[P007] Poster

12:10 PM - 1:00 PM JST(3:10 AM - 4:00 AM UTC)

[P04-495] Biocatalytic Precision Control of Post-Translational Modifications for Engineering Functional Biomolecules

○Noriho Kamiya^1,2 (1. Department of Applied Chemistry, Graduate School of Engineering, Kyushu University (Japan), 2. Center for Future Chemistry, Kyushu University (Japan))

Keywords:

Bioconjugation,Biopharmaceutical,Bond-forming enzyme,Lipid,Transglutaminase

Biomolecules are fundamental components of living systems and have been widely utilized across diverse sectors of the bioindustry. Engineering biomolecules through chemical and physical approaches is of great interest, as it expands their functional potential in biotechnology. In this context, enzyme-catalyzed post-translational modification (PTM) offers a powerful strategy for constructing designer bioconjugates with high selectivity under mild conditions. We have focused on microbial transglutaminase (MTG), an enzyme that catalyzes the formation of covalent bonds between glutamine (Gln) and lysine (Lys) residues, to develop functional bioconjugates. These include lipid–protein conjugates as artificial antifungal proteins and antibody–drug conjugates as representative biopharmaceuticals.To achieve precise control of modification sites and substrate specificity, we designed fusion proteins incorporating an engineered MTG zymogen (EzMTG), which retains crosslinking activity in its zymogen state. Fusion of EzMTG to the N-terminus of protein G generated EzMTG–pG, which enabled site-selective labeling of a Lys residue on the heavy chain of a clinically relevant IgG antibody (trastuzumab) using a fluorescent and drug-labeled Gln donor substrates. Notably, reversing the domain orientation (pG–EzMTG) resulted in a distinct labeling profile on IgG and its Fab fragment. These findings demonstrate that domain arrangement within fusion proteins plays a critical role in directing substrate preference and achieving site-selective conjugation via proximity effects.To further expand this approach, integrating AI-driven protein design with high-throughput screening (HTS) technologies is crucial. In this context, we have developed a screening platform that combines enzyme-mediated hydrogelation with fluorescence-activated droplet sorting (FADS) to identify recombinant MTG variants with enhanced catalytic activity via cell-free protein synthesis.Overall, our work highlights how biocatalysis, combined with rational design and high-throughput technologies, enables precise control of PTMs and opens new avenues for functional biomolecule engineering and sustainable biomanufacturing.