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Artificial metabolic cells for biomanufacturing of bio-based chiral fine chemicals

Periodic Reporting for period 2 - METACELL (Artificial metabolic cells for biomanufacturing of bio-based chiral fine chemicals )

Reporting period: 2021-07-01 to 2022-12-31

One of the major challenges of sustainable chemistry is expanding the palette of bio-based chemicals that can replace, or at least ameliorate, the exploitation of fuel-based chemicals. Cell-free metabolic engineering using soluble enzymes is an emerging and versatile approach that seeks to increase the selectivity and productivity of chemical biomanufacturing processes. However, soluble and isolated enzymes present major issues in terms of efficiency, stability and re-usability that hamper industrial applications. To solve these problems, enzymes can be rationally immobilized on smart materials resulting in robust, efficient and self-sufficient heterogeneous biocatalysts, but immobilization is still restricted to simple enzyme cascades. METACELL mission is to develop self-sufficient artificial metabolic cells (AMCs) by immobilizing complex metabolic networks on hierarchical porous materials. To this aim, the solid surfaces must play an active role in the chemical process rather than just being the mere immobilization support. This integrative proposal will exploit protein engineering, surface chemistry, bio-organic chemistry and protein immobilization tools for the successful development of 1) a cell-free artificial metabolism, 2) innovative engineering tools to modify both enzyme and material surfaces and 3) continuous synthesis of industrially relevant fine chemicals catalyzed by AMCs packed into flow reactors. The resulting technology of METACELL will serve as a prototyping platform to test artificial biosynthetic pathways with application in combinatorial chemistry (e.g drug discovery). METACELL may also offer long-term solutions for the on-demand production of drugs at the point of care In addition to the technological outputs, METACELL will also provide essential information to understand how the spatial organization of multi-enzyme systems affect the performance of in vitro biosynthetic pathways confined into artificial chassis (solid materials)
During the first 30 months of METACELL we have successfully implemented a modular cell-free metabolism to transform simple raw materials into -hydroxy acids using free enzymes. The surfaces of some of these enzymes have been engineered to control their orientation when immobilized on porous materials, aiming at maximizing the activity and stability of the resulting heterogeneous biocatalysts. Through this approach, we have elicited the most suitable regions through which these enzymes must be immobilized to achieve a high stabilization without reducing their catalytic efficiency. Besides controlling the enzyme orientation, we have also controlled the spatial organization of multi-enzyme systems co-immobilized on the same carrier. Remarkably, we have observed that the spatial arrangement of up to 5 enzymes across the porous surface of microbeads has a terrific impact on the overall kinetic behavior of the biosynthetic cascade catalyzed by that heterogeneous multi-enzyme system. Finally, we have also co-localized enzymes and cofactors within the same porous carrier, demonstrating that the confined cofactors are available for and used by the co-immobilized enzymes. We deeply study this phenomenon through time-lapse fluorescence microscopy and image analytics to understand the thermodynamics and kinetics that rule the interactions between the co-immobilized enzymes and cofactors. Through this approach, we show our capacity to fabricate self-sufficient heterogeneous biocatalysts that require no exogenous cofactor supply as both enzymes and cofactors are confined into the solid phase. All in all, we have set the basis to co-immobilize several enzymes and cofactors on porous materials to fabricate artificial metabolic cells that involve a complex enzymatic network. Until now, we have illustrated this concept with a minimalist system where two co-immobilized enzymes and one cofactor have been re-used in consecutive batch cycles but also integrated into different architectures (membranes, packed-bed reactors, and monoliths) for the continuous production of fine chemicals (i.enantiopurere -hydroxy acids) without exogenous addition of costly cofactors (i.e NADH).
The achievements obtained during the first 30 months of the project have meant a step forwards in the design of immobilized multi-enzyme systems for applied biocatalysis. This project has allowed us to face a series of challenges at different levels that go from the discovery of new-to-nature enzymatic networks that transform simple and monofunctional molecules into more complex and multifunctional ones to the development of a set of tools to engineer the immobilization of the enzymes forming part of those networks. As result, we have achieved more stable and active heterogeneous biocatalysts for which we envision a bright future in chemical biomanufacturing. Finally, we have advanced in understanding the structure/function relationships of immobilized enzymes within the porous surface. This intraparticle information has been decisive to unveil the impact of enzyme density and spatial distribution on the biocatalyst´s functionality. This innovative methodology opens an enormous opportunity to perform in operando studies of immobilized enzymes under different reaction configurations (stirred tank, packed bed reactor, fluidized reactor…).

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