Spatially-Directed Evolution

Evolution is an incredible mechanism for optimizing the function of organisms, and their composite biomolecular reactions, to survive in novel environments. This process usually happens on time scales that are well past the limits of human observation, however the rapid replication of bacteria serves as a foundation on which evolution can be, and currently is, studied. Over a large spatial gradient, the real-time evolution of E. coli to develop antibiotic resistance has been observed [1]. The nature of bacterial replication allows for the rapid acquisition of mutations, many of which are deleterious or neutral. Alternatively, some may allow for survival in hostile environments, in this case exposure to antibiotics. We aim to utilize this same principle to develop an improved isoform of the enzyme Rubisco.

Rubisco as a Target

Rubisco is the most abundant enzyme on Earth, catalyzing the carbon fixation reaction in photosynthesis. It could be assumed that an enzyme with such a fundamental and long-standing role in the development of life would have been optimized long ago. However, Rubisco has an extremely low rate of enzymatic activity and binds competitively with oxygen, leading to the photorespiration pathway which is less metabolically efficient and actually releases carbon dioxide. Both of these characteristics are potential points of improvements, but for purposes of this project we focus on enhancing the overall catalytic rate of Rubisco which should increase net carbon fixation.

Rubisco-Dependent Metabolic Engineering

The first challenge in pursuing this project was developing a strain of E. coli that was dependent on the function of Rubisco for survival. To accomplish this, we performed a double transformation of cyanobacterial Rubisco and Phosphoribulokinase A (prkA) into DH5 alpha E. coli [2]. The prkA, promoted by the presence of arabinose, diverts the endogenous glycolytic pathway to produce Ribulose bisphosphate (RuBP) which is the substrate for Rubisco and toxic to the cell. Through this logic, higher concentrations of arabinose should promote greater translation of prkA, in turn producing more RuBP which would induce cell death unless Rubisco can mutate to handle the greater enzymatic load. After a successful transformation, we observed this phenomenon under an increasing pressure of arabinose, with a clear minimum inhibitory concentration (MIC) of arabinose appearing. Implementing the stepwise selection gradient described above with arabinose as the selection pressure in place of antibiotics, we hope to direct the evolution of our metabolically engineered E. coli toward a more enzymatically active Rubisco.