Faculty Profiles - HORI Katsutoshi

A novel inverse membrane bioreactor for efficient bioconversion from methane gas to liquid methanol using a microbial gas-phase reaction Reviewed International journal Open Access

Chen Yan-Yu, Ishikawa Masahito, Hori Katsutoshi

BIOTECHNOLOGY FOR BIOFUELS AND BIOPRODUCTS Vol. 16 ( 1 ) page： 16 2023.2

Authorship：Last author,　Corresponding author Language：English Publishing type：Research paper (scientific journal) Publisher：Biotechnology for Biofuels and Bioproducts

ackground: Methane (CH4), as one of the major energy sources, easily escapes from the supply chain into the atmosphere, because it exists in a gaseous state under ambient conditions. Compared to carbon dioxide (CO2), CH4 is 25 times more potent at trapping radiation; thus, the emission of CH4 to the atmosphere causes severe global warming and climate change. To mitigate CH4 emissions and utilize them effectively, the direct biological conversion of CH4 into liquid fuels, such as methanol (CH3OH), using methanotrophs is a promising strategy. However, supplying biocatalysts in an aqueous medium with CH4 involves high energy consumption due to vigorous agitation and/or bubbling, which is a serious concern in methanotrophic processes, because the aqueous phase causes a very large barrier to the delivery of slightly soluble gases. Results: An inverse membrane bioreactor (IMBR), which combines the advantages of gas-phase bioreactors and membrane bioreactors, was designed and constructed for the bioconversion of CH4 into CH3OH in this study. In contrast to the conventional membrane bioreactor with bacterial cells that are immersed in an aqueous phase, the filtered cells were placed to face a gas phase in the IMBR to supply CH4 directly from the gas phase to bacterial cells. Methylococcus capsulatus (Bath), a representative methanotroph, was used to demonstrate the bioconversion of CH4 to CH3OH in the IMBR. Cyclopropanol was supplied from the aqueous phase as a selective inhibitor of methanol dehydrogenase, preventing further CH3OH oxidation. Sodium formate was added as an electron donor to generate NADH, which is necessary for CH3OH production. After optimizing the inlet concentration of CH4, the mass of cells, the cyclopropanol concentration, and the gas flow rate, continuous CH3OH production can be achieved over 72 h with productivity at 0.88 mmol L−1 h−1 in the IMBR, achieving a longer operation period and higher productivity than those using other types of membrane bioreactors reported in the literature. Conclusions: The IMBR can facilitate the development of gas-to-liquid (GTL) technologies via microbial processes, allowing highly efficient mass transfer of substrates from the gas phase to microbial cells in the gas phase and having the supplement of soluble chemicals convenient.

DOI： 10.1186/s13068-023-02267-6

Identification of the adhesive domain of AtaA from Acinetobacter sp. Tol 5 and its application in immobilizing Escherichia coli Reviewed International coauthorship International journal Open Access

Yoshimoto Shogo, Aoki Sota, Ohara Yuki, Ishikawa Masahito, Suzuki Atsuo, Linke Dirk, Lupas Andrei N., Hori Katsutoshi

FRONTIERS IN BIOENGINEERING AND BIOTECHNOLOGY Vol. 10 page： 1095057 2023.1

Authorship：Last author,　Corresponding author Language：English Publishing type：Research paper (scientific journal) Publisher：Frontiers in Bioengineering and Biotechnology

Cell immobilization is an important technique for efficiently utilizing whole-cell biocatalysts. We previously invented a method for bacterial cell immobilization using AtaA, a trimeric autotransporter adhesin from the highly sticky bacterium Acinetobacter sp. Tol 5. However, except for Acinetobacter species, only one bacterium has been successfully immobilized using AtaA. This is probably because the heterologous expression of large AtaA (1 MDa), that is a homotrimer of polypeptide chains composed of 3,630 amino acids, is difficult. In this study, we identified the adhesive domain of AtaA and constructed a miniaturized AtaA (mini-AtaA) to improve the heterologous expression of ataA. In-frame deletion mutants were used to perform functional mapping, revealing that the N-terminal head domain is essential for the adhesive feature of AtaA. The mini-AtaA, which contains a homotrimer of polypeptide chains from 775 amino acids and lacks the unnecessary part for its adhesion, was properly expressed in E. coli, and a larger amount of molecules was displayed on the cell surface than that of full-length AtaA (FL-AtaA). The immobilization ratio of E. coli cells expressing mini-AtaA on a polyurethane foam support was significantly higher compared to the cells with or without FL-AtaA expression, respectively. The expression of mini-AtaA in E. coli had little effect on the cell growth and the activity of another enzyme reflecting the production level, and the immobilized E. coli cells could be used for repetitive enzymatic reactions as a whole-cell catalyst.

