WP1
For DC1, the project has successfully demonstrated the application of the luxAB-based bioluminescence assay. This assay contributes to a deeper understanding of AlkB function, paving the way for future enzyme engineering and industrial applications. For DC2, efforts have focused on leveraging AI and machine learning to optimize the thermostability of an archaeal PylRS, identifying nine variants predicted to have enhanced stability. These variants are currently undergoing experimental validation. For DC3, the project has successfully demonstrated the in vivo synthesis of target NCAA. The broader application of these findings depends on the scalability of the biosynthetic pathways and the feasibility of in vivo incorporation.
WP2
For DC4, Si-NCAA and metal-free radical hydrosilylation reactions have shown promise in biocatalysis, therapeutics, and imaging. Potential applications include site-selective enzyme immobilization, enzyme stabilization for industrial use, and in vivo applications of protein conjugates. For DC5, the project has validated azidohomoalanine as an NCAA for site-selective protein immobilization. This technology has the potential to impact industrial biocatalysis, offering heterogeneous biocatalysts with improved properties, facilitating enzyme integration into industrial processes. The expertise developed during the first year will support an industrial secondment at Enginzyme in 2025, where the results are expected to attract interest from our industrial associate partner. For DC6, the project marks the first successful incorporation of NCAA into fibroblast growth factors via selective pressure incorporation, making it the second known example of such modifications in these proteins. Further work on incorporating NCAA with click-chemistry functional groups will expand research and application opportunities in cell culture and spatial biology.
WP3
For DC7, the project has demonstrated that Nδ-methylhistidine can be successfully incorporated as an active site ligand in a subgroup of non-heme iron enzymes, revealing critical first-sphere residue interactions that influence enzyme activity and stability. Notably, the observed differences in relative activity between axial and equatorial histidine mutants provide insights into designing more complex coordination motifs in related enzymatic classes. For DC8, the project has uncovered new-to-nature enzymatic activities with potential applications in sustainable chemical synthesis. While currently in the development phase, the commercial viability of this discovery hinges on the catalytic efficiency of optimized enzymes and the scalability of bond-forming reactions. For DC9, while bioinformatics tools exist for protein design, few are tailored for use with NCAA. This project is pioneering in that regard, offering a novel computational perspective on protein design. Although still in its early stages, the anticipated scaffold design results will contribute to the development of new computational tools for NCAA-based protein engineering. Finally, DC10 has successfully synthesized several tertiary amine-containing NCAA. While the immediate commercial value lies in the NCAA themselves, their full potential will emerge through further optimization and application development.
