A Map of the Rubisco Biochemical Landscape
Research on the Functional Mapping of Rubisco Enzyme
Background Introduction
Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) is the most abundant enzyme on Earth, responsible for the carbon dioxide fixation process in photosynthesis. However, Rubisco’s catalytic efficiency is low, and it is prone to side reactions with oxygen, limiting the efficiency of photosynthesis. Although scientists have long attempted to improve Rubisco’s catalytic performance through engineering, progress has been slow due to the difficulty in efficiently measuring its complex biochemical parameters, such as catalytic rate, carbon dioxide affinity, and specificity. In recent years, with the development of high-throughput screening technologies and machine learning methods, scientists have begun to systematically explore the sequence-function relationships of Rubisco to identify potential pathways for improving its performance.
This study was conducted by a collaborative team of scientists from University of California Berkeley, Howard Hughes Medical Institute, Nanyang Technological University, and other institutions, and was published in Nature in 2024. The research team developed a high-throughput screening method based on engineered Escherichia coli, systematically mapping the sequence-function landscape of Rubisco and revealing the diversity of its biochemical functions and potential engineering improvements.
Research Process and Experimental Methods
1. Construction of the Research Object: Rubisco-Dependent E. coli Strain
The research team first constructed a Rubisco-dependent E. coli strain, Δrpi, by knocking out the rpi gene (ribose-5-phosphate isomerase gene). This strain cannot grow on glycerol as the sole carbon source unless it expresses functional Rubisco. Rubisco rescues growth by converting ribulose-1,5-bisphosphate (RuBP) into 3-phosphoglycerate, which re-enters central carbon metabolism. This design directly links Rubisco enzyme activity to the growth rate of the strain, laying the foundation for subsequent high-throughput screening.
2. Construction and Screening of the Mutant Library
The research team selected Form II Rubisco from Rhodospirillum rubrum as the model enzyme and constructed a mutant library containing 8,760 single-amino acid mutants. The specific steps are as follows: - Mutant Library Design: The Rubisco gene was divided into 11 fragments, and all possible single-amino acid mutants were designed and synthesized for each fragment. - Mutant Library Construction: The mutant fragments were inserted into vectors using Golden Gate assembly, and random barcodes were introduced for subsequent sequencing analysis. - High-Throughput Screening: The mutant library was transformed into the Δrpi strain and screened for growth under different carbon dioxide concentrations. The relative growth rates (i.e., “fitness”) of each mutant were calculated by analyzing barcode abundance before and after screening using short-read sequencing (Illumina).
3. Inference of Enzyme Kinetic Parameters
To further analyze the biochemical properties of Rubisco mutants, the research team varied the carbon dioxide concentration in the culture environment and fitted the data to the Michaelis-Menten kinetic model to infer the maximum rate (Vmax) and carbon dioxide half-saturation constant (Kc) for each mutant. The specific methods are as follows: - Carbon Dioxide Titration Experiment: The mutant library was cultured under different carbon dioxide concentrations, and the growth fitness of each mutant was measured. - Kinetic Parameter Fitting: The growth fitness data were fitted to the Michaelis-Menten equation to calculate the Vmax and Kc for each mutant. - Validation Experiments: The Kc and Vmax of some mutants were validated through in vitro enzyme activity assays, confirming the reliability of the screening results.
4. Data Analysis and Structure-Function Relationships
The research team explored the relationship between the functional changes of Rubisco mutants and their structure through multiple sequence alignment and structural analysis. Key aspects of the data analysis include: - Mutational Tolerance: The tolerance of each amino acid site to mutations was assessed, revealing that some highly conserved sites exhibit high tolerance to mutations. - Function-Improving Mutations: A few mutations that significantly improve carbon dioxide affinity were identified, such as V266T and A102Y. - Structure-Function Relationships: Structural analysis revealed that these function-improving mutations are located in key regions of the Rubisco dimer interface, potentially altering catalytic performance by influencing enzyme conformation or electrostatic environment.
Main Research Findings
1. Comprehensive Characterization of the Mutant Library
The research team successfully constructed and screened a Rubisco mutant library containing 8,760 single-amino acid mutants, covering 99% of possible mutations. The screening results showed: - 72% of the mutants had a negative impact on Rubisco function. - 0.14% of the mutants exhibited higher fitness than the wild type, but these improvements were primarily related to protein expression levels rather than catalytic rate enhancement.
2. Discovery of Function-Improving Mutations
Through carbon dioxide titration experiments, the research team identified several mutations that significantly improve Rubisco’s carbon dioxide affinity, such as V266T and A102Y. The Kc values of these mutants were 2-3 times lower than that of the wild type, indicating higher carbon dioxide affinity. In vitro enzyme activity assays further validated the biochemical properties of these mutants, confirming the trade-off between catalytic rate and carbon dioxide affinity.
3. Revelation of Structure-Function Relationships
Structural analysis revealed that function-improving mutations are located in key regions of the Rubisco dimer interface, which may alter catalytic performance by influencing enzyme conformation or electrostatic environment. For example, the V266T and A102Y mutations are located near the C2 symmetry axis of the dimer interface and may enhance affinity by affecting the path of carbon dioxide entry into the active site.
Research Conclusions and Significance
1. Scientific Value
This study, through high-throughput screening and systematic analysis, comprehensively mapped the sequence-function landscape of Rubisco for the first time, revealing the diversity of its biochemical functions and potential engineering improvements. The research found that, although Rubisco’s functional space is constrained by various biochemical trade-offs, significant performance improvements can still be achieved through single-amino acid mutations. This discovery provides an important theoretical basis for further engineering optimization of Rubisco.
2. Application Value
Rubisco is a key enzyme in photosynthesis, and its performance directly affects the carbon fixation efficiency and crop yield of plants. The function-improving mutations discovered in this study offer new insights for designing more efficient Rubisco, with potential applications in crop improvement and bioenergy development.
3. Research Highlights
- High-Throughput Screening Method: Developed a high-throughput screening method based on engineered E. coli, enabling systematic characterization of Rubisco mutants.
- Discovery of Function-Improving Mutations: Identified multiple mutations that significantly improve Rubisco’s carbon dioxide affinity, breaking through traditional limitations.
- Revelation of Structure-Function Relationships: Revealed the molecular mechanisms of function-improving mutations through structural analysis, providing new targets for Rubisco engineering optimization.
Summary
This study, through innovative high-throughput screening methods and systematic analysis, comprehensively revealed the sequence-function relationships of Rubisco and identified multiple mutations that significantly improve its performance. This provides an important theoretical and experimental foundation for Rubisco engineering optimization and photosynthesis research. The results not only deepen our understanding of Rubisco’s function but also offer new possibilities for crop improvement and bioenergy development.