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A sustainable society was proposed more than 50 years ago. However, it is yet to be realised. For example, the production of ammonia, an important chemical widely used in the agriculture, steel, chemical, textile, and pharmaceutical industries, still depends on fossil fuels. Recently, biological approaches to achieve sustainable ammonia production have been gaining attention. Moreover, unlike chemical methods, biological approaches have a lesser environmental impact because ammonia can be produced under mild conditions of normal temperature and pressure. Therefore, in previous studies, nitrogen fixation by nitrogenase, including enzymatic ammonia production using food waste, has been attempted. Additionally, the production of crops using nitrogen-fixing bacteria has been implemented in the industry as one of the most promising approaches to achieving a sustainable ammonia economy. Thus, in this review, we described previous studies on biological ammonia production and showed the prospects for realising a sustainable society.
Yukio Watanabe; Wataru Aoki; Mitsuyoshi Ueda. Sustainable Biological Ammonia Production towards a Carbon-Free Society. Sustainability 2021, 13, 9496 .
AMA StyleYukio Watanabe, Wataru Aoki, Mitsuyoshi Ueda. Sustainable Biological Ammonia Production towards a Carbon-Free Society. Sustainability. 2021; 13 (17):9496.
Chicago/Turabian StyleYukio Watanabe; Wataru Aoki; Mitsuyoshi Ueda. 2021. "Sustainable Biological Ammonia Production towards a Carbon-Free Society." Sustainability 13, no. 17: 9496.
Yeast cell surface display (YSD) has been used to engineer various proteins, including antibodies. Directed evolution, which subjects a gene to iterative rounds of mutagenesis, selection and amplification, is useful for protein engineering. In vivo continuous mutagenesis, which continuously diversifies target genes in the host cell, is a promising tool for accelerating directed evolution. However, combining in vivo continuous evolution and YSD is difficult because mutations in the gene encoding the anchor proteins may inhibit the display of target proteins on the cell surface. In this study, we have developed a modified YSD method that utilises SpyTag/SpyCatcher-based in vivo protein ligation. A nanobody fused with a SpyTag of 16 amino acids and an anchor protein fused with a SpyCatcher of 113 amino acids are encoded by separate gene cassettes and then assembled via isopeptide bond formation. This system achieved a high display efficiency of more than 90%, no intercellular protein ligation events, and the enrichment of target cells by cell sorting. These results suggested that our system demonstrates comparable performance with conventional YSD methods; therefore, it can be an appropriate platform to be integrated with in vivo continuous evolution.
Kaho Kajiwara; Wataru Aoki; Naoki Koike; Mitsuyoshi Ueda. Development of a yeast cell surface display method using the SpyTag/SpyCatcher system. Scientific Reports 2021, 11, 1 -10.
AMA StyleKaho Kajiwara, Wataru Aoki, Naoki Koike, Mitsuyoshi Ueda. Development of a yeast cell surface display method using the SpyTag/SpyCatcher system. Scientific Reports. 2021; 11 (1):1-10.
Chicago/Turabian StyleKaho Kajiwara; Wataru Aoki; Naoki Koike; Mitsuyoshi Ueda. 2021. "Development of a yeast cell surface display method using the SpyTag/SpyCatcher system." Scientific Reports 11, no. 1: 1-10.
The high-valent iron-oxo species formed in the non-heme diiron enzymes have high oxidative reactivity and catalyze difficult chemical reactions. Although the hydroxylation of inert methyl groups is an industrially promising reaction, utilizing non-heme diiron enzymes as such a biocatalyst has been difficult. Here we show a three-component monooxygenase system for the selective terminal hydroxylation of α-aminoisobutyric acid (Aib) into α-methyl-D-serine. It consists of the hydroxylase component, AibH1H2, and the electron transfer component. Aib hydroxylation is the initial step of Aib catabolism inRhodococcus wratislaviensis C31-06, which has been fully elucidated through a proteome analysis. The crystal structure analysis revealed that AibH1H2 forms a heterotetramer of two amidohydrolase superfamily proteins, of which AibHm2 is a non-heme diiron protein and functions as a catalytic subunit. The Aib monooxygenase was demonstrated to be a promising biocatalyst that is suitable for bioprocesses in which the inert C–H bond in methyl groups need to be activated.
