Faculty Profiles - HORI Katsutoshi

Papers - HORI Katsutoshi

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人工細胞と固体表面をつなぐ接着ナノファイバー～新規生体触媒の開発を目指して～ Invited

石川　聖人,堀　克敏

バイオサイエンスとインダストリー Vol. 78 ( 5 ) page： 408 - 409 2020.9

Native display of a huge homotrimeric protein fiber on the cell surface after precise domain deletion Reviewed International coauthorship International journal

S. Aoki, S. Yoshimoto, M. Ishikawa, D. Linke, A. Lupas, and K. Hori

J. Biosci. Bioeng Vol. 129 ( 4 ) page： 412 - 417 2020.4

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

AtaA, a trimeric autotransporter adhesin from Acinetobacter sp. Tol 5, exhibits nonspecific, high adhesiveness to abiotic surfaces. For identification of the functional domains of AtaA, precise design of domain-deletion mutants is necessary so as not to cause undesirable structural distortion. Here, we designed and constructed three types of AtaA mutants from which the same domain, FGG1, was deleted. The first mutant was designed to preserve the periodicity of hydrophobic residues in the coiled-coil segments sandwiching the deleted region. After the deletion, the protein was properly displayed on the cell surface and had the same adhesive function as the wild type. Transmission electron microscopy (TEM) imaging and circular dichroism (CD) spectroscopy showed that its isolated passenger domain had the same fiber structure as in the AtaA wild type. In contrast, a mutant designed to disturb the coiled-coil periodicity at the deletion site failed to reach the cell surface. Although secretion occurred for the mutant designed with a flexible connector between the coiled coils, the cells exhibited a decrease in adhesiveness. Furthermore, TEM imaging of the mutant fibers showed bending at the fiber tip and changes in their CD spectrum indicated a decrease in secondary structure content. Thus, we succeeded to natively display the huge homotrimeric fiber structure of AtaA on the cell surface after precise deletion of a domain, maintaining the proper folding state and adhesive function by preserving its coiled-coil periodicity. This strategy enables us to construct various domain-deletion mutants of AtaA without structural distortion for complete functional mapping.

DOI： 10.1016/j.jbiosc.2019.09.022

Gas-phase bioproduction of a high-value-added monoterpenoid (E)-geranic acid by metabolically engineered Acinetobacter sp. Tol 5 Reviewed International journal

Atsushi Usami, Masahito Ishikawa, Katsutoshi Hori

Green Chemistry Vol. 22 ( 4 ) page： 1258 - 1268 2020.2

Authorship：Last author,　Corresponding author Language：English Publishing type：Research paper (scientific journal) Publisher：Royal Society of Chemistry

Gas-phase bioproduction, in which immobilized biocatalysts are employed and chemical reactions are performed in a gas phase, has attracted researchers' attention as a green process. However, there is difficulty in the employment of whole cell catalysts for gas-phase bioproduction due to the lack of a suitable cell immobilization method. Acinetobacter sp. Tol 5 is a unique bacterium, which is remarkably sticky and can be easily immobilized onto various material surfaces through the adhesive bacterionanofiber protein AtaA. In this study, we demonstrate the gas-phase bioproduction of (E)-geranic acid (GA), a high-value-added monoterpenoid, from geraniol using immobilized Tol 5 transformant cells, into which a gene involved in a (E)-GA synthetic pathway was introduced. Time course analysis of the liquid-phase bioproduction of (E)-GA revealed the inherent metabolism of Tol 5 involved in the degradation of (E)-GA. By disrupting the fadD4-ortholog gene, which encodes a key enzyme of the (E)-GA degradation, we successfully generated a (E)-GA-accumulating strain, Tol 5 ΔfadD4 (pGeoA). The immobilized cells of this mutant strain on a polyurethane support enabled the production of (E)-GA with a passive supply of gaseous geraniol in a batch gas-phase reaction. A major fraction of the (E)-GA, which was produced, was adsorbed onto the polyurethane support but easily extracted into ethanol, a safe solvent without environmental impact. This is the first example of gas-phase bioproduction of a complex and high-value-added compound. Tol 5 is a highly promising platform for gas-phase bioproduction.

