[3S04m] 11. The Society for Biotechnology - Part 1

11:45 AM - 12:00 PM JST(2:45 AM - 3:00 AM UTC)

[3SBT-07-KL] From Natural Enzyme Condensates to Designer Assemblies for Tunable Biocatalysis

○Natsuko Miura¹ (1. Osaka Metropolitan University (Japan))

Keywords:

Enzyme assembly,Saccharomyces cerevisiae,Metabolic engineering,Hypoxic culture

[Purpose] Reversible enzyme condensation has emerged as a potential mechanism for metabolic regulation in living cells. We previously found that, under hypoxic conditions, more than 20 enzymes including glycolytic enzymes reversibly assemble into metabolic enzyme transient assembling (META) bodies in yeast cells, and that this assembly is associated with enhanced metabolic efficiency and cell survival. Based on these findings, this study aimed to clarify the regulatory basis of enzyme condensate formation and to explore whether such assemblies can be artificially designed and applied to tunable biocatalysis. [Method] To investigate the mechanism of assembly formation, we developed a small-scale hypoxic culture system suitable for high-throughput screening of candidate molecules involved in condensate regulation. Using this platform, we analyzed the assembly behavior of metabolic enzymes under controlled oxygen conditions. We further examined the amino acid sequences of assembly-forming enzymes to identify short peptide motifs responsible for condensation. These motifs were fused to heterologous proteins and enzymes to test whether artificial assemblies could be induced in a programmable manner. The effects of induced assembly on enzyme reactions were then evaluated under different oxygen conditions. [Results] The screening platform enabled systematic analysis of hypoxia-responsive enzyme condensation in yeast and facilitated identification of factors associated with assembly formation. We identified peptide motifs capable of promoting protein assembly when fused to target proteins. Importantly, these motifs allowed the construction of designer enzyme assemblies beyond naturally assembling proteins. By applying them to arbitrary enzymes, we succeeded in inducing artificial assemblies and demonstrated that enzyme reactions could be modulated tunably, independently of ambient oxygen concentration. [Consideration] These results suggest that the molecular principles underlying natural enzyme condensates can be translated into a practical engineering strategy for controlling biocatalysis. The ability to confer assembly-forming properties on target enzymes using short peptide motifs indicates that enzyme condensation is not merely a native stress response, but also a designable platform for reaction control. This concept may broaden the scope of enzyme engineering by providing a new layer of regulation distinct from conventional approaches based on expression level, localization, or protein scaffold design.[Conclusion] Natural enzyme condensates in yeast provide a useful framework for the development of designer assemblies for tunable biocatalysis. Our findings establish a basis for expanding enzyme condensates from a biological phenomenon to an engineering platform with potential applications in future biotechnology and biomanufacturing.