Makoto Hibi; Dai Fukuda; Chihiro Kenchu; Masutoshi Nojiri; Ryotaro Hara; Michiki Takeuchi; Shunsuke Aburaya; Wataru Aoki; Kimihiko Mizutani; Yoshihiko Yasohara; Mitsuyoshi Ueda; Bunzo Mikami; Satomi Takahashi; Jun Ogawa. A three-component monooxygenase from Rhodococcus wratislaviensis may expand industrial applications of bacterial enzymes. Communications Biology 2021, 4, 1 -10.
AMA StyleMakoto Hibi, Dai Fukuda, Chihiro Kenchu, Masutoshi Nojiri, Ryotaro Hara, Michiki Takeuchi, Shunsuke Aburaya, Wataru Aoki, Kimihiko Mizutani, Yoshihiko Yasohara, Mitsuyoshi Ueda, Bunzo Mikami, Satomi Takahashi, Jun Ogawa. A three-component monooxygenase from Rhodococcus wratislaviensis may expand industrial applications of bacterial enzymes. Communications Biology. 2021; 4 (1):1-10.
Chicago/Turabian StyleMakoto Hibi; Dai Fukuda; Chihiro Kenchu; Masutoshi Nojiri; Ryotaro Hara; Michiki Takeuchi; Shunsuke Aburaya; Wataru Aoki; Kimihiko Mizutani; Yoshihiko Yasohara; Mitsuyoshi Ueda; Bunzo Mikami; Satomi Takahashi; Jun Ogawa. 2021. "A three-component monooxygenase from Rhodococcus wratislaviensis may expand industrial applications of bacterial enzymes." Communications Biology 4, no. 1: 1-10.
Ammonia is critical for agricultural and chemical industries. The extracellular production of ammonia by yeast (Saccharomyces cerevisiae) using cell surface engineering can be efficient approach because yeast can avoid growth deficiencies caused by knockout of genes for ammonia assimilation. In this study, we produced ammonia outside the yeast cells by displaying an l-amino acid oxidase with a wide substrate specificity derived from Hebeloma cylindrosporum (HcLAAO) on yeast cell surfaces. The HcLAAO-displaying yeast successfully produced 12.6 m m ammonia from a mixture of 20 proteinogenic amino acids (the theoretical conversion efficiency was 63%). We also succeeded in producing ammonia from a food processing waste, soybean residues (okara) derived from tofu production. The conversion efficiency was 88.1%, a higher yield than reported in previous studies. Our study demonstrates that ammonia production outside of yeast cells is a promising strategy to utilize food processing wastes.
Yukio Watanabe; Wataru Aoki; Mitsuyoshi Ueda. Improved ammonia production from soybean residues by cell surface-displayed l-amino acid oxidase on yeast. Bioscience, Biotechnology, and Biochemistry 2020, 1 .
AMA StyleYukio Watanabe, Wataru Aoki, Mitsuyoshi Ueda. Improved ammonia production from soybean residues by cell surface-displayed l-amino acid oxidase on yeast. Bioscience, Biotechnology, and Biochemistry. 2020; ():1.
Chicago/Turabian StyleYukio Watanabe; Wataru Aoki; Mitsuyoshi Ueda. 2020. "Improved ammonia production from soybean residues by cell surface-displayed l-amino acid oxidase on yeast." Bioscience, Biotechnology, and Biochemistry , no. : 1.
Ribosomes are the sophisticated machinery that is responsible for protein synthesis in a cell. Recently, quantitative mass spectrometry (qMS) have been successfully applied for understanding the dynamics of protein complexes. Here, we developed a highly specific and reproducible method to quantify all ribosomal proteins (r-proteins) by combining selected reaction monitoring (SRM) and isotope labeling. We optimized the SRM methods using purified ribosomes and Escherichia coli lysates and verified this approach as detecting 41 of the 54 r-proteins separately synthesized in E. coli S30 extracts. The SRM methods will enable us to utilize qMS as a highly specific analytical tool in the research of E. coli ribosomes, and this methodology have potential to accelerate the understanding of ribosome biogenesis, function, and the development of engineered ribosomes with additional functions.
