RubisCO selection using the vigorously aerobic and metabolically versatile bacterium Ralstonia eutropha.

Area of Science:

• Biotechnology and Synthetic Biology
• Environmental Science and Climate Change
• Microbiology and Enzymology

Background:

• Atmospheric carbon dioxide (CO2) capture is crucial for mitigating global warming and increasing carbon availability.
• Ralstonia eutropha is an aerobic bacterium utilizing the Calvin-Benson-Bassham (CBB) cycle and ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) for CO2 fixation.
• Improving RubisCO's CO2 capture efficiency is a key target for biotechnological applications.

Purpose of the Study:

• To develop a host strain of Ralstonia eutropha for bioprospecting and engineering of RubisCO enzymes with enhanced CO2-capture capabilities.
• To identify key amino acid substitutions that improve RubisCO's substrate specificity and CO2 fixation efficiency in aerobic environments.

Main Methods:

• Construction of a host strain of R. eutropha by deleting endogenous RubisCO genes.
• Complementation of the host strain with native or heterologous RubisCO genes.
• Mutagenesis and suppressor selection to identify beneficial amino acid substitutions in RubisCO.

Main Results:

• Identified specific amino acid substitutions in a hydrophobic region of RubisCO that enhance its interaction with CO2 over O2.
• The engineered R. eutropha RubisCO demonstrated optimal CO2 fixation rates in a high-oxygen environment, outcompeting oxygen's inhibitory effect.
• The structure-function properties of R. eutropha RubisCO are similar to plant enzymes, enabling analogous enzyme engineering strategies.

Conclusions:

• Ralstonia eutropha serves as a robust host for directed evolution and artificial selection of RubisCO enzymes.
• This approach holds significant potential for improving biological CO2 utilization in aerobic conditions, aiding climate change mitigation.
• The engineered RubisCO variants can be applied to enhance carbon capture in various biotechnological processes.