DOI： 10.1039/c9gc03478a

Process Description of an Unconventional Biofilm Formation by Bacterial Cells Autoagglutinating through Sticky, Long, and Peritrichate Nanofibers Reviewed International journal Open Access

Furuichi Yoshihide, Yoshimoto Shogo, Inaba Tomohiro, Nomura Nobuhiko, Hori Katsutoshi

Environmental Science & Technology Vol. 54 ( 4 ) page： 2520 - 2529 2020.2

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

In this study, we elucidated the formation process of an unconventional biofilm formed by a bacterium autoagglutinating through sticky, long, and peritrichate nanofibers. Understanding the mechanisms of biofilm formation is essential to control microbial behavior and improve environmental biotechnologies. Acinetobacter sp. Tol 5 autoagglutinate through the interaction of the long, peritrichate nanofiber protein AtaA, a trimeric autotransporter adhesin. Using AtaA, without cell growth or extracellular polymeric substances production, Tol 5 cells quickly form an unconventional biofilm. The process forming this unconventional biofilm started with cell-cell interactions, proceeded to cell clumping, and led to the formation of large cell aggregates. The cell-cell interaction was described by Derjaguin-Landau-Verwey-Overbeek (DLVO) theory based on a new concept, which considers two independent interactions between two cell bodies and between two AtaA fiber tips forming a discontinuous surface. If cell bodies cannot collide owing to an energy barrier at low ionic strengths but approach within the interactive distance of AtaA fibers, cells can agglutinate through their contact. Cell clumping proceeds following the cluster-cluster aggregation model, and an unconventional biofilm containing void spaces and a fractal nature develops. Understanding its formation process would extend the utilization of various types of biofilms, enhancing environmental biotechnologies.

DOI： 10.1021/acs.est.9b06577

Evaluation of methane degradation performance in microbial gas-phase reactions using effectively immobilized methanotrophs Reviewed International journal Open Access

Yan-Yu Chen, Masahito Ishikawa, Risa Suzuki, Hidehiro Ito, Toshiaki Kamachi, Katsutoshi Hori

Biochemical Engineering Journal Vol. 154 page： 107441 2020.2

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

The removal of atmospheric methane by methanotrophs is a potential strategy for mitigating global warming. However, due to the low mass transfer of methane to methanotrophic cells, the conventional bioprocesses require active gas-supply with high energy consumption. This study aimed to evaluate methane degradation performance in microbial gas-phase reactions using effectively immobilized Methylococcus capsulatus (Bath) cells. Cell immobilization was achieved by freeze-thaw treatment and a simple filtration process. The methane degradation rate was much faster in the gas-phase reaction than in the aqueous-phase reaction with stirring. The efficiency of methane degradation was almost the same between the gas-phase reaction using gel-entrapped cells and the static aqueous reaction using a cell suspension. The results of this study will facilitate the development of a high-efficiency and low-energy bioprocess for methane removal.

DOI： 10.1016/j.bej.2019.107441

Extracellular electron transfer mediated by a cytocompatible redox polymer to study the crosstalk among the mammalian circadian clock, cellular metabolism, and cellular redox state Reviewed International journal Open Access

Masahito Ishikawa, Kazuki Kawai, Masahiro Kaneko, Kenya Tanaka, Shuji Nakanishi, Katsutoshi Hori

RSC ADVANCES Vol. 10 ( 3 ) page： 1648 - 1657 2020.1

Language：English Publishing type：Research paper (scientific journal) Publisher：Royal Society of Chemistry

The circadian clock is an endogenous biological timekeeping system that controls various physiological and cellular processes with a 24 h rhythm. The crosstalk among the circadian clock, cellular metabolism, and cellular redox state has attracted much attention. To elucidate this crosstalk, chemical compounds have been used to perturb cellular metabolism and the redox state. However, an electron mediator that facilitates extracellular electron transfer (EET) has not been used to study the mammalian circadian clock due to potential cytotoxic effects of the mediator. Here, we report evidence that a cytocompatible redox polymer pMFc (2-methacryloyloxyethyl phosphorylcholine-co-vinyl ferrocene) can be used as the mediator to study the mammalian circadian clock. EET mediated by oxidized pMFc (ox-pMFc) extracted intracellular electrons from human U2OS cells, resulting in a longer circadian period. Analyses of the metabolome and intracellular redox species imply that ox-pMFc receives an electron from glutathione, thereby inducing pentose phosphate pathway activation. These results suggest novel crosstalk among the circadian clock, metabolism, and redox state. We anticipate that EET mediated by a redox cytocompatible polymer will provide new insights into the mammalian circadian clock system, which may lead to the development of new treatments for circadian clock disorders.