Yuishin Kosaka; Wataru Aoki; Megumi Mori; Shunsuke Aburaya; Yuta Ohtani; Hiroyoshi Minakuchi; Mitsuyoshi Ueda. Selected reaction monitoring for the quantification of Escherichia coli ribosomal proteins. PLOS ONE 2020, 15, e0236850 .
AMA StyleYuishin Kosaka, Wataru Aoki, Megumi Mori, Shunsuke Aburaya, Yuta Ohtani, Hiroyoshi Minakuchi, Mitsuyoshi Ueda. Selected reaction monitoring for the quantification of Escherichia coli ribosomal proteins. PLOS ONE. 2020; 15 (12):e0236850.
Chicago/Turabian StyleYuishin Kosaka; Wataru Aoki; Megumi Mori; Shunsuke Aburaya; Yuta Ohtani; Hiroyoshi Minakuchi; Mitsuyoshi Ueda. 2020. "Selected reaction monitoring for the quantification of Escherichia coli ribosomal proteins." PLOS ONE 15, no. 12: e0236850.
The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system has enabled efficient, markerless genome editing in a wide range of organisms. However, there is an off-target effect and a limit to the area of precise editing. Bases that can be precisely edited are limited to within the 20-base pair gRNA-targeting site and protospacer adjacent motif (PAM) sequence. We have developed a CRISPR nickase system that can perform a precise genome-wide base editing in Saccharomyces cerevisiae using a single Cas9 nickase. This system can precisely edit a broader genomic region by the avoidance of double-strand break (DSB) and subsequent non-homologous end joining (NHEJ). Furthermore, unintended mutations were not found at off-target sites in this system. In combination with yeast gap repair cloning, precise genome editing of yeast cells can be performed in 5 days. Here, we describe the methods for precise and convenient genome editing using this novel CRISPR nickase system.
Kouichi Kuroda; Mitsuyoshi Ueda. CRISPR Nickase-Mediated Base Editing in Yeast. Methods in Molecular Biology 2020, 27 -37.
AMA StyleKouichi Kuroda, Mitsuyoshi Ueda. CRISPR Nickase-Mediated Base Editing in Yeast. Methods in Molecular Biology. 2020; ():27-37.
Chicago/Turabian StyleKouichi Kuroda; Mitsuyoshi Ueda. 2020. "CRISPR Nickase-Mediated Base Editing in Yeast." Methods in Molecular Biology , no. : 27-37.
Ammonia is used as a fertilizer for agriculture, chemical raw material, and carrier for transporting hydrogen, and with economic development, the demand for ammonia has increased. The Haber–Bosch process, which is the main method for producing ammonia, can produce ammonia with high efficiency. However, since it consumes a large amount of fossil energy, it is necessary to develop an alternative method for producing ammonia with less environmental impact. Ammonia production from food by-products is an appealing production process owing to unused resource usage, including waste, and mild reaction conditions. However, when food by-products and biomass are used as feedstocks, impurities often reduce productivity. Using metabolic profiling, glucose was identified as a potential inhibitor of ammonia production from impure food by-products. We constructed the recombinant Escherichia coli, in which glucose uptake was reduced by ptsG gene disruption and amino acid catabolism was promoted by glnA gene disruption. Ammonia production efficiency from okara, a food by-product, was improved in this strain; 35.4 mM ammonia was produced (47% yield). This study might provide a strategy for efficient ammonia production from food by-products.
Yuki Tatemichi; Kouichi Kuroda; Takeharu Nakahara; Mitsuyoshi Ueda. Efficient ammonia production from food by-products by engineered Escherichia coli. AMB Express 2020, 10, 1 -9.
AMA StyleYuki Tatemichi, Kouichi Kuroda, Takeharu Nakahara, Mitsuyoshi Ueda. Efficient ammonia production from food by-products by engineered Escherichia coli. AMB Express. 2020; 10 (1):1-9.
