Periodic Reporting for period 1 - KITZYME (Development of rationally designed enzyme kits)
Okres sprawozdawczy: 2024-07-01 do 2025-12-31
Enzymes exhibit high efficiency, specificity, selectivity, biodegradability, non-toxicity, and the ability to function effectively under gentle biological conditions. These qualities render enzymes a sustainable and eco-friendly substitute for traditional catalysts within industrial settings. However, harnessing enzymes for industrial applications often necessitates extensive and costly experimental engineering efforts. Computational methods hold promise as potential solutions, but these have not yet demonstrated the ability to rapidly design highly efficient enzymes that mimic those found in Nature. As opposed to other methods, our computational approach developed in the previous ERC-STG can introduce active site and distal mutations that modulate the enzyme conformational dynamics, achieving increases in catalytic efficiency of up to 1000-fold. This project aimed to exploit the proprietary technology developed in ERC-STG and ERC-POC for generating a set of patentable rationally designed enzyme kits focused on stereoselective carbon-carbon bond formation. KITZYME aimed to create a spin-off for the exploitation of the new set of rationally designed enzyme kits as well as the proprietary technology developed for enzyme optimization. Both the enzyme kits and our technology formed the cornerstone of the project, for providing industries with a cost-effective, scalable, and environmentally sustainable solution. Beyond the scientific goals, the project was explicitly oriented toward impact, aiming to establish a clear pathway from technological innovation to industrial application through intellectual property protection, strategic partnerships, and the creation of a dedicated spin-off company. In doing so, the project aimed to contribute significantly to the adoption of greener manufacturing processes, reduce development costs and timelines for industrial biocatalysts, and strengthen the transfer of academic innovation into the market, ultimately supporting a more sustainable and competitive chemical sector.
The project focused on the development and validation of a computational pipeline for the rational design of biocatalysts capable of promoting stereoselective carbon–carbon bond formation. The computational pipeline combined molecular dynamics simulations with correlation-based Shortest Path Map (SPM) analysis to identify catalytically relevant positions within enzyme active sites and beyond. These positions were systematically explored through a combination of in silico design and experimental screening, enabling the generation of multiple enzyme variants with modulated reactivity profiles. Experimental validation was carried out using a diverse panel of electrophiles and donor substrates, allowing the assessment of catalytic activity, substrate scope, and stereochemical outcome. Several designed variants displayed measurable and reproducible activity, demonstrating that the computationally identified positions can be exploited to tune enzymatic reactivity and expand substrate acceptance beyond previously reported limitations. Thermal stability assays were conducted to evaluate the impact of the introduced modifications, revealing differences in stability and mutational tolerance across the studied enzyme families. Importantly, a subset of variants retained a significant proportion of activity relative to their corresponding wild-type counterparts while maintaining comparable stability, highlighting their suitability as starting points for further optimization. Overall, the project delivered a validated design strategy, a collection of promising enzyme variants, and a comprehensive experimental dataset describing activity, stability, and substrate scope.
The project advances the state of the art by demonstrating the effectiveness of a rational, physics-based approach to enzyme engineering for stereoselective carbon–carbon bond formation, a transformation of high relevance for sustainable chemical synthesis. Beyond generating individual active variants, the work provides a rational design pipeline that can be applied to other enzyme classes and reaction types. The results reveal previously unexploited catalytic potential within known biocatalytic scaffolds and show that targeted mutations can unlock reactivity toward structurally diverse substrates. This expands the accessible chemical space of enzymatic transformations and supports the broader adoption of biocatalysis in fine chemical and pharmaceutical manufacturing. Additional rounds of computational refinement and experimental optimization will be required to enhance activity, selectivity, and operational robustness under industrially relevant conditions. Demonstration at larger scale will be a key next step. Complementary efforts in intellectual property protection, regulatory alignment, and access to investment and commercial partnerships will be essential to translate the results into market-ready enzyme solutions.