Degree Type

Dissertation

Date of Award

2011

Degree Name

Doctor of Philosophy

Department

Biochemistry, Biophysics and Molecular Biology

First Advisor

Basil J. Nikolau

Abstract

Biosynthesis of very long chain fatty acids (VLCFAs) is an integral process for plants, animals & yeast. VLCFAs serve in a variety of biological processes (e.g. protein trafficking, membrane stabilization, precursors for lipid second messengers) and in plants, are components of cuticular waxes, suberin, sphingolipids, phospholipids, seed oils, and GPI anchors. VLCFAs are synthesized by the fatty acid elongase system (FAE), which is composed of four integral membrane proteins: 3-ketoacyl-CoA synthase (KCS), 3-ketoacyl-CoA reductase (KCR), 3-hydroxyacyl-CoA dehydratase (HCD), & an enoyl-CoA reductase using fatty acid primers provided by de novo fatty acid synthesis.

The study of FAE, particularly the role of paralogous genes encoding each of its catalytic components, can be difficult due to its integral membrane nature. We have therefore built a platform to study the FAE system in yeast: a more tractable system for functionally evaluating the enzymological role of each paralog. The paralogs include previously characterized maize KCRs (glossy8a and glossy8b) and in silico-identified maize genes encoding the remaining components of the system. We have shown each component gene can individually complement yeast lacking a functional copy of the ortholog, and are working towards the complete reconstitution of this FAE within a single yeast strain that lacks endogenous elongase. This strain series has been used to further our understanding of why maize retains two ZmKCR paralogs and understand their roles within the FAE multienzyme system. Additionally, these strains can now be used to complete the reconstitution of maize FAE in a system free of the endogenous yeast FAE. Bioengineering this system will not only be a powerful platform to characterize the interactions and functional significance of the FAE components, but also provides the biosynthetic machinery to produce long chain substrate for the functional characterization of the cuticular wax biosynthetic genes. Understanding this pathway is a useful tool for the production of potential candidates for next generation fuels.

DOI

https://doi.org/10.31274/etd-180810-433

Copyright Owner

Alexis Ann Campbell

Language

en

Date Available

2012-04-06

File Format

application/pdf

File Size

144 pages

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