DOI： 10.3389/fbioe.2022.1095057

微生物飼料を活用した次世代陸上養殖の展開

田丸　浩、堀　克敏

月刊バイオインダストリー Vol. 40 ( 1 ) page： 51 - 57 2023.1

小型化接着タンパク質による大腸菌の固定化と微生物反応への応用; バイオサイエンスとインダストリー

吉本　将悟、堀　克敏

バイオサイエンスとインダストリー Vol. 40 page： 51 - 57 2023

Perturbation by Antimicrobial Bacteria of the Epidermal Bacterial Flora of Rainbow Trout in Flow-Through Aquaculture Reviewed International journal Open Access

Nakatani Hajime, Yamada Naoki, Hashimoto Naoki, Okazaki Fumiyoshi, Arakawa Tomoko, Tamaru Yutaka, Hori Katsutoshi

BIOLOGY-BASEL Vol. 11 ( 8 ) page： 1249 2022.8

Authorship：Last author,　Corresponding author Language：English Publishing type：Research paper (scientific journal) Publisher：MDPI

The bacterial flora of the epidermal mucus of fish is closely associated with the host’s health and susceptibility to pathogenic infections. In this study, we analyzed the epidermal mucus bacteria of rainbow trout (Oncorhynchus mykiss) reared in flow-through aquaculture under environmental perturbations. Over ~2 years, the bacteria present in the skin mucus and water were analyzed based on the 16S rDNA sequences. The composition of the mucus bacterial community showed significant monthly fluctuations, with frequent changes in the dominant bacterial species. Analysis of the beta- and alpha-diversity of the mucus bacterial flora showed the fluctuations of the composition of the flora were caused by the genera Pseudomonas, Yersinia, and Flavobacterium, and some species of Pseudomonas and Yersinia in the mucus were identified as antimicrobial bacteria. Examination of the antimicrobial bacteria in the lab aquarium showed that the natural presence of antimicrobial bacteria in the mucus and water, or the purposeful addition of them to the rearing water, caused a transition in the mucus bacteria community composition. These results demonstrate that specific antimicrobial bacteria in the water or in epidermal mucus comprise one of the causes of changes in fish epidermal mucus microflora.

DOI： 10.3390/biology11081249

The extracellular juncture domains of Type 5 autotransporters Reviewed International coauthorship

Weikum, J; Kulakova, A; Tesei, G; Yoshimoto, S; Jægerum, LV; Schütz, M; Hori, K; Skepö, M; Harris, P; Leo, JC; Morth, JP

ACTA CRYSTALLOGRAPHICA A-FOUNDATION AND ADVANCES Vol. 78 page： E5 - E5 2022.8

Fabrication of a highly protective 3D-printed mask and evaluation of its viral filtration efficiency using a human head mannequin Reviewed International journal Open Access

Ohara Yuki, Kanie Junichi, Hori Katsutoshi

HARDWAREX Vol. 11 page： e00314 2022.4

Authorship：Last author,　Corresponding author Language：English Publishing type：Research paper (scientific journal) Publisher：Elsevier

Facemasks are one of the most effective and low-cost prophylactics for COVID-19. In the spring 2020, when a severe shortage of facemasks occurred worldwide, various types of 3D-printed masks were designed and proposed. However, the protective effects conferred by most of these masks were not experimentally evaluated. Here, we provide a new simple design of 3D-printed mask and evaluate its protective effect in a viral filtration test using a human head mannequin. The developed mask can be constructed with a low-cost 3D printer, with an approximate production cost of US $4. This mask has three parts: the main part, wearing parts, and a piece of non-woven fabric filter. The volume of the filter, which needs to be changed daily, was reduced to approximately 1/10 of that of commercially available surgical masks used in this study. The developed mask is fabricated from polylactic acid, a biodegradable plastic, and its surface contour contacting the face may be adjusted after softening the material with hot water at 60–80 °C. The viral filtration efficiency of the developed mask was found to be over 80%. This performance is better than that of commercially available facemasks, such as surgical masks and cloth masks, and equal to those of KN95 and KF94.

