Optimizing SynBio Systems to Express Bryophyte Rubiscos in E. coli

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is the notoriously inefficient carbon-fixing enzyme involved in photosynthesis. Despite its importance, it has a slow catalytic rate, poor ability to differentiate between CO2 and O2, and tendency to be inhibited. Therefore, engineering Rubisco for increased efficiency is of great interest for improving crop yield. One such way is to study Rubiscos from other plants that may have better efficiency, which may include bryophyte Rubiscos. Bryophytes are a lineage of plants composed of liverworts, hornworts, and mosses that diverged from a common ancestor with other land plants around 500 million years ago. Not only do they primarily inhabit moist environments, leading to a decreased concentration of bioavailable CO2 substrate, but they also have restrictive anatomy, further limiting uptake of CO2. Under these conditions, bryophyte Rubiscos may have evolved improved efficiency with increased CO2 specificity. To better understand these poorly studied Rubiscos, we optimized an E. coli Synthetic Biology (SynBio) system to produce high yields of pure bryophyte Rubiscos, which was crucial due to difficulties isolating Rubisco from plants. We focused on Physcomitrium patens (moss), Phaeoceros carolinianus (hornwort), and Marchantia polymorpha (liverwort). We successfully purified the first moss Rubisco and ran the first qualitative analysis of its kinetics. Although our purification of liverwort and hornwort Rubiscos were unsuccessful, it allowed us to hypothesize further methods to further optimize the SynBio system using cognate Rubisco chaperones and chaperonins. Using bioinformatics tools such as multiple-sequence alignments, AlphaFold, and ChimeraX, we formulated hypotheses for further experimentation on a possible Rubisco linker protein in Phaeoceros carolinianus. We found that the SynBio system is a useful tool for purifying bryophyte Rubiscos, however it does not produce pure Rubisco for all bryophytes and therefore, different Rubiscos may require alternative methods for characterization.

These past 10 weeks working with my wonderful mentor, Dan Hong Loh, have been truly formative to my undergraduate experience and career as a scientist. Dan Hong always made sure to explain the procedures we were performing and encouraged me to think critically about the work I was doing. When I began in my lab, I had never really interacted with a STEM graduate student, however, this experience has thankfully taught me a lot about graduate school and research. The Gunn Lab as a whole has been a wonderful, welcoming place that has truly pushed me to grow and think inquisitively as a scientist, all while having lots of fun! My experience and interactions with Dan Hong and the rest of the Gunn Lab have left me with a strong desire to pursue graduate school and research.