DOI： 10.1039/c9ra10023g

接着性人工細胞触媒のボトムアップ創製 Invited

石川　聖人，堀　克敏

生物工学 Vol. 98 page： 651 - 654 2020

接着タンパク質・接着微生物を利用した効率的バイオプロセスの構築 Invited

石川　聖人，堀　克敏

酵素工学ニュース Vol. 84 page： 13 - 17 2020

Bottom-up Creation of an Artificial Cell Covered with the Adhesive Bacterionanofiber Protein AtaA Reviewed International journal Open Access

Kosaku Noba, Masahito Ishikawa, Atsuko Uyeda, Takayoshi Watanabe, Takahiro Hohsaka, Shogo Yoshimoto, Tomoaki Matsuura, Katsutoshi Hori

JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Vol. 141 ( 48 ) page： 19058 - 19066 2019.12

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

The bacterial cell surface structure has important roles for various cellular functions. However, research on reconstituting bacterial cell surface structures is limited. This study aimed to bottom-up create a cell-sized liposome covered with AtaA, the adhesive bacterionanofiber protein localized on the cell surface of Acinetobacter sp. Tol 5, without the use of the protein secretion and assembly machineries. Liposomes containing a benzylguanine derivative-modified phospholipid were decorated with a truncated AtaA protein fused to a SNAP-tag expressed in a soluble fraction in Escherichia coli. The obtained liposome showed a similar surface structure and function to that of native Tol 5 cells and adhered to both hydrophobic and hydrophilic solid surfaces. Furthermore, this artificial cell was able to drive an enzymatic reaction in the adhesive state. The developed artificial cellular system will allow for analysis of not only AtaA, but also other cell surface proteins under a cell-mimicking environment. In addition, AtaA-decorated artificial cells may inspire the development of biotechnological applications that require immobilization of cells onto a variety of solid surfaces, in particular, in environments where the use of genetically modified organisms is prohibited.

DOI： 10.1021/jacs.9b09340

Control of AtaA-mediated bacterial immobilization by casein hydrolysates. Reviewed International journal

Y. Ohara, S. Yoshimoto, K. Hori

Journal of bioscience and bioengineering Vol. 128 ( 5 ) page： 544 - 550 2019.11

Language：English Publishing type：Research paper (scientific journal)

Acinetobacter sp. Tol 5 exhibits an autoagglutinating nature and high adhesiveness to various abiotic surfaces through its bacterionanofiber protein AtaA. We have developed new bacterial immobilization methods utilizing the high adhesiveness of AtaA. We previously reported that salt is essential for the adhesiveness of AtaA. In the current study, we unexpectedly found that Tol 5 cells were not immobilized onto polyurethane foam support during growth in LB medium although AtaA was properly expressed and displayed onto the cell surface. The adhesion of Tol 5 resting cells was not affected by sugars but drastically inhibited by yeast extract and casein hydrolysates such as tryptone and casamino acids technical grade (CA-T). Some amino acids, which are major components of CA-T, partially inhibited the adhesion of Tol 5 cells. Experimental results suggested that oligopeptides might effectively inhibit the cell adhesion. Immobilized cells onto the support through AtaA were detached in CA-T solution. Also, the detached cells could be re-immobilized onto the support without impairing of their adhesiveness by replacing CA-T solution to a basal salt medium. Microscopic observation revealed that breaking of AtaA-mediated cell–cell interaction is important for the detachment of Tol 5 cells from the support. CA-T also inhibited AtaA-mediated autoagglutination and dispersed cell clumps through AtaA. This is the first report on adhesion inhibitors against AtaA and suggests that casein hydrolysates like CA-T would be a powerful tool for controlling AtaA-mediated bacterial immobilization.

