Date of Award

Spring 5-4-2026

Document Type

Thesis

Degree Name

Bachelor of Arts

Department

Chemistry

First Advisor

Dr. Kerry Rouhier

Abstract

Plants co-opt primary metabolic pathways to synthesize a wide range of specialized metabolites that enhance their durability and survival rates, including agriculturally- and medicinally-useful defense compounds. Prior research points to tomato and other Solanaceae plants using the 2C fatty acid synthesis pathway in specialized trichome tissues to produce natural pesticides called acylsugars. A gene-duplicated and trichome tissue-expressed 3-ketoacyl-acyl carrier protein (ACP) synthase III (KASIII) pathway initiator enzyme is likely responsible for the selective structural composition of the acyl chains of acylsugar pesticides. I propose that this isozyme will have different acyl-CoA substrate preferences as compared to the canonically-expressed KASIII to corroborate this biosynthetic specialization. However, KASIII is notoriously difficult to characterize because of the limitations when visualizing the malonyl-ACP substrate conversion to the 3-ketoacyl-ACP product. The traditional separatory technique of polyacrylamide gel electrophoresis was found to show little difference between substrate and product resolution when functionalizing Solanum lycopersicum ACP. Adopting described LC-MS/MS methodology allowed for verification that both malonyl-ACP substrate and 3-ketoacyl-ACP KASIII product were generated from a two-step, “one-pot” reaction in vitro. The KASIII isozymes could then be characterized via urea-PAGE and spectrophotometric analyses. The S. lycopersicum canonically-expressed KASIII substantially prefers shorter chain substrates, specifically acetyl-CoA. The trichome-enriched KASIIIs of S. lycopersicum, S. nigrum, and Physalis philadelphica all showed similar acyl-CoA preference patterns, preferring intermediate lengths. Using a combination of targeted LC-MS/MS, gel-based, and spectrophotometric techniques, provides for better analysis of the reactions within the type II fatty acid synthesis pathway. This will provide novel insights as to where acyl chain elongation diverges for acylsugar metabolism, and allow for bioengineering advancements in an important crop family.

Rights Statement

All rights reserved. This copy is provided to the Kenyon Community solely for individual academic use. For any other use, please contact the copyright holder for permission.

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