InVivoRuBisCO · In vivo Directed Evolution of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase in Saccharomyces cerevisiae Using an Orthogonal DNA Replication System
Horizon 2020 — Marie Skłodowska-Curie Actions
- Duration
- 2020-12-01 → 2022-11-30
- EU contribution
- €178,320
- Participants
- 1
- Scheme
- MSCA-IF
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Results in brief
In vivo Directed Evolution of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase in Saccharomyces cerevisiae Using an Orthogonal DNA Replication System
Global warming can be greatly mitigated by replacing fossil fuels with renewable solar biofuels. The most abundant renewable fuel is bioethanol (EtOH) produced from yeast fermentation. First generation biofuel systems remain economically non-viable because of the low cost of fossil fuels, high costs of substrate refinement, and competition from agricultural feedstock. Second generation biofuels also remain non-viable given the limited capability of industrially relevant yeasts to ferment 5-carbon sugars like xylose. In a new approach to renewable fuel production, the sun is used to drive the water splitting reaction to produce hydrogen (H2) that can be coupled to carbon dioxide (CO2) using engineered bacteria to furnish liquid biofuels from only sunlight, water, and air. Dubbed the “Bionic Leaf,” this system is part of a larger class of third generation biorefineries. All CO2 converted by a Bionic Leaf must transit through the enzyme Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase (RuBisCO). RuBisCO is the arbiter of photosynthesis and links carbon dioxide into the biosphere and is the most abundant protein on earth. The proper expression and function of this enzyme is dependent on the activity of multiple molecular partners that facilitate folding and activation. The RuBisCo enzyme, across all swathes of life, suffers from slow reactivity and poor selectivity for its primary substrate (CO2), making it the rate-limiting step for plant and cell growth and solar fuel synthesis. 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. Therefore, a substantial breakthrough in this regard would be to develop an improved RuBisCO with enhanced turnover rate and selectivity. Our general research objective is to create a Bionic Leaf using an industrially relevant strain of S. cerevisiae. The work we can be broken down into 3 specific aims: 1.) Perform in vivo, continuous directed evolution of RuBisCO using an orthogonal DNA replication system; 2.) Incorporate an oxygen tolerant hydrogenase into yeast to enable cellular reducing equivalents to be derived from H2; 3.) Design and build a bionic leaf bioreactor employing the newly constructed yeast strain expressing RuBisCO and hydrogenase. Production of a Hydrogenase and RuBisCO expressing yeast that can grow and generate ethanol using only CO2 and H2 would be a revolutionary advance for biofuel production. Finally, this new organism will be employed in a Bionic Leaf to produce ethanol in a 100% renewable fashion, using only sunlight, water, and air. This successful completion of this research will enable to the full scale transition of society to net-carbon-zero fuels and help to remediate damage done to the environment by combustion of fossil fuels.
Data: CORDIS, © European Union
Project objective
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.
Original text from CORDIS.
Participants
- VIB VZW · ZWIJNAARDE - GENTCoordinatorBelgium
Links
Data: CORDIS, © European Union