DOI： 10.1016/j.jbiosc.2019.04.019

接着性バクテリオナノファイバーによる微生物のプロセスの革新 Invited Reviewed

堀　克敏

環境バイオテクノロジー学会誌 Vol. 19 page： 21 - 28 2019.3

On-fiber display of a functional peptide at sites distant from the cell surface using a long bacterionanofiber of a trimeric autotransporter adhesin Reviewed International journal

Hajime Nakatani, Junichi Kanie, Katsutoshi Hori

BIOTECHNOLOGY AND BIOENGINEERING Vol. 116 ( 2 ) page： 239 - 249 2019.2

Language：English Publishing type：Research paper (scientific journal) Publisher：Wiley

In the cell surface display system, the distance of a surface-displayed molecule from the cell surface should influence its functionality due to the interference by other surface structures. For the purpose of developing this distance-variable surface display system, we utilized a long fibrous adhesin, Acinetobacter trimeric autotransporter adhesin (AtaA) of the strain Tol 5. We constructed His-tagged full-length and shorter AtaA fibers designed by N-terminal deletion and expressed them in the ΔataA mutant. Immunoelectron microscopy clearly showed that they formed fibers on the cell surface and the His-tag was displayed on the fiber tip located at fixed distances from the cell surface. N-terminal deletion of AtaA shortened the distance between the His-tag and the cell surface, as designed. Time-course analyses of the cell-to-Ni-Sepharose beads binding revealed that cells producing the longer fibers bound more rapidly to the beads. The His-tagged AtaA derivatives were also displayed on Escherichia coli cells, and a similar tendency was shown; the His-tag on the longer fiber was more functional than that on the shorter one. Thus, we developed an on-fiber display system of a functional peptide using a long trimeric autotransporter adhesin (TAA) fiber, which can vary the distance between the displayed molecule and the cell surface.

DOI： 10.1002/bit.26857

The Acinetobacter trimeric autotransporter adhesin Ata controls key virulence traits of Acinetobacter baumannii Reviewed International coauthorship International journal Open Access

Marko Weidensdorfer, Masahito Ishikawa, Katsutoshi Hori, Dirk Linke, Bardya Djahanschiri, Ruben Iruegas, Ingo Ebersberger, Sara Riedel-Christ, Giulia Enders, Laura Leukert, Peter Kraiczy, Florian Rothweiler, Jindrich Cinatl, Juergen Berger, Katharina Hipp, Volkhard A. J. Kempf, Stephan Goettig

VIRULENCE Vol. 10 ( 1 ) page： 68 - 81 2019.1

Language：English Publishing type：Research paper (scientific journal)

Acinetobacter baumannii is a Gram-negative pathogen that causes a multitude of nosocomial infections. The Acinetobacter trimeric autotransporter adhesin (Ata) belongs to the superfamily of trimeric autotransporter adhesins which are important virulence factors in many Gram-negative species. Phylogenetic profiling revealed that ata is present in 78% of all sequenced A. baumannii isolates but only in 2% of the closely related species A. calcoaceticus and A. pittii. Employing a markerless ata deletion mutant of A. baumannii ATCC 19606 we show that adhesion to and invasion into human endothelial and epithelial cells depend on Ata. Infection of primary human umbilical cord vein endothelial cells (HUVECs) with A. baumannii led to the secretion of interleukin (IL)-6 and IL-8 in a time- and Ata-dependent manner. Furthermore, infection of HUVECs by WT A. baumannii was associated with higher rates of apoptosis via activation of caspases-3 and caspase-7, but not necrosis, in comparison to ∆ata. Ata deletion mutants were furthermore attenuated in their ability to kill larvae of Galleria mellonella and to survive in larvae when injected at sublethal doses. This indicates that Ata is an important multifunctional virulence factor in A. baumannii that mediates adhesion and invasion, induces apoptosis and contributes to pathogenicity in vivo.

