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

Project description

How yeast can support renewable energy goals

RuBisCO, likely the most abundant protein on Earth, is a key enzyme present in many photosynthetic organisms including many bacteria, algae, and plants. It helps these organisms transform atmospheric carbon dioxide into energy-rich molecules such as glucose. Expressing RuBisCO in yeast, where it is not natively found, has also shown to enhance ethanol production by recapturing CO2 released during fermentation. Harnessing the activity of RuBisCO to sequester CO2 and produce bioethanol fuels remains a challenge due to the enzymeʻs exceedingly slow rate of reaction and poor selectivity for CO2, which has impeded productivity. Extensive efforts have been devoted to enhance enzyme activity using prokaryotic bacteria but have not been fruitful. InVivoRuBisCO is turning to in vivo-directed evolution in eukaryotic yeast (S. cerevisiae) to quickly and significantly boost RuBisCO activity. Success will enable industrially relevant levels of bioethanol production capable of contributing to the EU's sustainable future.

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.

Fields of science (EuroSciVoc)

Keywords

Programme(s)

Topic(s)

Funding Scheme

MSCA-IF - Marie Skłodowska-Curie Individual Fellowships (IF)

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Call for proposal

(opens in new window) H2020-MSCA-IF-2018

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Coordinator

VIB VZW
Net EU contribution
€ 178 320,00
Address
SUZANNE TASSIERSTRAAT 1
9052 ZWIJNAARDE - GENT
Belgium
Region
Vlaams Gewest Prov. Oost-Vlaanderen Arr. Gent
Activity type
Research Organisations
Links
Total cost
€ 178 320,00