Chicago/Turabian StyleYuki Tatemichi; Kouichi Kuroda; Takeharu Nakahara; Mitsuyoshi Ueda. 2020. "Efficient ammonia production from food by-products by engineered Escherichia coli." AMB Express 10, no. 1: 1-9.
Neurons are categorised into many subclasses, and each subclass displays different morphology, expression patterns, connectivity and function. Changes in protein synthesis are critical for neuronal function. Therefore, analysing protein expression patterns in individual neuronal subclass will elucidate molecular mechanisms for memory and other functions. In this study, we used neuronal subclass-selective proteomic analysis with cell-selective bio-orthogonal non-canonical amino acid tagging. We selected Caenorhabditis elegans as a model organism because it shows diverse neuronal functions and simple neural circuitry. We performed proteomic analysis of all neurons or AFD subclass neurons that regulate thermotaxis in C. elegans. Mutant phenylalanyl tRNA synthetase (MuPheRS) was selectively expressed in all neurons or AFD subclass neurons, and azido-phenylalanine was incorporated into proteins in cells of interest. Azide-labelled proteins were enriched and proteomic analysis was performed. We identified 4,412 and 1,834 proteins from strains producing MuPheRS in all neurons and AFD subclass neurons, respectively. F23B2.10 (RING-type domain-containing protein) was identified only in neuronal cell-enriched proteomic analysis. We expressed GFP under the control of the 5′ regulatory region of F23B2.10 and found GFP expression in neurons. We expect that more single-neuron specific proteomic data will clarify how protein composition and abundance affect characteristics of neuronal subclasses.
Shunsuke Aburaya; Yuji Yamauchi; Takashi Hashimoto; Hiroyoshi Minakuchi; Wataru Aoki; Mitsuyoshi Ueda. Neuronal subclass-selective proteomic analysis in Caenorhabditis elegans. Scientific Reports 2020, 10, 1 -9.
AMA StyleShunsuke Aburaya, Yuji Yamauchi, Takashi Hashimoto, Hiroyoshi Minakuchi, Wataru Aoki, Mitsuyoshi Ueda. Neuronal subclass-selective proteomic analysis in Caenorhabditis elegans. Scientific Reports. 2020; 10 (1):1-9.
Chicago/Turabian StyleShunsuke Aburaya; Yuji Yamauchi; Takashi Hashimoto; Hiroyoshi Minakuchi; Wataru Aoki; Mitsuyoshi Ueda. 2020. "Neuronal subclass-selective proteomic analysis in Caenorhabditis elegans." Scientific Reports 10, no. 1: 1-9.
Ribosomes are the sophisticated machinery that is responsible for protein synthesis in a cell. Recently, quantitative mass spectrometry (qMS) based on data-dependent acquisition (DDA) have been widely used to understand the biogenesis and function of ribosomes. However, DDA-based qMS sometimes does not provide the reproducible and quantitatively reliable analysis that is needed for high-throughput hypothesis testing. To overcome this problem, we developed a highly sensitive, specific, and accurate method to quantify all ribosomal-proteins by combining selected reaction monitoring (SRM) and stable isotope labeling. We optimized the SRM methods using purified ribosomes and Escherichia coli lysates, and verified this approach as a high-throughput analytical tool by detecting 41 of the 54 r-proteins separately synthesized in E. coli S30 extracts. The SRM methods will enable us to utilize qMS as a high-throughput hypothesis testing tool in the research of E. coli ribosomes, and they have potential to accelerate the understanding of ribosome biogenesis, function, and the development of ribosome engineering.
Yuishin Kosaka; Wataru Aoki; Megumi Mori; Shunsuke Aburaya; Yuta Ohtani; Hiroyoshi Minakuchi; Mitsuyoshi Ueda. Selected reaction monitoring for the quantification of Escherichia coli ribosomal proteins. 2020, 1 .
AMA StyleYuishin Kosaka, Wataru Aoki, Megumi Mori, Shunsuke Aburaya, Yuta Ohtani, Hiroyoshi Minakuchi, Mitsuyoshi Ueda. Selected reaction monitoring for the quantification of Escherichia coli ribosomal proteins. . 2020; ():1.