DOI： 10.1080/21505594.2018.1558693

Efficient Counterselection for Methylococcus capsulatus (Bath) by Using a Mutated pheS Gene Reviewed International journal Open Access

Masahito Ishikawa, Sho Yokoe, Souichiro Kato, Katsutoshi Hori

APPLIED AND ENVIRONMENTAL MICROBIOLOGY Vol. 84 ( 23 ) page： e01875-18 2018.12

Language：English Publishing type：Research paper (scientific journal) Publisher：American Society for Microbiology

Methylococcus capsulatus (Bath) is a representative gammaproteobacterial methanotroph that has been studied extensively in diverse research fields. The sacB gene, which encodes levansucrase, causing cell death in the presence of sucrose, is widely used as a counterselectable marker for disruption of a target gene in Gram-negative bacteria. However, sacB is not applicable to all Gram-negative bacteria, and its efficiency for the counterselection of M. capsulatus (Bath) is low. Here, we report the construction of an alternative counterselectable marker, pheS*, by introduction of two point mutations (A306G and T252A) into the pheS gene from M. capsulatus (Bath), which encodes the α-subunit of phenylalanyl-tRNA synthetase. The transformant harboring pheS* on an expression plasmid showed sensitivity to 10 mM p-chloro-phenylalanine, whereas the transformant harboring an empty plasmid showed no sensitivity, indicating the availability of pheS* as a counterselectable marker in M. capsulatus (Bath). To validate the utility of the pheS* marker in counterselection, we attempted to obtain an unmarked mutant of xoxF, a gene encoding the major subunit of Xox methanol dehydrogenase, which we failed to obtain by counterselection using the sacB marker. PCR, immunodetection using an anti-XoxF antiserum, and a cell growth assay in the absence of calcium demonstrated successful disruption of the xoxF gene in M. capsulatus (Bath). The difference in counterselection efficiencies of the markers indicated that pheS* is more suitable than sacB for counterselection in M. capsulatus (Bath). This study provides a new genetic tool enabling efficient counterselection in M. capsulatus (Bath).

DOI： 10.1128/AEM.01875-18

Extracellular Electron Transfer via Outer Membrane Cytochromes in a Methanotrophic Bacterium Methylococcus capsulatus (Bath) Reviewed International journal Open Access

Kenya Tanaka, Sho Vokoe, Kensuke Igarashi, Motoko Takashino, Masahito Ishikawa, Katsutoshi Hori, Shuji Nakanishi, Souichiro Kato

FRONTIERS IN MICROBIOLOGY Vol. 9 ( NOV ) page： 2905 2018.11

Language：English Publishing type：Research paper (scientific journal) Publisher：Frontiers

Electron exchange reactions between microbial cells and solid materials, referred to as extracellular electron transfer (EET), have attracted attention in the fields of microbial physiology, microbial ecology, and biotechnology. Studies of model species of iron-reducing, or equivalently, current-generating bacteria such as Geobacter spp. and Shewanella spp. have revealed that redox-active proteins, especially outer membrane c-type cytochromes (OMCs), play a pivotal role in the EET process. Recent (meta)genomic analyses have revealed that diverse microorganisms that have not been demonstrated to have EET ability also harbor OMC-like proteins, indicating that EET via OMCs could be more widely preserved in microorganisms than originally thought. A methanotrophic bacterium Methylococcus capsulatus (Bath) was reported to harbor multiple OMC genes whose expression is elevated by Cu starvation. However, the physiological role of these genes is unknown. Therefore, in this study, we explored whether M. capsulatus (Bath) displays EET abilities via OMCs. In electrochemical analysis, M. capsulatus (Bath) generated anodic current only when electron donors such as formate were available, and could reduce insoluble iron oxides in the presence of electron donor compounds. Furthermore, the current-generating and iron-reducing activities of M. capsulatus (Bath) cells that were cultured in a Cu-deficient medium, which promotes high levels of OMC expression, were higher than those cultured in a Cu-supplemented medium. Anodic current production by the Cu-deficient cells was significantly suppressed by disruption of MCA0421, a highly expressed OMC gene, and by treatment with carbon monoxide (CO) gas (an inhibitor of c-type cytochromes). Our results provide evidence of EET in M. capsulatus (Bath) and demonstrate the pivotal role of OMCs in this process. This study raises the possibility that EET to solid compounds is a novel survival strategy of methanotrophic bacteria.

