Periodic Reporting for period 1 - EvoZyme (Development of a synthetic diversification platform for targeted EVOLUTION of ENZYMES supporting a sustainable bioeconomy – EvoZyme)
Periodo di rendicontazione: 2024-01-01 al 2025-12-31
Context and Motivation
Climate change and the urgent need to transition from a fossil-based economy to a circular bioeconomy have driven the search for sustainable biotechnological solutions. A key strategy to achieve this transition is the development of microbial production strains capable of utilizing CO2-derived one-carbon (C1) substrates, such as formate, to generate valuable bioproducts. One of the most promising metabolic pathways enabling this is the reductive glycine pathway (rGlyP), which is highly efficient, energy-conserving, and relatively easy to engineer. However, despite recent advances in metabolic engineering, the growth performance of engineered formatotrophic strains remains suboptimal for industrial applications.
Traditional methods for optimizing these synthetic pathways rely on adaptive laboratory evolution (ALE), which involves random mutation and natural selection. While effective, ALE is time-consuming and often introduces unwanted off-target mutations, making it an inefficient method for precise pathway optimization. Recent advancements in directed evolution and synthetic biology have provided new tools for improving enzyme function and pathway efficiency, but these tools have primarily been optimized for Escherichia coli and are challenging to implement in other bacterial hosts.
Overall Objectives
The EvoZyme project aims to address these challenges by developing a synthetic diversification platform that enables the targeted evolution of enzymes in multiple bacterial hosts. The platform will allow for precise and accelerated genetic diversification of key metabolic enzymes, optimizing microbial growth and productivity in a way that circumvents the limitations of traditional ALE. The project is structured around two primary objectives:
Development of a synthetic diversification platform – A system capable of hypermutating a gene of interest (GOI) in a targeted manner while allowing seamless transfer of the evolved GOI to various bacterial hosts. This ensures pathway optimization without introducing off-target effects in the host genome.
Optimization of the reductive glycine pathway (rGlyP) through targeted evolution of the glycine cleavage system (GCS) – The GCS is the core enzyme complex responsible for key metabolic conversions within rGlyP. EvoZyme will apply the diversification platform to improve the efficiency of the GCS, enabling enhanced formatotrophic growth in industrially relevant microbes (E. coli, Cupriavidus necator, Pseudomonas putida).
By achieving these objectives, EvoZyme will pave the way for a new approach to enzyme engineering, establishing a broadly applicable diversification tool that could optimize other critical biosynthetic pathways beyond rGlyP.
Pathway to Impact
The EvoZyme project is expected to significantly advance synthetic metabolism by providing a novel, adaptable platform for improving enzymatic functions in diverse microbial hosts. The key expected impacts include:
Scientific and Technological Impact:
• Introduction of a highly efficient and versatile enzyme evolution tool for synthetic biology and metabolic engineering.
• Generation of optimized formatotrophic strains capable of efficiently converting CO2-derived substrates into biomass and valuable bioproducts.
• Improved understanding of the molecular mechanisms underlying enzyme optimization, with applications in industrial biotechnology.
Economic and Industrial Impact:
• Acceleration of biotechnological strain development, reducing the time and cost associated with optimizing metabolic pathways.
• Enhancement of biomanufacturing processes, improving the feasibility of using engineered microbes for large-scale production of bio-based chemicals.
• Potential commercialization of the diversification platform as a broadly applicable tool for enzyme engineering in academia and industry.
Societal and Environmental Impact:
• Contribution to a sustainable bioeconomy, reducing dependency on fossil fuels and mitigating CO2 emissions.
• Support for achieving multiple United Nations Sustainable Development Goals (SDGs), particularly those related to climate action, sustainable industry, and responsible consumption.
• Increased public engagement in synthetic biology through open science practices and science communication initiatives.
Setting the Scene for EvoZyme
The EvoZyme project operates at the intersection of biotechnology, synthetic biology, and metabolic engineering, tackling one of the key barriers to a bio-based circular economy—the efficient engineering of microbial metabolism. By creating a powerful and tunable enzyme diversification platform, EvoZyme will not only improve biotechnological production processes but also provide an essential tool for future research in metabolic engineering and synthetic metabolism. With a strong focus on scientific innovation, industrial applicability, and environmental sustainability, EvoZyme has the potential to transform how microbial strains are engineered, paving the way for a new era of sustainable bioproduction.
Climate change and the urgent need to transition from a fossil-based economy to a circular bioeconomy have driven the search for sustainable biotechnological solutions. A key strategy to achieve this transition is the development of microbial production strains capable of utilizing CO2-derived one-carbon (C1) substrates, such as formate, to generate valuable bioproducts. One of the most promising metabolic pathways enabling this is the reductive glycine pathway (rGlyP), which is highly efficient, energy-conserving, and relatively easy to engineer. However, despite recent advances in metabolic engineering, the growth performance of engineered formatotrophic strains remains suboptimal for industrial applications.