Chicago/Turabian StyleYuishin Kosaka; Wataru Aoki; Megumi Mori; Shunsuke Aburaya; Yuta Ohtani; Hiroyoshi Minakuchi; Mitsuyoshi Ueda. 2020. "Selected reaction monitoring for the quantification of Escherichia coli ribosomal proteins." , no. : 1.
Ammonia is an essential substance for agriculture and the chemical industry. The intracellular production of ammonia in yeast (Saccharomyces cerevisiae) by metabolic engineering is difficult because yeast strongly assimilates ammonia, and the knockout of genes enabling this assimilation is lethal. Therefore, we attempted to produce ammonia outside the yeast cells by displaying a glutaminase (YbaS) from Escherichia coli on the yeast cell surface. YbaS-displaying yeast successfully produced 3.34 g/L ammonia from 32.6 g/L glutamine (83.2% conversion rate), providing it at a higher yield than in previous studies. Next, using YbaS-displaying yeast, we also succeeded in producing ammonia from glutamine in soybean residues (okara) produced as food waste from tofu production. Therefore, ammonia production outside cells by displaying ammonia-lyase on the cell surface is a promising strategy for producing ammonia from food waste as a novel energy resource, thereby preventing food loss.
Yukio Watanabe; Kouichi Kuroda; Yuki Tatemichi; Takeharu Nakahara; Wataru Aoki; Mitsuyoshi Ueda. Construction of engineered yeast producing ammonia from glutamine and soybean residues (okara). AMB Express 2020, 10, 1 -8.
AMA StyleYukio Watanabe, Kouichi Kuroda, Yuki Tatemichi, Takeharu Nakahara, Wataru Aoki, Mitsuyoshi Ueda. Construction of engineered yeast producing ammonia from glutamine and soybean residues (okara). AMB Express. 2020; 10 (1):1-8.
Chicago/Turabian StyleYukio Watanabe; Kouichi Kuroda; Yuki Tatemichi; Takeharu Nakahara; Wataru Aoki; Mitsuyoshi Ueda. 2020. "Construction of engineered yeast producing ammonia from glutamine and soybean residues (okara)." AMB Express 10, no. 1: 1-8.
Yeast surface display is a powerful technology used to isolate and engineer proteins to improve their activity, specificity, and stability. In this method, gene expression is regulated by promoters, and secretion efficiency is affected by secretion signals. Furthermore, both the accessibility and activity of the displayed proteins are affected by the length of anchor proteins. The ideal promoter, secretion signal, and anchor protein combination depend on the proteins of interest. In this study, we optimized a yeast surface display suitable for nanobody evaluation. We designed five display systems that used different combinations of promoters, secretion signals, and anchor proteins. Anti-hen egg-white lysozyme nanobody was used as the model nanobody. The amount of nanobodies displayed on yeast cells, the number of antigens bound to the displayed nanobodies, and the display efficiency were quantified. Overall, we improved the yeast display system for nanobody engineering and proposed its optimization.
Kaho Kajiwara; Wataru Aoki; Mitsuyoshi Ueda. Evaluation of the yeast surface display system for screening of functional nanobodies. AMB Express 2020, 10, 1 -10.
AMA StyleKaho Kajiwara, Wataru Aoki, Mitsuyoshi Ueda. Evaluation of the yeast surface display system for screening of functional nanobodies. AMB Express. 2020; 10 (1):1-10.
Chicago/Turabian StyleKaho Kajiwara; Wataru Aoki; Mitsuyoshi Ueda. 2020. "Evaluation of the yeast surface display system for screening of functional nanobodies." AMB Express 10, no. 1: 1-10.
Occupying less than 1% of the seas, coral reefs are estimated to harbor ∼25% of all marine species. However, the destruction of coral reefs has intensified in the face of global climate changes, such as rising seawater temperatures, which induce the overproduction of reactive oxygen species harmful to corals. Although reef-building corals form complex consortia with bacteria and photosynthetic endosymbiotic algae of the family Symbiodiniaceae , the functional roles of coral-associated bacteria remain largely elusive. By manipulating the Symbiodiniaceae bacterial community, we demonstrated that a bacterium that produces an antioxidant carotenoid could mitigate thermal and light stresses in cultured Symbiodiniaceae isolated from a reef-building coral. Therefore, this study illuminates the unexplored roles of coral-associated bacteria under stressful conditions.
