A Rapid Method for Detecting Normal or Modified Plant and Algal Carbonic Anhydrase Activity Using Saccharomyces cerevisiae
Abstract
:1. Introduction
2. Results
2.1. The S. cerevisiae Strain ΔCA Has a High-CO2-Dependent Growth Phenotype
2.2. ΔCA Can Be Used as a Heterologous Complementation System to Detect CA Activity of Normal and Modified CAs
2.3. Plant and Algal Carbonic Anhydrases Show CA Activity in ΔCA-Based Heterologous Complementation System
2.4. ΔCA-Based Heterologous Complementation System Can Be Used to Quantify CA Activity Using Wilbur–Anderson Assay and MIMS
3. Discussion
4. Materials and Methods
4.1. S. cerevisiae Strain and Growth Conditions
4.2. Generation of S. cerevisiae Carbonic Anhydrase Knock-Out
4.3. Genetic Constructs and Vectors
4.4. S. cerevisiae CA Knock-Out Transformation
4.5. High-CO2-Dependence Growth Assay
4.6. Immunological Detection of Expressed Proteins in S. cerevisiae
4.7. Analysis of Neutral Lipid Synthesis Using Radiolabel 14C-Acetic Acid
4.8. Carbonic Anhydrase Activity Assay
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Biochemical Trait | ΔCA-hCA-YCO | ΔCA-hCA-YCO AcV5 | ΔCA-hCA-Atex | ΔCA-hCA-Atex AcV5 | ΔCA-hCA-YCO eGFP | ΔCA-hCA-Atex eGFP | ΔCA-EV | ΔCA-ScCA |
---|---|---|---|---|---|---|---|---|
Specific activity | 4.7 ± 0.5 WAU mg−1 | 3.9 ± 0.4 WAU mg−1 | 1.9 ± 0.2 WAU mg−1 | 0.9 ± 0.1 WAU mg−1 | 2.5 ± 0.3 WAU mg−1 | 1.4 ± 0.2 WAU mg−1 | 0.9 ± 0.1 WAU mg−1 | 1.2 ± 0.1 WAU mg−1 |
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Rai, A.K.; DiMario, R.J.; Kasili, R.W.; Groszmann, M.; Cousins, A.B.; Donze, D.; Moroney, J.V. A Rapid Method for Detecting Normal or Modified Plant and Algal Carbonic Anhydrase Activity Using Saccharomyces cerevisiae. Plants 2022, 11, 1882. https://doi.org/10.3390/plants11141882
Rai AK, DiMario RJ, Kasili RW, Groszmann M, Cousins AB, Donze D, Moroney JV. A Rapid Method for Detecting Normal or Modified Plant and Algal Carbonic Anhydrase Activity Using Saccharomyces cerevisiae. Plants. 2022; 11(14):1882. https://doi.org/10.3390/plants11141882
Chicago/Turabian StyleRai, Ashwani K., Robert J. DiMario, Remmy W. Kasili, Michael Groszmann, Asaph B. Cousins, David Donze, and James V. Moroney. 2022. "A Rapid Method for Detecting Normal or Modified Plant and Algal Carbonic Anhydrase Activity Using Saccharomyces cerevisiae" Plants 11, no. 14: 1882. https://doi.org/10.3390/plants11141882