Biology subjects

Kagawa, A.

Publications and source records attributed to Kagawa, A..

5 recordsLinked to original sources

bioRxiv2026

Selective Recognition, Covalent Linkage and Chemical Exchange of Artificial Cells through Engineered α-Hemolysin

Bottom-up synthetic biology aims not only to build individual artificial cells but also to connect them into higher-order systems that communicate with each other. Reported artificial cells communicate through partner recognition, physical connection, and chemical signaling that are typically implemented by separate molecular species. Here, we engineer the pore-forming protein -hemolysin (AH) with the SpyTag-SpyCatcher (ST-SC) pair to integrate partner recognition, covalent linkage between cells, and nanopore-mediated connection of their lumens into a single protein system. Fusion of ST or SC to AH preserved heptamer assembly and pore formation, and allowed the two heptamers (AH-ST and AH-SC) to be covalently joined. Cryo-electron microscopy resolved the resulting head-to-head 14-mer assembly at 2.20 [A]. Calcein leakage and single-channel recordings confirmed that the engineered pores remain functional. Mixing ST- and SC-displaying giant unilamellar vesicles (GUVs) resulted in partner-specific association between the two vesicle populations. Finally, photo-triggered release of a newly synthesized caged ethidium bromide (EtBr) in sender GUVs produced EtBr accumulation in receiver GUVs that required direct physical linkage between the two GUVs. This platform provides a modular route toward artificial-cell assemblies in which both connectivity and cargo exchange are programmable.

synthetic biology↗

bioRxiv2026

Elucidating the Bell-Shaped Dependence of Protein Translation Activity on EF-Tu Concentration in a Reconstituted Cell-Free System Using a Mechanistic Model

Protein synthesis in cell-free protein synthesis systems often exhibits non-intuitive input-output relationships. In the PURE system, a reconstituted cell-free system, protein production peaked at low elongation factor Tu (EF-Tu) concentrations and decreased at higher concentrations, resulting in a characteristic bell-shaped profile. Here, we investigated the origin of this behavior using a detailed mechanistic model of translation in the PURE system, designated as ePURE, which describes reaction dynamics of hundreds of molecular species and reactions. Our computational analysis suggested that excess EF-Tu sequesters the initiator tRNA (tRNAfMet) into non-productive EF-Tu{middle dot}GTP{middle dot}Met-tRNAfMet complexes, thereby depleting the pool of initiator tRNA available for translation initiation. This suppression arises from competition for a limited molecular resource rather than from direct inhibition. Based on this mechanism, we predicted that increasing the concentrations of tRNAfMet and methionyl-tRNA formyl-transferase would eliminate the bell-shaped dependence, and experimentally confirmed this prediction. Under these modified conditions, the bell-shaped response disappeared and protein production was enhanced. These findings demonstrate how mechanistic computational models can reveal hidden constraints underlying non-intuitive input-output relationships in complex biochemical networks and guide the rational optimization of cell-free protein synthesis systems.

synthetic biology↗

bioRxiv2025

A universal polyphosphate kinase powers in vitro transcription

Polyphosphate kinases (PPKs) catalyze phosphoryl transfer between polyphosphates and nucleotides. Polyphosphates are a cost-effective source of phosphorylating power, making polyphosphate kinases an attractive enzyme for nucleotide production and regeneration. However, at present, applications that require the simultaneous utilization of diverse nucleotides, such as RNA transcription, remain out of reach due to the restricted substrate profiles of known PPK enzymes. Here, we present the discovery and characterization of a universal PPK capable of efficiently phosphorylating all eight common ribonucleotides: purines and pyrimidines, monophosphates and diphosphates to triphosphates. Under optimal reaction conditions, approximately 70% triphosphate conversion was observed for all common ribonucleotides, with only limited over-phosphorylation. At elevated temperatures, however, production of adenine-capped polyphosphates up to a 30-mer was achieved. An analysis of mutant and chimeric enzymes revealed a rugged functional landscape, particularly for non-adenine nucleotides. Finally, we demonstrated the biotechnological potential of a universal PPK enzyme with a one-pot assay for PPK-powered in vitro transcription.

synthetic biology↗

bioRxiv2020

Hyperactivation of L-lactate oxidase by liquid-liquid phase separation

Liquid droplets formed by liquid-liquid phase separation are attracting attention as functional states of proteins in living cells. Liquid droplets are thought to activate enzymatic reactions by assembling the required molecules. Thus, liquid droplets usually increase the affinity of an enzyme to its substrates, leading to decreased KM values. In this study, we demonstrate a new mechanism of enzyme activation in the droplets using Llactate oxidase (LOX). In the presence of poly-L-lysine (PLL), LOX formed droplets with diameters of hundreds of nanometers to tens of micrometers, stabilized by electro-static interaction. The enzyme activity of LOX in the droplets was significantly enhanced by a fourfold decrease in KM and a tenfold increase in kcat. To our knowledge, this represents the first report for increasing kcat by the formation of the liquid droplet. Interestingly, the conformation of LOX changed in the liquid droplet, probably leading to increased kcat value. Understanding enzyme activation in the droplets provides essential information about enzyme function in living cells in addition to biotechnology applications.

biochemistry↗

bioRxiv2020

Foliar water uptake as a source of hydrogen and oxygen in plant biomass

Introductory biology lessons around the world typically teach that plants absorb water through their roots, but, unfortunately, absorption of water through leaves and subsequent transport and use of this water for biomass formation remains a field limited mostly to specialists. Recent studies have identified foliar water uptake as a significant but still unquantified net water source for terrestrial plants. The growing interest in the development of a new model that includes foliar uptake of liquid water to explain hydrogen and oxygen isotope ratios in leaf water and tree rings requires a method for distinguishing between these two water sources. I therefore devised a method utilizing two different heavy waters (HDO and H218O) to simultaneously label both foliar-uptake water and root-uptake water and quantify their relative contributions to plant biomass. Using this new method, I here present evidence that, in the case of well-watered Cryptomeria japonica, hydrogen and oxygen incorporated into new leaf cellulose in the rainy season derives mostly from foliar-uptake water, while that of new root cellulose derives mostly from root-uptake water, and new branch xylem is somewhere in between. Abandoning the assumption that these elements are supplied from soil water alone may have vast implications in fields ranging from isotope dendroclimatology, silviculture, to biogeochemistry.

plant biology↗