Keisuke Motone; Toshiyuki Takagi; Shunsuke Aburaya; Natsuko Miura; Wataru Aoki; Mitsuyoshi Ueda. A Zeaxanthin-Producing Bacterium Isolated from the Algal Phycosphere Protects Coral Endosymbionts from Environmental Stress. mBio 2020, 11, 1 .
AMA StyleKeisuke Motone, Toshiyuki Takagi, Shunsuke Aburaya, Natsuko Miura, Wataru Aoki, Mitsuyoshi Ueda. A Zeaxanthin-Producing Bacterium Isolated from the Algal Phycosphere Protects Coral Endosymbionts from Environmental Stress. mBio. 2020; 11 (1):1.
Chicago/Turabian StyleKeisuke Motone; Toshiyuki Takagi; Shunsuke Aburaya; Natsuko Miura; Wataru Aoki; Mitsuyoshi Ueda. 2020. "A Zeaxanthin-Producing Bacterium Isolated from the Algal Phycosphere Protects Coral Endosymbionts from Environmental Stress." mBio 11, no. 1: 1.
Unlike other snakes, most species ofRhabdophispossess glands in their dorsal skin, sometimes limited to the neck, known as nucho-dorsal and nuchal glands, respectively. Those glands contain powerful cardiotonic steroids known as bufadienolides, which can be deployed as a defense against predators. Bufadienolides otherwise occur only in toads (Bufonidae) and some fireflies (Lampyrinae), which are known or believed to synthesize the toxins. The ancestral diet ofRhabdophisconsists of anuran amphibians, and we have shown previously that the bufadienolide toxins of frog-eating species are sequestered from toads consumed as prey. However, one derived clade, theRhabdophis nuchalisGroup, has shifted its primary diet from frogs to earthworms. Here we confirm that the worm-eating snakes possess bufadienolides in their nucho-dorsal glands, although the worms themselves lack such toxins. In addition, we show that the bufadienolides ofR. nuchalisGroup species are obtained primarily from fireflies. Although few snakes feed on insects, we document through feeding experiments, chemosensory preference tests, and gut contents that lampyrine firefly larvae are regularly consumed by these snakes. Furthermore, members of theR. nuchalisGroup contain compounds that resemble the distinctive bufadienolides of fireflies, but not those of toads, in stereochemistry, glycosylation, acetylation, and molecular weight. Thus, the evolutionary shift in primary prey among members of theR. nuchalisGroup has been accompanied by a dramatic shift in the source of the species’ sequestered defensive toxins.
Tatsuya Yoshida; Rinako Ujiie; Alan H. Savitzky; Teppei Jono; Takato Inoue; Naoko Yoshinaga; Shunsuke Aburaya; Wataru Aoki; Hirohiko Takeuchi; Li Ding; Qin Chen; Chengquan Cao; Tein-Shun Tsai; Anslem de Silva; Dharshani Mahaulpatha; Tao Thien Nguyen; Yezhong Tang; Naoki Mori; Akira Mori. Dramatic dietary shift maintains sequestered toxins in chemically defended snakes. Proceedings of the National Academy of Sciences 2020, 117, 5964 -5969.
AMA StyleTatsuya Yoshida, Rinako Ujiie, Alan H. Savitzky, Teppei Jono, Takato Inoue, Naoko Yoshinaga, Shunsuke Aburaya, Wataru Aoki, Hirohiko Takeuchi, Li Ding, Qin Chen, Chengquan Cao, Tein-Shun Tsai, Anslem de Silva, Dharshani Mahaulpatha, Tao Thien Nguyen, Yezhong Tang, Naoki Mori, Akira Mori. Dramatic dietary shift maintains sequestered toxins in chemically defended snakes. Proceedings of the National Academy of Sciences. 2020; 117 (11):5964-5969.
