Toki Taira

Recently, the relationship between phase‐separation behavior and biological functions in both natural and artificial systems has garnered significant attention. An aqueous two‐phase system (ATPS) composed of polyethylene glycol (PEG) and dextran (Dex), traditionally utilized for protein purification, has emerged as a model for liquid–liquid phase separation (LLPS). In this study, we found that Cstalk, a giant trimeric fibrous protein with a length of approximately 40 nm, preferentially partitions to the Dex‐rich phase in PEG/Dex ATPS. Consequently, conjugation of a protein of interest (POI) to the giant protein scaffold by the SpyTag‐SpyCatcher system enabled the recruitment of POIs into the Dex‐rich phase. Given that the Dex phase acts as a mimic of a polysaccharide‐rich fungal cell wall, we exploited the potential of Cstalk to deliver antifungal chitinase to the cell wall of Trichoderma viride . Precise control of the conjugation ratio of chitinase to Cstalk showed the tunable antifungal activity against the growth of T. viride depending on the enzyme loading. Amidst the urgent need for novel antifungal strategies, our results demonstrated a new design principle of antifungal therapeutics by leveraging the unique phase‐selective partitioning property and the tunable protein loading capacity of a giant fibrous protein scaffold.

Indigo dyeing is a traditional microbial fermentation process practiced worldwide using plants that produce indican, a precursor of indigo. In Okinawa Prefecture, Japan, indigo dyeing is traditionally performed using a muddy suspension containing indigo, prepared from Strobilanthes cusia. In this region, the addition of a fully fermented dye liquid, Tomodane, is believed to promote fermentation, although its microbial basis remains unclear. In this study, we developed a laboratory-scale indigo fermentation system with biological replication to investigate the effects of Tomodane supplementation on the dyeing intensity, bacterial community dynamics, and indigo particle size. Fermentation supplemented with Tomodane showed an earlier onset of fabric dyeing than fermentation without Tomodane. Microbial community analyses revealed that the bacterial communities in Tomodane-supplemented fermentation converged more rapidly toward a stable community structure. Additionally, bacterial taxa putatively associated with extracellular electron transfer (EET), a process relevant to indigo reduction, were more abundant in the bacterial community at earlier fermentation stages in the presence of Tomodane. Indigo particle size decreased more rapidly during Tomodane-supplemented fermentation, coinciding with an earlier dyeing onset. These results suggest that Tomodane facilitates indigo fermentation by facilitating the early establishment of a functionally competent microbial community capable of efficient indigo reduction.

Lipid‐based formulation of antifungal small drugs is used to mitigate drug toxicity while retaining effective antifungal activity. Herein, the effect of artificial cholesterylation of is studied, both of which were site‐specifically modified using a microbial transglutaminase‐reactive Q‐tag (LysM‐Q and CatD‐Q). This produced protein–lipid conjugates (LysM‐cholesteryl [Chol] or ‐palmitoyl [Pal] and CatD‐Chol or ‐Pal). Although lipidation of these chitinase domains enhances the antifungal activity against Trichoderma viride , palmitoylated proteins (LysM‐Pal and CatD‐Pal) show stronger antifungal effects than cholesterylated proteins (LysM‐Chol and CatD‐Chol) both in the absence and presence of amphotericin B. Further characterization with lipidated LysM‐fused green fluorescent protein (LysM‐muGFP‐Pal and ‐Chol) shows that the cholesterylation of proteins promotes distribution to dextran‐rich phase in an aqueous two‐phase system consisting of polyethylene glycol and dextran. Fluorescence microscopic analyses of the phase‐separated giant unilamellar vesicles reveal a distinct anchoring behavior between LysM‐muGFP‐Pal and ‐Chol to lipid membranes, which is possibly correlated with aggregate formation. These results highlight that the chemical properties of the lipid moiety are crucial to achieve effective enhancement of antifungal activity through artificial lipidation.

The antifungal activity of a small chitin-binding domain from antifungal chitinase was enhanced by the artificial lipidation with a specific alkyl chain length and structure in the presence of amphotericin B.

Candida albicans is a prevalent fungal pathogen that displays antibiotic resistance. The polyene antifungal amphotericin B (AmB) has been the gold standard because of its broad antifungal spectra, and its liposomal formulation, AmBisome, has been used widely and clinically in treating fungal infections. Herein, we explored enhancing the antifungal activity of AmBisome by integrating a small chitin-binding domain (LysM) of chitinase A derived from Pteris ryukyuensis. LysM conjugated with a lipid (LysM–lipid) was initially prepared through microbial transglutaminase (MTG)-mediated peptide tag-specific conjugation of LysM with a lipid–peptide substrate. The AmBisome formulation modified with LysM–lipid conjugates had a size distribution that was comparable to the native liposomes but an increased zeta potential, indicating that LysM–lipid conjugates were anchored to AmBisome. LysM–lipid-modified AmBisome exhibited long-term stability at 4 °C while retaining the capacity to bind chitin. Nevertheless, the antifungal efficacy of LysM–lipid-modified AmBisome against C. albicans was modest. We then redesigned a new LysM–lipid conjugate by introducing a peptide linker containing a thrombin digestion (TD) site at the C-terminus of LysM (LysM–TD linker–lipid), thereby facilitating the liberation of the LysM domain from AmBisome upon the addition of thrombin. This new AmBisome formulation anchored with LysM–TD linker–lipid exhibited superior performance in suppressing C. albicans growth in the presence of thrombin compared with the LysM–lipid formulation. These results provide a platform to design stimuli-responsive AmBisome formulations that respond to external environments and thus advance the treatment of pathogenic fungi infections.

