A Rapid Method for Detecting Normal or Modified Plant and Algal Carbonic Anhydrase Activity Using Saccharomyces...

Area of Science:

• Biotechnology
• Plant Science
• Biochemistry

Background:

• Improving photosynthesis in C3 plants is crucial for agriculture.
• Cyanobacterial and algal CO2-concentrating mechanisms (CCMs) offer potential strategies.
• Understanding carbonic anhydrase (CA) and bicarbonate transporters is key for CCM component integration.

Purpose of the Study:

• To develop a heterologous complementation system for evaluating CCM components.
• To create a Saccharomyces cerevisiae carbonic anhydrase knock-out (ΔCA) strain for screening CA activity.
• To assess the utility of the ΔCA strain for investigating modified CAs and transporters.

Main Methods:

• Generated a Saccharomyces cerevisiae CA knock-out (ΔCA) strain with a high-CO2-dependent growth phenotype.
• Complemented the ΔCA strain with human, Chlamydomonas reinhardtii, and Arabidopsis thaliana CAs.
• Utilized Wilbur–Anderson assay and MIMS for probing CA activity.

Main Results:

• The ΔCA strain exhibits limited growth at ambient CO2 levels due to bicarbonate unavailability.
• Complementation with various CAs restored growth at ambient CO2, demonstrating functional expression.
• The system successfully screened CAs from different organisms, validating its screening capability.

Conclusions:

• The developed ΔCA yeast strain serves as a valuable tool for screening carbonic anhydrase activity.
• This system facilitates the assessment of CCM components for potential transfer into terrestrial C3 plants.
• It enables efficient evaluation of modified CAs before their introduction into plant systems for photosynthesis enhancement.