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

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Global warming can be greatly mitigated by replacing fossil fuels with renewable solar energy. In one approach, researchers have combined solar water splitting with bacteria capable of consuming atmospheric CO2 and solar derived hydrogen to produce solar fuels. All CO2 fixed by these bacteria must go through the enzyme Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase (RuBisCO). RuBisCO is the arbiter of photosynthesis and is the primary conduit linking carbon into the biosphere. However, the enzyme suffers from slow reactivity and poor selectivity for its primary substrate (CO2), making it the rate-limiting step for solar fuel synthesis and cell growth. To create an efficient and effective solar fuels process, any participatory enzymes 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 with enhanced turnover rate and selectivity. 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 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. This fellowship will enable the necessary research to usher in a clean and renewable future.