Main conclusion Each β-1,3-glucanase with antifungal activity or yeast lytic activity hydrolyzes different structures of β-1,3-glucans in the fungal cell wall, respectively. Abstract Plants express several glycoside hydrolases that target chitin and β-glucan in fungal cell walls and inhibit pathogenic fungal infection. An antifungal β-1,3-glucanase was purified from gazyumaru (Ficus microcarpa) latex, designated as GlxGluA, and the corresponding gene was cloned and expressed in Escherichia coli. The sequence shows that GlxGluA belongs to glycoside hydrolase family 17 (GH17). To investigate how GlxGluA acts to degrade fungal cell wall β-glucan, it was compared with β-1,3-glucanase with different substrate specificities. We obtained recombinant β-1,3-glucanase (designated as CcGluA), which belongs to GH64, from the bacterium Cellulosimicrobium cellulans. GlxGluA inhibited the growth of the filamentous fungus Trichoderma viride but was unable to lyse the yeast Saccharomyces cerevisiae. In contrast, CcGluA lysed yeast cells but had a negligible inhibitory effect on the growth of filamentous fungi. GlxGluA degraded the cell wall of T. viride better than CcGluA, whereas CcGluA degraded the cell wall of S. cerevisiae more efficiently than GlxGluA. These results suggest that the target substrates in fungal cell walls differ between GlxGluA (GH17 class I β-1,3-glucanase) and CcGluA (GH64 β-1,3-glucanase).

Enzymatic reaction offers site-specific conjugation of protein units to form protein conjugates or protein polymers with intrinsic functions. Herein, we report horseradish peroxidase (HRP)- and microbial transglutaminase (MTG)-catalyzed orthogonal conjugation reactions to create antifungal protein polymers composed of Pteris ryukyuensis chitinase-A (ChiA) and its two domains, catalytic domain, CatD, and chitin-binding domain, LysM₂. We engineered the ChiA and CatD by introducing a peptide tag containing tyrosine (Y-tag) at N-termini and a peptide tag containing lysine and tyrosine (KY-tag) at C-termini to construct Y-ChiA-KY and Y-CatD-KY. Also, LysM₂ with Y-tag and KY-tag (Y-LysM₂-KY) or with a glutamine-containing peptide tag (Q-tag) (LysM₂-Q) were constructed. The proteins with Y-tag and KY-tag were efficiently polymerized by HRP reaction through the formation of dityrosine bonds at the tyrosine residues in the peptide tags. The Y-CatD-KY polymer was further treated by MTG to orthogonally graft LysM₂-Q to the KY-tag via isopeptide formation between the side chains of the glutamine and lysine residues in the peptide tags to form LysM₂-grafted CatD polymer. The LysM₂-grafted CatD polymer exhibited significantly higher antifungal activity than the homopolymer of Y-ChiA-KY and the random copolymer of Y-CatD-KY and Y-LysM₂-KY, demonstrating that the structural differences of artificial chitinase polymers have a significant impact on the antifungal activity. This strategy of polymerization and grafting reaction of protein can contribute to the further research and development of functional protein polymers for specific applications in various fields in biotechnology.

Main conclusion A chitin-binding domain could contribute to the antifungal ability of chitinase through its affinity to the fungal lateral wall by hydrophobic interactions. Abstract Complementary DNA encoding the antifungal chitinase of gazyumaru (Ficus microcarpa), designated GlxChiB, was cloned and expressed in Escherichia coli cells. The results of cDNA cloning showed that the precursor of GlxChiB has an N-terminal endoplasmic reticulum targeting signal and C-terminal vacuolar targeting signal, whereas mature GlxChiB is composed of an N-terminal carbohydrate-binding module family-18 domain (CBM18) and a C-terminal glycoside hydrolase family-19 domain (GH19) with a short linker. To clarify the role of the CBM18 domain in the antifungal activity of chitinase, the recombinant GlxChiB (wild type) and its catalytic domain (CatD) were used in quantitative antifungal assays under different ionic strengths and microscopic observations against the fungus Trichoderma viride. The antifungal activity of the wild type was stronger than that of CatD under all ionic strength conditions used in this assay; however, the antifungal activity of CatD became weaker with increasing ionic strength, whereas that of the wild type was maintained. The results at high ionic strength further verified the contribution of the CBM18 domain to the antifungal ability of GlxChiB. The microscopic observations clearly showed that the wild type acted on both the tips and the lateral wall of fungal hyphae, while CatD acted only on the tips. These results suggest that the CBM18 domain could contribute to the antifungal ability of chitinase through its affinity to the fungal lateral wall by hydrophobic interactions.