Date of Defense

12-3-2025

Date of Graduation

12-2025

Department

Biological Sciences

First Advisor

Todd Barkman

Second Advisor

Frederick Stull

Abstract

Bixin, a key component in annatto, is a commercially important natural pigment made by the small tree known as Bixa orellana, achiote, and/or the lipstick tree (Faria et al., 2020). Despite this pigment’s use in many products, such as in dairy products and pharmaceuticals, its biosynthetic pathway seemingly remains largely a mystery (Rivera-Madrid et al., 2013). While Bouvier and colleagues identified the family of enzymes responsible for bixin’s biosynthesis, the enzymes they claimed to have identified within the genome remain unsupported by other research groups (Bouvier et al., 2003; Cárdenas-Conejo et al., 2023; Moreira et al., 2023). In this experiment, we sought to better characterize some of the promising SABATH enzymes in the genome of Bixa orellana to uncover the enzyme responsible for the final methylation to generate bixin from norbixin. We conducted bioinformatic and phylogenetic analyses, to identify potential genes that could be involved in methylation of norbixin to bixin. From the candidates identified, we then heterologously expressed protein to determine enzyme substrate preferences. We used radioactive assays to assess enzyme activity with particular substrates and verified product formation using gas chromatography-mass spectrometry (GC-MS), where possible. Multiple active enzymes were obtained, including one capable of methylating norbixin to bixin, suggesting it may be the enzyme carrying out the final step in the biosynthetic pathway. Additionally, we tested Bouvier and colleagues' methyltransferase to assess whether it was able to interact with norbixin and other common SABATH enzyme substrates and observed no activity.

Access Setting

Honors Thesis-Restricted

Restricted to Campus until

2-12-2028

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Available for download on Saturday, February 12, 2028

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