Traditional methods for optimizing these synthetic pathways rely on adaptive laboratory evolution (ALE), which involves random mutation and natural selection. While effective, ALE is time-consuming and often introduces unwanted off-target mutations, making it an inefficient method for precise pathway optimization. Recent advancements in directed evolution and synthetic biology have provided new tools for improving enzyme function and pathway efficiency, but these tools have primarily been optimized for Escherichia coli and are challenging to implement in other bacterial hosts.
Overall Objectives
The EvoZyme project aims to address these challenges by developing a synthetic diversification platform that enables the targeted evolution of enzymes in multiple bacterial hosts. The platform will allow for precise and accelerated genetic diversification of key metabolic enzymes, optimizing microbial growth and productivity in a way that circumvents the limitations of traditional ALE. The project is structured around two primary objectives:
Development of a synthetic diversification platform – A system capable of hypermutating a gene of interest (GOI) in a targeted manner while allowing seamless transfer of the evolved GOI to various bacterial hosts. This ensures pathway optimization without introducing off-target effects in the host genome.
Optimization of the reductive glycine pathway (rGlyP) through targeted evolution of the glycine cleavage system (GCS) – The GCS is the core enzyme complex responsible for key metabolic conversions within rGlyP. EvoZyme will apply the diversification platform to improve the efficiency of the GCS, enabling enhanced formatotrophic growth in industrially relevant microbes (E. coli, Cupriavidus necator, Pseudomonas putida).
By achieving these objectives, EvoZyme will pave the way for a new approach to enzyme engineering, establishing a broadly applicable diversification tool that could optimize other critical biosynthetic pathways beyond rGlyP.
Pathway to Impact
The EvoZyme project is expected to significantly advance synthetic metabolism by providing a novel, adaptable platform for improving enzymatic functions in diverse microbial hosts. The key expected impacts include:
Scientific and Technological Impact:
• Introduction of a highly efficient and versatile enzyme evolution tool for synthetic biology and metabolic engineering.
• Generation of optimized formatotrophic strains capable of efficiently converting CO2-derived substrates into biomass and valuable bioproducts.
• Improved understanding of the molecular mechanisms underlying enzyme optimization, with applications in industrial biotechnology.
Economic and Industrial Impact:
• Acceleration of biotechnological strain development, reducing the time and cost associated with optimizing metabolic pathways.
• Enhancement of biomanufacturing processes, improving the feasibility of using engineered microbes for large-scale production of bio-based chemicals.
• Potential commercialization of the diversification platform as a broadly applicable tool for enzyme engineering in academia and industry.
Societal and Environmental Impact:
• Contribution to a sustainable bioeconomy, reducing dependency on fossil fuels and mitigating CO2 emissions.
• Support for achieving multiple United Nations Sustainable Development Goals (SDGs), particularly those related to climate action, sustainable industry, and responsible consumption.
• Increased public engagement in synthetic biology through open science practices and science communication initiatives.
Setting the Scene for EvoZyme
The EvoZyme project operates at the intersection of biotechnology, synthetic biology, and metabolic engineering, tackling one of the key barriers to a bio-based circular economy—the efficient engineering of microbial metabolism. By creating a powerful and tunable enzyme diversification platform, EvoZyme will not only improve biotechnological production processes but also provide an essential tool for future research in metabolic engineering and synthetic metabolism. With a strong focus on scientific innovation, industrial applicability, and environmental sustainability, EvoZyme has the potential to transform how microbial strains are engineered, paving the way for a new era of sustainable bioproduction.
Despite being conducted over a reduced duration of 11 months instead of 24 months, the EvoZyme project successfully achieved its first objective (O1): the development of a synthetic diversification platform for the targeted evolution of enzymes. The platform was designed to enable precise hypermutation of a gene of interest (GOI) while minimizing off-target effects and ensuring its transferability to different bacterial hosts.
Development and Benchmarking of the Diversification Platform (O1 – Successfully Completed)
The main goal of O1 was to construct and validate a high-efficiency targeted mutagenesis system while maintaining host genome integrity. This was accomplished through the creation of two distinct diversification strains based on Escherichia coli ST18:
• ST18:MutT (Targeted Diversification) – Engineered to utilize MutaT7 technology, a fusion of a nucleobase deaminase with T7 RNA polymerase, enabling site-specific hypermutation of a GOI under the control of a T7 promoter. This system provides precise and controlled mutagenesis of pathway enzymes without affecting the host genome.
• ST18:MutR (Random Diversification) – A strain incorporating a mutagenesis plasmid (MP) system, which induces random mutagenesis across the entire cellular genome. This offers an alternative approach for pathway optimization, allowing for broader evolutionary exploration.
Both strains were successfully constructed, genetically validated, and subjected to initial characterization experiments. These experiments confirmed the functionality and efficiency of the diversification platform, demonstrating its potential as a versatile tool for enzyme evolution.