DOI： 10.1016/j.ohx.2022.e00314

細菌接着タンパク質と微生物固定化への利用 Invited

堀　克敏

バイオインダストリー Vol. 39 ( 3 ) page： 11 - 18 2022.3

Single-cell adhesion force mapping of a highly sticky bacterium in liquid Reviewed International journal

Satoshi Ishii, Shogo Yoshimoto, Katsutoshi Hori

JOURNAL OF COLLOID AND INTERFACE SCIENCE Vol. 606 ( Pt 1 ) page： 628 - 634 2022.1

Authorship：Last author,　Corresponding author Language：English Publishing type：Research paper (scientific journal) Publisher：Journal of Colloid and Interface Science

The sticky bacterium Acinetobacter sp. Tol 5 adheres to various material surfaces via its cell surface nanofiber protein, AtaA. This adhesiveness has only been evaluated based on the amount of cells adhering to a surface. In this study, the adhesion force mapping of a single Tol 5 cell in liquid using the quantitative imaging mode of atomic force microscopy (AFM) revealed that the adhesion of Tol 5 was near 2 nN, which was 1–2 orders of magnitude higher than that of other adhesive bacteria. The adhesion force of a cell became stronger with the increase in AtaA molecules present on the cell surface. Many fibers of peritrichate AtaA molecules simultaneously interact with a surface, strongly attaching the cell to the surface. The adhesion force of a Tol 5 cell was drastically reduced in the presence of 1% casamino acids but not in deionized water (DW), although both liquids decrease the adhesiveness of Tol 5 cells, suggesting that DW and casamino acids inhibit the cell approaching step and the subsequent direct interaction step of AtaA with surfaces, respectively. Heterologous production of AtaA provided non-adhesive Acinetobacter baylyi ADP1 cells with a strong adhesion force to AFM tip surfaces of silicon and gold.

DOI： 10.1016/j.jcis.2021.08.039

Recent advances in research on biointerfaces: From cell surfaces to artificial interfaces Invited Reviewed International journal

K. Hori, S. Yoshimoto, T. Yoshino, T. Zako, G. Hirao, S. Fujita, C. Nakamura, A. Yamagishi, and N. Kamiya

Journal of Bioscience and Bioengineering Vol. 133 ( 3 ) page： 195 - 207 2022.1

Authorship：Lead author,　Corresponding author Language：English Publishing type：Research paper (scientific journal)

Biointerfaces are regions where biomolecules, cells, and organic materials are exposed to environmental media or come in contact with other biomaterials, cells, and inorganic/organic materials. In this review article, six research topics on biointerfaces are described to show examples of state-of-art research approaches. First, biointerface design of nanoparticles for molecular detection is described. Functionalized gold nanoparticles can be used for sensitive detection of various target molecules, including chemical compounds and biomolecules, such as DNA, proteins, cells, and viruses. Second, the interaction between bacterial cell surfaces and material surfaces, including the introduction of advances in analytical methods and theoretical calculations, are explained as well as their applications to bioprocesses. Third, bioconjugation technologies for localizing functional proteins at biointerfaces are introduced, in particular, by focusing the potential of enzymes as a catalytic tool for designing different types of bioconjugates that function at biointerfaces. Forth topics is focusing on lipideprotein interaction in cell membranes as natural biointerfaces. Examples of membrane lipid engineering are introduced, and it is mentioned how their compositional profiles affect membrane protein functions. Fifth topic is the physical method for molecular delivery across the biointerface being developed currently, such as highly efficient nanoinjection, electroporation, and nanoneedle devices, in which the key is how to perforate the cell membrane. Final topic is the chemical design of lipid- or polymer-based RNA delivery carriers and their behavior on the cell interface, which are currently attracting attention as RNA vaccine technologies targeting COVID-19. Finally, future directions of biointerface studies are presented.

DOI： 10.1016/j.jbiosc.2021.12.004

ウイルス除去効果の高い樹脂製立体マスクの開発 Invited

堀　克敏

繊維機械学会誌月刊せんい Vol. 74 ( 11 ) page： 593 - 598 2021.11

Complete Genome Sequence of the Highly Adhesive Bacterium Acinetobacter sp. Strain Tol 5 Reviewed International journal Open Access

Masahito Ishikawa, Katsutoshi Hori

Microbiology Resource Announcements Vol. 10 ( 35 ) page： e0056721 2021.9

Authorship：Last author,　Corresponding author Language：English Publishing type：Research paper (scientific journal) Publisher：American Society for Microbiology

Acinetobacter sp. strain Tol 5 is a nonpathogenic Gram-negative bacterium with biotechnological and environmental applications. Here, we report the complete genome sequence of Acinetobacter sp. strain Tol 5, which has a genome size of 4,799,506 bp and a G1C content of 38.1%.

