Periodic Reporting for period 1 - BiocatCodeExpander (Genetic code expansion for biocatalysis and enzyme engineering doctoral network)
Reporting period: 2023-01-01 to 2024-12-31
The expansion of the genetic code through the incorporation of non-canonical amino acids (NCAA) opens up a range of exciting biotechnological possibilities, including protein imaging, spectroscopy, and enzyme engineering. However, the application of NCAA in enzyme engineering has been limited by the inefficiency or lack of flexibility in current incorporation methods. This DN proposal seeks to address these limitations by fostering a collaborative, multidisciplinary approach, bringing together expertise in biocatalysis, synthetic and computational biology. Through the training of 10 DCs at the cutting edge of these fields, the project will enhance NCAA incorporation tools, facilitating site-selective protein conjugation, high-throughput screening, and the design of new-to-nature reactions. The newly designed biocatalysts developed through this research will not only contribute to scientific innovation but also ensure the next generation of researchers are equipped to drive sustainable and environmentally friendly production of chemicals and pharmaceuticals.
WP1 – Tools for NCAA Incorporation
WP1 aims to increase both the efficiency and diversity of NCAA incorporation. DC1 developed a luxAB-based bioluminescence assay for high-throughput screening of alkane monooxygenase variants. The method was tested with various enzymes and substrates and the best alkane monooxygenase was identified. DC2 worked on designing a new expression plasmid and in-silico engineering thermostable aminoacyl-tRNA synthetases. To address cost challenges in NCAA production, DC3 optimized a cyanobacterial enzyme cascade for synthesizing L-homophenylalanine and L-homotyrosine from LB medium. In vivo incorporation studies were conducted during a secondment at the University of Manitoba.
WP2 – Novel Strategies for Coupling & Immobilization
The work in this WP centered on developing strategies for enzyme coupling and immobilization. DC4 developed a multigram-scale synthesis of Si-containing L-lysine derivatives and a metal-free radical hydrosilylation reaction compatible with proteins. DC5 worked on site-directed enzyme immobilization, developing a battery of conjugation chemistries based on natural AA and NCAA and creating a small library of genetically modified protein variants with single-site NCAA modifications. DC6 focused on stabilizing growth factors, expressing fibroblast growth factors with non-canonical proline derivatives, which exhibited enhanced thermostability.
WP3 – Enzyme Engineering with NCAA
WP3 explores how NCAA can introduce novel interactions and functionalities into enzymes through directed evolution and rational design. DC7 investigated Nδ-methylhistidine substitutions in two dioxygenases, successfully purifying and characterizing new variants. DC8 focused on engineering of tandem-fused 4-oxalocrotonate tautomerase by incorporating different catalytic NCAA at the catalytic residue position Pro-1 and the non-catalytic residue position Pro-68. DC9 used computational tools such as FuncLib and AlphaFold2 to design enzyme scaffolds tailored for NCAA incorporation. DC10 explored synthesis of 7 tertiary-amine-containing NCAA, successfully upscaling 2 variants and sharing them with DC2 for further research.
WP1 aims to increase both the efficiency and diversity of NCAA incorporation. DC1 developed a luxAB-based bioluminescence assay for high-throughput screening of alkane monooxygenase variants. The method was tested with various enzymes and substrates and the best alkane monooxygenase was identified. DC2 worked on designing a new expression plasmid and in-silico engineering thermostable aminoacyl-tRNA synthetases. To address cost challenges in NCAA production, DC3 optimized a cyanobacterial enzyme cascade for synthesizing L-homophenylalanine and L-homotyrosine from LB medium. In vivo incorporation studies were conducted during a secondment at the University of Manitoba.
WP2 – Novel Strategies for Coupling & Immobilization
The work in this WP centered on developing strategies for enzyme coupling and immobilization. DC4 developed a multigram-scale synthesis of Si-containing L-lysine derivatives and a metal-free radical hydrosilylation reaction compatible with proteins. DC5 worked on site-directed enzyme immobilization, developing a battery of conjugation chemistries based on natural AA and NCAA and creating a small library of genetically modified protein variants with single-site NCAA modifications. DC6 focused on stabilizing growth factors, expressing fibroblast growth factors with non-canonical proline derivatives, which exhibited enhanced thermostability.
WP3 – Enzyme Engineering with NCAA
WP3 explores how NCAA can introduce novel interactions and functionalities into enzymes through directed evolution and rational design. DC7 investigated Nδ-methylhistidine substitutions in two dioxygenases, successfully purifying and characterizing new variants. DC8 focused on engineering of tandem-fused 4-oxalocrotonate tautomerase by incorporating different catalytic NCAA at the catalytic residue position Pro-1 and the non-catalytic residue position Pro-68. DC9 used computational tools such as FuncLib and AlphaFold2 to design enzyme scaffolds tailored for NCAA incorporation. DC10 explored synthesis of 7 tertiary-amine-containing NCAA, successfully upscaling 2 variants and sharing them with DC2 for further research.
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.
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.