Bryophyte Rubisco Expression and Assembly in E. coli

Ribulose-1,5-bisphosphate carboxylase oxygenase (Rubisco) catalyzes the fixation of carbon dioxide (CO2) in the Calvin-Benson-Bassham cycle of photosynthesis, which is the primary manner for carbon acquisition into the biosphere. Alternatively, the enzyme fixes oxygen gas, producing a toxic byproduct that is metabolized in photorespiration, expending energy and releasing previously fixed CO2. Additionally, Rubisco is notoriously slow, requiring plants to overexpress the enzyme to meet carbon needs. Finding a more efficient Rubisco can allow us to create more efficient plants with increased crop yields. An E. coli expression system can act as an exploratory method to study new Rubiscos without complications of plant sampling and cultivation. However, Rubisco has a complex biogenesis process that requires eight auxiliary proteins for assembly. Therefore, we coexpress Rubisco and its auxiliary proteins in E. coli to produce a functional Rubisco enzyme. To reduce costs and increase efficiency, we wanted to see if auxiliary proteins from one species could produce a functional Rubisco from a related species. Using an E. coli expression system, we attempted to express and assemble Rubisco from the liverwort Marchantia polymorpha and the moss Physcomitrium patens using auxiliary proteins from the hornwort Anthoceros agrestis. Bryophytes are a monophyletic clade of plants that consist of hornworts, liverworts, and mosses. They are an understudied clade, and little is known about the Rubiscos of these species. This approach was successful in assembling P. patens Rubisco, but at a lower yield and purity than a hornwort Rubisco control, suggesting cognate auxiliary proteins are critical to Rubisco biogenesis

My time at BTI and Cornell University has greatly helped me down a path towards a professional career in scientific research. I have been so grateful for the numerous resources I was provided through the program to ensure my success now and in the future. Direct mentorship for my project from graduate students, postdocs, and principal investigators has helped me grow as a researcher, guiding my understanding and preparation for graduate studies and the future. My research project has allowed me to use methods I hadn’t had experience in but will readily use in my future studies. Furthermore, outside of research, I am grateful to have participated in weekly seminars with faculty, a bioinformatics course, and a Cornell Graduate Studies information session, all which have been helpful tools to guide my future.