Periodic Reporting for period 4 - METACELL (Artificial metabolic cells for biomanufacturing of bio-based chiral fine chemicals )
Okres sprawozdawczy: 2024-07-01 do 2025-12-31
One of the major challenges in sustainable chemistry is expanding the palette of bio-based chemicals that can replace, or at least reduce, the exploitation of fossil fuel–based chemicals. Cell-free metabolic engineering using soluble enzymes is an emerging and versatile approach aimed at increasing the selectivity and productivity of chemical biomanufacturing processes. However, soluble and isolated enzymes face significant limitations in efficiency, stability, and reusability, which hinder their industrial application.
To address these challenges, enzymes can be rationally immobilized on smart materials, yielding robust, efficient, and self-sufficient heterogeneous biocatalysts. Nevertheless, current immobilization strategies are largely limited to simple enzyme cascades. The mission of METACELL is to develop self-sufficient artificial metabolic cells (AMCs) by immobilizing complex metabolic networks onto hierarchical porous materials. To achieve this goal, solid surfaces must play an active role in the chemical process rather than serving merely as passive immobilization supports. These AMCs envision very productive and robust multi-functional heterogeneous biocatalysts to be implemented under industrial settings.
This integrative proposal will combine protein engineering, surface chemistry, bio-organic chemistry, and protein immobilization strategies to enable: (1) the development of a cell-free artificial metabolism; (2) innovative engineering tools to modify both enzyme and material surfaces; and (3) the continuous synthesis of industrially relevant fine chemicals catalyzed by AMCs packed into flow reactors. The resulting METACELL technology will serve as a prototyping platform for testing artificial biosynthetic pathways towards the manufacturing of fine chemicals with applications in polymer, food, and medicinal chemistry. Furthermore, the AMCs herein developed may contribute to expanding the biochemical toolbox of biorefineries. Beyond its technological outcomes, METACELL will generate fundamental insights into how the spatial organization of multi-enzyme systems influences the performance of in vitro biosynthetic pathways confined within artificial chassis based on solid materials.
To address these challenges, enzymes can be rationally immobilized on smart materials, yielding robust, efficient, and self-sufficient heterogeneous biocatalysts. Nevertheless, current immobilization strategies are largely limited to simple enzyme cascades. The mission of METACELL is to develop self-sufficient artificial metabolic cells (AMCs) by immobilizing complex metabolic networks onto hierarchical porous materials. To achieve this goal, solid surfaces must play an active role in the chemical process rather than serving merely as passive immobilization supports. These AMCs envision very productive and robust multi-functional heterogeneous biocatalysts to be implemented under industrial settings.
This integrative proposal will combine protein engineering, surface chemistry, bio-organic chemistry, and protein immobilization strategies to enable: (1) the development of a cell-free artificial metabolism; (2) innovative engineering tools to modify both enzyme and material surfaces; and (3) the continuous synthesis of industrially relevant fine chemicals catalyzed by AMCs packed into flow reactors. The resulting METACELL technology will serve as a prototyping platform for testing artificial biosynthetic pathways towards the manufacturing of fine chemicals with applications in polymer, food, and medicinal chemistry. Furthermore, the AMCs herein developed may contribute to expanding the biochemical toolbox of biorefineries. Beyond its technological outcomes, METACELL will generate fundamental insights into how the spatial organization of multi-enzyme systems influences the performance of in vitro biosynthetic pathways confined within artificial chassis based on solid materials.
During the whole execution of METACELL, we have successfully implemented a modular approach to assemble cell-free immobilized biosynthetic pathways that transform simple raw materials into α- and ω-substituted β-hydroxy acids using free enzymes. As a result, we have fabricated self-sufficient and robust artificial metabolic cells (AMCs), which integrate complex metabolic networks into hierarchically structured porous materials as artificial chassis. The resulting self-sufficient AMCs have been exploited for the manufacturing of chiral fine chemicals. AMCs fabrication has involved the assembly of different functional modules (enzymes, cofactors, and sensors) into solid materials (artificial chassis).
The main milestones achieved during this research endeavor are summarized below:
1. New cell-free oxidative enzyme cascades for β- and ω-hydroxy acid synthesis.
2. Engineered enzyme surfaces with His clusters for controlled, site-directed immobilization.
3. High-throughput screening for multi-enzyme co-immobilization.
4. Demonstration that enzyme spatial distribution and crowding inside porous supports critically impact cascade performance.
5. A new generation of self-sufficient heterogeneous biocatalysts that operate without external supply of exogeneous cofactors.
6. Implementation of multi-functional and self-sufficient heterogeneous biocatalysts in continuous-flow packed-bed, porous membrames and 3D-printed monolitich bioreactors.
The main milestones achieved during this research endeavor are summarized below:
1. New cell-free oxidative enzyme cascades for β- and ω-hydroxy acid synthesis.
2. Engineered enzyme surfaces with His clusters for controlled, site-directed immobilization.
3. High-throughput screening for multi-enzyme co-immobilization.
4. Demonstration that enzyme spatial distribution and crowding inside porous supports critically impact cascade performance.
5. A new generation of self-sufficient heterogeneous biocatalysts that operate without external supply of exogeneous cofactors.
6. Implementation of multi-functional and self-sufficient heterogeneous biocatalysts in continuous-flow packed-bed, porous membrames and 3D-printed monolitich bioreactors.
The achievements attained during the execution of METACELL represent a significant step forward in the design of immobilized multi-enzyme systems for applied biocatalysis. This project enabled us to address a series of challenges at multiple levels, ranging from the discovery of new-to-nature enzymatic networks capable of transforming simple, monofunctional molecules into more complex and multifunctional compounds to the development of a comprehensive set of tools for engineering the immobilization of the enzymes that constitute these networks.
As a result, we have obtained more stable and highly active heterogeneous biocatalysts, which we anticipate will have a strong impact on future chemical biomanufacturing processes. In parallel, we have advanced the understanding of structure–function relationships of immobilized enzymes within porous materials. This intraparticle-level information has been crucial in revealing how enzyme density and spatial distribution influence overall biocatalyst performance.
The innovative methodologies developed within METACELL open new opportunities to perform operando studies of immobilized enzymes under diverse reaction configurations, including stirred-tank reactors, packed-bed reactors, and fluidized-bed reactors. Furthermore, we have demonstrated the feasibility of co-immobilizing enzymes and cofactors, significantly enhancing process cost efficiency. Overall, this project has delivered novel methodologies for the immobilization of complex enzyme systems and generated fundamental knowledge that enables a more rational and predictable design of immobilized biocatalysts.
As a result, we have obtained more stable and highly active heterogeneous biocatalysts, which we anticipate will have a strong impact on future chemical biomanufacturing processes. In parallel, we have advanced the understanding of structure–function relationships of immobilized enzymes within porous materials. This intraparticle-level information has been crucial in revealing how enzyme density and spatial distribution influence overall biocatalyst performance.
The innovative methodologies developed within METACELL open new opportunities to perform operando studies of immobilized enzymes under diverse reaction configurations, including stirred-tank reactors, packed-bed reactors, and fluidized-bed reactors. Furthermore, we have demonstrated the feasibility of co-immobilizing enzymes and cofactors, significantly enhancing process cost efficiency. Overall, this project has delivered novel methodologies for the immobilization of complex enzyme systems and generated fundamental knowledge that enables a more rational and predictable design of immobilized biocatalysts.