In vivo Directed Evolution of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase in Saccharomyces cerevisiae Using an Orthogonal DNA Replication System

To create an efficient and effective solar fuels process that combines solar electricity with biological organisms, any participatory enzymes in the cells need to have sufficiently high turnover rates to match the input of solar energy. A substantial breakthrough in this regard would be to develop an improved RuBisCO enzyme, which fixes CO2 from the atmosphere, with enhanced turnover rate and selectivity. In nature, RuBisCO has slow turnover rates (1-5 s–1) and poor selectivity for CO2 versus O2. To date, directed evolution of RuBisCO has been hindered by traditional laboratory evolution techniques with prokaryotic hosts and yielded only marginal improvements in enzyme activity. To overcome evolutionary slowness and host inefficacies, I propose the in vivo directed evolution of RuBisCO in eukaryotic S. cerevisiae. This work will be divided into three parts: 1.) Designing a host yeast organism that is dependent on RuBisCO activity through specific gene additions and deletions 2.) Develop a continuous directed evolution experiment to increase CO2 fixation, fidelity, and catalytic rate by applying selective pressures; and 3.) Isolate, propagate, and assay the resultant mutants, and test practical applications. The results of this work will serve to dramatically increase bioethanol yields through CO2 re-uptake and help achieve the European Commission's goal of using 25% biofuels in the transportation sector by 2030.