Conclusion and Next Steps
The successful development of the diversification platform (O1) represents a major milestone in the EvoZyme project. While further characterization is required to refine and expand its applications, the platform has already demonstrated significant potential for enzyme engineering and metabolic pathway optimization.
The research will continue under the leadership of the beneficiary, who has now taken on the role of Assistant Professor at the University of Groningen. Future work will focus on further characterizing the mutagenesis efficiency, host compatibility, and practical applications of the diversification platform in industrial and synthetic biology contexts. The EvoZyme project has already laid a strong foundation for advancing next-generation enzyme engineering strategies, with promising implications for biotechnology, metabolic engineering, and sustainable bioeconomy development.
Development and Benchmarking of the Diversification Platform (O1 – Successfully Completed)
The main goal of O1 was to construct and validate a high-efficiency targeted mutagenesis system while maintaining host genome integrity. This was accomplished through the creation of two distinct diversification strains based on Escherichia coli ST18:
• ST18:MutT (Targeted Diversification) – Engineered to utilize MutaT7 technology, a fusion of a nucleobase deaminase with T7 RNA polymerase, enabling site-specific hypermutation of a GOI under the control of a T7 promoter. This system provides precise and controlled mutagenesis of pathway enzymes without affecting the host genome.
• ST18:MutR (Random Diversification) – A strain incorporating a mutagenesis plasmid (MP) system, which induces random mutagenesis across the entire cellular genome. This offers an alternative approach for pathway optimization, allowing for broader evolutionary exploration.
Both strains were successfully constructed, genetically validated, and subjected to initial characterization experiments. These experiments confirmed the functionality and efficiency of the diversification platform, demonstrating its potential as a versatile tool for enzyme evolution.
Conclusion and Next Steps
The successful development of the diversification platform (O1) represents a major milestone in the EvoZyme project. While further characterization is required to refine and expand its applications, the platform has already demonstrated significant potential for enzyme engineering and metabolic pathway optimization.
The research will continue under the leadership of the beneficiary, who has now taken on the role of Assistant Professor at the University of Groningen. Future work will focus on further characterizing the mutagenesis efficiency, host compatibility, and practical applications of the diversification platform in industrial and synthetic biology contexts. The EvoZyme project has already laid a strong foundation for advancing next-generation enzyme engineering strategies, with promising implications for biotechnology, metabolic engineering, and sustainable bioeconomy development.
The EvoZyme project successfully developed a synthetic diversification platform that integrates directed evolution methods into a versatile and accessible platform strain. While the core mutagenesis techniques are based on published methods, EvoZyme enhances their usability and applicability across diverse bacterial hosts, addressing key limitations of current approaches.
Key Advances
• Streamlined and Accessible Diversification Platform: EvoZyme consolidates targeted (MutaT7-based) and random (mutagenesis plasmid) diversification into a single, easy-to-use system within E. coli ST18.
• Broad Host Compatibility: The platform allows evolved genes to be easily transferred and tested in multiple industrially relevant bacteria (E. coli, Pseudomonas putida, Cupriavidus necator), expanding its applicability.
• Minimized Off-Target Effects: The separation of mutation and selection phases reduces unintended genomic changes, making the system more precise and reliable.
Next Steps for Further Uptake
To maximize impact, further work is needed to:
• Characterize and optimize the platform across additional bacterial species.
• Enhance screening methods to streamline the identification of improved enzyme variants.
• Explore industry collaborations to facilitate broader adoption in biotechnology and metabolic engineering.
Conclusion
EvoZyme provides a practical and scalable tool for enzyme evolution, making directed mutagenesis more accessible to both academic and industrial researchers. While additional research is required to refine and expand its applications, the platform has strong potential to support biotechnological advancements in metabolic pathway optimization and enzyme engineering.
Key Advances
• Streamlined and Accessible Diversification Platform: EvoZyme consolidates targeted (MutaT7-based) and random (mutagenesis plasmid) diversification into a single, easy-to-use system within E. coli ST18.
• Broad Host Compatibility: The platform allows evolved genes to be easily transferred and tested in multiple industrially relevant bacteria (E. coli, Pseudomonas putida, Cupriavidus necator), expanding its applicability.
• Minimized Off-Target Effects: The separation of mutation and selection phases reduces unintended genomic changes, making the system more precise and reliable.
Next Steps for Further Uptake
To maximize impact, further work is needed to:
• Characterize and optimize the platform across additional bacterial species.
• Enhance screening methods to streamline the identification of improved enzyme variants.
• Explore industry collaborations to facilitate broader adoption in biotechnology and metabolic engineering.
Conclusion
EvoZyme provides a practical and scalable tool for enzyme evolution, making directed mutagenesis more accessible to both academic and industrial researchers. While additional research is required to refine and expand its applications, the platform has strong potential to support biotechnological advancements in metabolic pathway optimization and enzyme engineering.