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PhD Defense

ESS Oral Defense: Alysha Lee "Sterol Biosynthesis in Methylococcales and Myxococcota"

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Stanford University

*** Ph.D. Thesis/ Oral Defense ***

Sterol Biosynthesis in Methylococcales and Myxococcota

Alysha Lee

Tuesday, June 18, 10:00am

Green Earth Sciences 365

Department of Earth System Science

Advisor: Dr. Paula Welander

Sterols, such as cholesterol, are a versatile class of lipids primarily associated with eukaryotes where they carry out essential functions to cell viability. The biosynthesis of these lipids can consist of over 20 enzymes which are broadly conserved throughout the eukaryotic domain. Much of the biosynthetic pathway is thought to have been present in the last eukaryotic ancestor (LECA). Additionally, fossilized sterols can be found in rocks up to 1.6 billion years old, providing a rich record of eukaryotic life across Earth’s history. However, bacteria are also capable of de novo sterol biosynthesis, challenging our understanding of the evolution of this pathway and complicating the use of these lipids as eukaryotic biomarkers. While the synthesis of sterols in bacteria presents an opportunity to study a well characterized and important molecule in a unique system, little is known about bacterial sterol biosynthesis and physiology. In this dissertation, we explore sterol biosynthesis in both Methylococcales and Myxococcota, identifying steps in biosynthesis unique to the bacterial domain and discovering a complexity in bacterial sterol production that rival eukaryotes. In Chapter One, we uncover a bacterial sterol isomerase responsible for generating the unusual 8(14) sterol unsaturation which characterizes the sterols produced by aerobic methanotrophs. We find this enzyme is also present in pathogenic sterol degrading bacteria and identify key residues involved in catalysis. In Chapter Two, we demonstrate the myxobacterium Enhygromyxa salina is capable of de novo cholesterol production, representing the first instance of endogenous bacterial cholesterol biosynthesis. We find this bacterium harbors an additional protein involved in C-4 demethylation, distinguishing this process in E. salina from both eukaryotes and other sterol producing bacteria. In Chapter Three, we identify two additional myxobacteria, Sandaracinus amylolyticus and Minicystis rosea, also capable of de novo cholesterol production. These two myxobacteria are more distantly related to E. salina and appear to synthesize cholesterol using a different biosynthetic pathway. We then explore the evolutionary history of the proteins involved in sterol synthesis within Myxococcota and provide evidence for both vertical and horizontal transfer of sterol biosynthesis in the different families that comprise this phylum. Finally in Chapter Four, we adapt a thermal proteome profiling approach to identify putative sterol binding proteins in E. salina and generate hypotheses regarding the potential role of these proteins in sterol biosynthesis, regulation, and physiology, providing a foundation for future investigation into these processes in bacteria. Altogether, this work provides further examples for the independent evolution of bacterial sterol biosynthesis enzymes, highlighting the importance of these steps in sterol physiology and suggesting a complicated evolutionary history underpinning the presence of these lipids in the bacterial domain.

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