DOI： 10.3389/fmicb.2018.02905

Heterologous expression of geraniol dehydrogenase for identifying the metabolic pathways involved in the biotransformation of citral by Acinetobacter sp Tol 5 Reviewed International journal Open Access

Usami Atsushi, Masahito Ishikawa, Katsutoshi Hori

BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY Vol. 82 ( 11 ) page： 2012 - 2020 2018.11

Language：English Publishing type：Research paper (scientific journal)

The biotransformation of citral, an industrially important monoterpenoid, has been extensively studied using many microbial biocatalysts. However, the metabolic pathways involved in its biotransformation are still unclear, because citral is a mixture of the trans-isomer geranial and the cis-isomer neral. Here, we applied the heterologous expression of geoA, a gene encoding geraniol dehydrogenase that specifically converts geraniol to geranial and nerol to neral, to identify the metabolic pathways involved in the biotransformation of citral. Acinetobacter sp. Tol 5 was employed in order to demonstrate the utility of this methodology. Tol 5 transformed citral to (1R,3R,4R)-1-methyl-4-(1-methylethenyl)-1,3-cyclohexanediol and geranic acid. Biotransformation of citral precursors (geraniol and nerol) by Tol 5 transformant cells expressing geoA revealed that these compounds were transformed specifically from geranial. Our methodology is expected to facilitate a better understanding of the metabolic pathways involved in the biotransformation of substrates that are unstable and include geometric isomers.

DOI： 10.1080/09168451.2018.1501263

Cell behavior of the highly sticky bacterium Acinetobacter sp Tol 5 during adhesion in laminar flows Reviewed International journal Open Access

Yoshihide Furuichi, Keita Iwasaki, Katsutoshi Hori

SCIENTIFIC REPORTS Vol. 8 ( 1 ) page： 8285 2018.5

Language：English Publishing type：Research paper (scientific journal) Publisher：Springer Nature

It is important to characterize how medically, industrially, or environmentally important bacteria adhere to surfaces in liquid flows in order to control their cell adhesion and subsequent biofilm formation. Acinetobacter sp. Tol 5 is a remarkably sticky bacterium that autoagglutinates through the adhesive nanofiber protein AtaA, which is applicable to cell immobilization in bioprocesses. In this study, the adhesion and behavior of Tol 5 cells in laminar flows were investigated using flow cell systems. Tol 5 cells autoagglutinated through AtaA and formed cell clumps during flowing. The cell clumps rather than single cells went downward due to gravity and adhered to the bottom surface. Under appropriate shear stress, a twin vortex was caused by a separated flow generated at the rear of the pre-immobilized cell clumps and carried the small cell clumps to this location, resulting in their stacking there. The rearward immobilized cell clumps developed into a large, stable aggregate with a streamlined shape, independent of cell growth. Cell clumps hardly ever developed under weak shear stress that could not generate a twin vortex and were broken up under excessively strong shear stress. These cell behaviors including the importance of clumping are interesting features in the bacterial adhesion processes.

DOI： 10.1038/s41598-018-26699-5

Immobilization of Enterobacter aerogenes by a Trimeric Autotransporter Adhesin, AtaA, and Its Application to Biohydrogen Production Reviewed International journal Open Access

Hajime Nakatani, Ding Nan, Yuki Ohara, Katsutoshi Hori

CATALYSTS Vol. 8 ( 4 ) page： 159 2018.4

Language：English Publishing type：Research paper (scientific journal)

Biological hydrogen production by microbial cells has been extensively researched as an energy-efficient and environmentally-friendly process. In this study, we propose a fast, easy method for immobilizing Enterobacter aerogenes by expressing ataA, which encodes the adhesive protein of Acinetobacter sp. Tol 5. AtaA protein on the E. aerogenes cells carrying the ataA gene was demonstrated by immunoblotting and flow cytometry. The AtaA-producing cells exhibited stronger adherence and auto-agglutination characteristics than wild-type cells, and were successfully immobilized (at approximately 2.5 mg/cm3) on polyurethane foam. Hydrogen production from the cell-immobilized polyurethane foams was monitored in repetitive batch reactions and flow reactor studies. The total hydrogen production in triple-repetitive batch reactions reached 0.6 mol/mol glucose, and the hydrogen production rate in the flow reactor was 42 mL·h−1·L−1. The AtaA production achieved simple and immediate immobilization of E. aerogenes on the foam, enabling repetitive and continuous hydrogen production. This report newly demonstrates the production of AtaA on the cell surfaces of bacterial genera other than Acinetobacter, and can simplify and accelerate the immobilization of whole-cell catalysts.