DOI： 10.1128/MRA.00567-21

Development of a Biocontained Toluene-Degrading Bacterium for Environmental Protection Reviewed International journal Open Access

Masahito Ishikawa, Takaaki Kojima, Katsutoshi Hori

MICROBIOLOGY SPECTRUM Vol. 9 ( 1 ) page： 1 - 11 2021.9

Authorship：Last author,　Corresponding author Language：English Publishing type：Research paper (scientific journal) Publisher：American Society for Microbiology

Biocontainment is a safeguard strategy for preventing uncontrolled proliferation of genetically engineered microorganisms (GEMs) in the environment. Biocontained GEMs are designed to survive only in the presence of a specific molecule. The design of a pollutant-degrading and pollutant-dependent GEM prevents its proliferation after cleaning the environment. In this study, we present a biocontained toluene-degrading bacterium based on Acinetobacter sp. Tol 5. The bamA gene, which encodes an essential outer membrane protein, was deleted from the chromosome of Tol 5 but complemented with a plasmid carrying a bamA gene regulated by the Pu promoter and the regulatory protein XylR. The resultant strain (PuBamA) degraded toluene, similarly to the wild-type Tol 5. Although the cell growth of the PuBamA strain was remarkably inhibited after toluene depletion, escape mutants emerged at a frequency of 1 per 5.3 × 10−7 cells. Analyses of escape mutants revealed that insertion sequences (ISs) carrying promoters were inserted between the Pu promoter and the bamA gene on the complemented plasmid. MinION deep sequencing of the plasmids extracted from the escape mutants enabled the identification of three types of ISs involved in the emergence of escape mutants, suggesting a strategy for reducing it. IMPORTANCE GEMs are beneficial for various applications, including environmental protection. However, the risks of GEM release into the environment have been debated for a long time. If a pollutant is employed as a specific molecule for a biocontainment system, GEMs capable of degrading pollutants are available for environmental protection. Nevertheless, to our knowledge, biocontained degraders for real pollutants have not been reported in academic journals so far. This is possibly due to the difficulty in the expression of enzymes for degrading pollutants in a tractable bacterium such as Escherichia coli. On the other hand, bacteria with enzymes for degrading pollutants are often intractable as a host of GEMs due to the shortage of tools for genetic manipulation. This study reports the feasibility of a biocontainment strategy for a toluene degrader. Our results provide useful insights into the construction of a GEM biocontainment system for environmental protection.

DOI： 10.1128/Spectrum.00259-21

界面微生物工学微生物固定化・細胞表層工学から膜タンパク質利用技術まで Invited

堀　克敏

化学と生物 Vol. 59 ( 8 ) page： 393 - 400 2021.8

Metabolic alteration of Methylococcus capsulatus str. Bath during a microbial gas-phase reaction Reviewed International journal Open Access

Yan-Yu Chen, Yuki Soma, Masahito Ishikawa, Masatomo Takahashi, Yoshihiro Izumi, Takeshi Bamba, Katsutoshi Hori

BIORESOURCE TECHNOLOGY Vol. 330 page： 125002 2021.6

Authorship：Last author,　Corresponding author Language：English Publishing type：Research paper (scientific journal) Publisher：Elsevier

This study demonstrates the metabolic alteration of Methylococcus capsulatus (Bath), a representative bacterium among methanotrophs, in microbial gas-phase reactions. For comparative metabolome analysis, a bioreactor was designed to be capable of supplying gaseous substrates and liquid nutrients continuously. Methane degradation by M. capsulatus (Bath) was more efficient in a gas-phase reaction operated in the bioreactor than in an aqueous phase reaction operated in a batch reactor. Metabolome analysis revealed remarkable alterations in the metabolism of cells in the gas-phase reaction; in particular, pyruvate, 2-ketoglutarate, some amino acids, xanthine, and hypoxanthine were accumulated, whereas 2,6-diaminopimelate was decreased. Based on the results of metabolome analysis, cells in the gas-phase reaction seemed to alter their metabolism to reduce the excess ATP and NADH generated upon increased availability of methane and oxygen. Our findings will facilitate the development of efficient processes for methane-based bioproduction with low energy consumption.

DOI： 10.1016/j.biortech.2021.125002