system, and production of caffeine and intermediate products such as theobromine was confirmed via LC-MS. One major benefit of cell-free systems is the ability to use toxic substrates that would normally be difficult to use in a cellular environment to synthesize novel products, however we found that TCS1 and its homologs were unable to use cofactors such as SAE, the ethyl analogue of SAM. Therefore, protein engineering is essential to expand the repertoire of SAM-dependent methyltransferases for synthesizing new products, as some enzymes are unable to use SAM analogues. A structure-guided approach was employed to modify the tyrosine O-methyltransferase MfnG to use SAE in addition to SAM while also reducing its preference for SAM. A series of MfnG mutants were generated, and one mutant exhibited significantly higher capacity to utilize SAE for tyrosine O-ethylation. Further investigation of this mutant was carried out through predicted structural analysis and molecular dynamics simulations. This work demonstrates the potential of cell-free systems for natural product synthesis and the power of protein engineering to expand the capabilities of SAM-dependent methyltransferases for novel product synthesis.
dc.format.mimetypeapplication/pdf
dc.identifier.citationDitzel, Alex. "Utilizing a Cell-free Protein Synthesis System and Engineered Methyltransferases for Natural Product Synthesis." (2023) Diss., Rice University. <a href="https://hdl.handle.net/1911/115146">https://hdl.handle.net/1911/115146</a>.
dc.identifier.urihttps://hdl.handle.net/1911/115146
dc.language.isoeng
dc.rightsCopyright is held by the author, unless otherwise indicated. Permission to reuse, publish, or reproduce the work beyond the bounds of fair use or other exemptions to copyright law must be obtained from the copyright holder.
dc.subjectCell-free
dc.subjectmethyltransferases
dc.subjectnatural products, protein engineering
dc.titleUtilizing a Cell-free Protein Synthesis System and Engineered Methyltransferases for Natural Product Synthesis
dc.typeThesis
dc.type.materialText
thesis.degree.departmentBiochemistry and Cell Biology
thesis.degree.disciplineNatural Sciences
thesis.degree.grantorRice University
thesis.degree.levelDoctoral
thesis.degree.nameDoctor of Philosophy
Files
Original bundle
Now showing 1 - 1 of 1
Thumbnail Image
Name:
DITZEL-DOCUMENT-2023.pdf
Size:
18.36 MB
Format:
Adobe Portable Document Format
Download
License bundle
Now showing 1 - 2 of 2
No Thumbnail Available
Name:
PROQUEST_LICENSE.txt
Size:
5.84 KB
Format:
Plain Text
Description:
Download
No Thumbnail Available
Name:
LICENSE.txt
Size:
2.6 KB
Format:
Plain Text
Description:
Download
Collections
Rice University Electronic Theses and Dissertations