DOI： 10.3390/catal8040159

Real-time monitoring of intracellular redox changes in Methylococcus capsulatus (Bath) for efficient bioconversion of methane to methanol Reviewed International journal Open Access

Masahito Ishikawa, Yuya Tanaka, Risa Suzuki, Kota Kimura, Kenya Tanaka, Kazuhide Kamiya, Hidehiro Ito, Souichiro Kato, Toshiaki Kamachi, Katsutoshi Hori, Shuji Nakanishi

BIORESOURCE TECHNOLOGY Vol. 241 page： 1157 - 1161 2017.10

Language：English Publishing type：Research paper (scientific journal)

This study aimed to develop a novel method for real-time monitoring of the intracellular redox states in a methanotroph Methylococcus capsulatus, using Peredox as a genetically encoded fluorescent sensor of the NADH:NAD+ ratio. As expected, the fluorescence derived from the Peredox-expressing M. capsulatus transformant increased by supplementation of electron donor compounds (methane and formate), while it decreased by specifically inhibiting the methanol oxidation reaction. Electrochemical measurements confirmed that the Peredox fluorescence reliably represents the intracellular redox changes. This study is the first to construct a reliable redox-monitoring method for methanotrophs, which will facilitate to develop more efficient methane-to-methanol bioconversion processes.

DOI： 10.1016/j.biortech.2017.05.107

Reversible bacterial immobilization based on the salt-dependent adhesion of the bacterionanofiber protein AtaA Reviewed International journal Open Access

Shogo Yoshimoto, Yuki Ohara, Hajime Nakatani, Katsutoshi Hori

MICROBIAL CELL FACTORIES Vol. 16 ( 1 ) page： 123 2017.7

Language：English Publishing type：Research paper (scientific journal)

Background: Immobilization of microbial cells is an important strategy for the efficient use of whole-cell catalysts because it simplifies product separation, enables the cell concentration to be increased, stabilizes enzymatic activity, and permits repeated or continuous biocatalyst use. However, conventional immobilization methods have practical limitations, such as limited mass transfer in the inner part of a gel, gel fragility, cell leakage from the support matrix, and adverse effects on cell viability and catalytic activity. We previously showed a new method for bacterial cell immobilization using AtaA, a member of the trimeric autotransporter adhesin family found in Acinetobacter sp. Tol 5. This approach is expected to solve the drawbacks of conventional immobilization methods. However, similar to all other immobilization methods, the use of support materials increases the cost of bioprocesses and subsequent waste materials. Results: We found that the stickiness of the AtaA molecule isolated from Tol 5 cells is drastically diminished at ionic strengths lower than 10 mM and that it cannot adhere in deionized water, which also inhibits cell adhesion mediated by AtaA. Cells immobilized on well plates and polyurethane foam in a salt solution were detached in deionized water by rinsing and shaking, respectively. The detached cells regained their adhesiveness in a salt solution and could rapidly be re-immobilized. The cells expressing the ataA gene maintained their adhesiveness throughout four repeated immobilization and detachment cycles and could be repeatedly immobilized to polyurethane foam by a 10-min shake in a flask. We also demonstrated that both bacterial cells and a support used in a reaction could be reused for a different type of reaction after detachment of the initially immobilized cells from the support and a subsequent immobilization step. Conclusions: We invented a unique reversible immobilization method based on the salt-dependent adhesion of the AtaA molecule that allows us to reuse bacterial cells and supports by a simple manipulation involving a deionized water wash. This mitigates problems caused by the use of support materials and greatly helps to enhance the efficiency and productivity of microbial production processes.

DOI： 10.1186/s12934-017-0740-7

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