Discipline: Biological Sciences
Ayana Mitchell - University of Maryland Baltimore County
Algal biofuels are an environmentally sustainable alternative to currently used and diminishing fossil fuels because they can be obtained directly from biomass derived from carbon dioxide (CO2) and sunlight. Increasing the photosynthetic activity of algae can increase their growth rate and biomass. One way to increase photosynthetic activity is to improve the Calvin cycle, a photosynthetic pathway of enzymatic reactions that convert CO2 and the energy of sunlight into sugars. Dual-function fructose-1,6-bisphosphatase/sedoheptulose-1,7-bisphosphatase (DFS) is a cyanobacterial enzyme that works to regenerate ribulose-1,5-bisphosphate (RuBP) in the regeneration phase of the Calvin cycle. Previously others found that overexpressing this cyanobacterial protein that possesses two Calvin cycle enzyme activities, could significantly improve plant biomass production. The goal of this project is to determine whether overexpressing this dual-function enzyme has the same effect in algae. We have generated C. reinhardtii transformants that contain the coding region for DFS (with flag-epitope tag) under the control of psbD and psbA 5’ and 3’ regulatory sequences, respectively, integrated into the chloroplast genome. We used western blotting to analyze transformants and found that they accumulate a flag-tagged protein that migrates at ~38 kDa, very near the expected size of 40 kDa for flag-tagged DFS. No flag protein of this size appears in untransformed controls so we conclude we have succeeded in expressing DFS in the C. reinhardtii chloroplast. We will proceed by comparing the growth of these transformants with wild C. reinhardtii. If expression of DFS leads to increased growth rate, then we would conclude that DFS carries out one or more rate-limiting steps in the Calvin cycle. If that is the case, we ultimately will overexpress DFS in a biotechnology production organism like Chlorella, in hopes of improving it as a biofuel-producing organism.
Funder Acknowledgement(s): These results were obtained as a part of the Research Experience and Mentoring (REM) program in the Department of Biological Sciences at the University of Maryland Baltimore County. This program is funded by a grant (REM supplement to NSF-EFRI-1332344) from the National Science Foundation (NSF) Directorate for Engineering (ENG) Office of Emerging Frontiers in Research and Innovation (EFRI).
Faculty Advisor: Stephen Miller, firstname.lastname@example.org
Role: I conducted the examination of the protein expression through western blot analysis. This portion of the project was important because it was the determining factor in whether or not DFS was correctly expressed in the algal cells. What we found was that DFS was correctly expressed in the chloroplast of C. reinhardtii.