Chicago/Turabian StyleTatsuya Yoshida; Rinako Ujiie; Alan H. Savitzky; Teppei Jono; Takato Inoue; Naoko Yoshinaga; Shunsuke Aburaya; Wataru Aoki; Hirohiko Takeuchi; Li Ding; Qin Chen; Chengquan Cao; Tein-Shun Tsai; Anslem de Silva; Dharshani Mahaulpatha; Tao Thien Nguyen; Yezhong Tang; Naoki Mori; Akira Mori. 2020. "Dramatic dietary shift maintains sequestered toxins in chemically defended snakes." Proceedings of the National Academy of Sciences 117, no. 11: 5964-5969.
Despite the promising clinical efficacy of the second-generation anaplastic lymphoma kinase (ALK) inhibitor alectinib in patients with ALK-rearranged lung cancer, some tumor cells survive and eventually relapse, which may be an obstacle to achieving a cure. Limited information is currently available on the mechanisms underlying the initial survival of tumor cells against alectinib. Using patient-derived cell line models, we herein demonstrate that cancer cells survive a treatment with alectinib by activating Yes-associated protein 1 (YAP1), which mediates the expression of the anti-apoptosis factors Mcl-1 and Bcl-xL, and combinatorial inhibition against both YAP1 and ALK provides a longer tumor remission in ALK-rearranged xenografts when compared with alectinib monotherapy. These results suggest that the inhibition of YAP1 is a candidate for combinatorial therapy with ALK inhibitors to achieve complete remission in patients with ALK-rearranged lung cancer.
Takahiro Tsuji; Hiroaki Ozasa; Wataru Aoki; Shunsuke Aburaya; Tomoko Yamamoto Funazo; Koh Furugaki; Yasushi Yoshimura; Masatoshi Yamazoe; Hitomi Ajimizu; Yuto Yasuda; Takashi Nomizo; Hironori Yoshida; Yuichi Sakamori; Hiroaki Wake; Mitsuyoshi Ueda; Young Hak Kim; Toyohiro Hirai. YAP1 mediates survival of ALK-rearranged lung cancer cells treated with alectinib via pro-apoptotic protein regulation. Nature Communications 2020, 11, 1 -16.
AMA StyleTakahiro Tsuji, Hiroaki Ozasa, Wataru Aoki, Shunsuke Aburaya, Tomoko Yamamoto Funazo, Koh Furugaki, Yasushi Yoshimura, Masatoshi Yamazoe, Hitomi Ajimizu, Yuto Yasuda, Takashi Nomizo, Hironori Yoshida, Yuichi Sakamori, Hiroaki Wake, Mitsuyoshi Ueda, Young Hak Kim, Toyohiro Hirai. YAP1 mediates survival of ALK-rearranged lung cancer cells treated with alectinib via pro-apoptotic protein regulation. Nature Communications. 2020; 11 (1):1-16.
Chicago/Turabian StyleTakahiro Tsuji; Hiroaki Ozasa; Wataru Aoki; Shunsuke Aburaya; Tomoko Yamamoto Funazo; Koh Furugaki; Yasushi Yoshimura; Masatoshi Yamazoe; Hitomi Ajimizu; Yuto Yasuda; Takashi Nomizo; Hironori Yoshida; Yuichi Sakamori; Hiroaki Wake; Mitsuyoshi Ueda; Young Hak Kim; Toyohiro Hirai. 2020. "YAP1 mediates survival of ALK-rearranged lung cancer cells treated with alectinib via pro-apoptotic protein regulation." Nature Communications 11, no. 1: 1-16.
Since G-protein coupled receptors (GPCRs) are linked to various diseases, screening of functional ligands against GPCRs is vital for drug discovery. In the present study, we developed a high-throughput functional cell-based assay by combining human culture cells producing a GPCR, yeast cells secreting randomized peptide ligands, and a droplet microfluidic device. We constructed a reporter human cell line that emits fluorescence in response to the activation of human glucagon-like peptide-1 receptor (hGLP1R). We then constructed a yeast library secreting an agonist of hGLP1R or randomized peptide ligands. We demonstrated that high-throughput identification of functional ligands against hGLP1R could be performed by co-culturing the reporter cells and the yeast cells in droplets. We identified functional ligands, one of which had higher activity than that of an original sequence. The result suggests that our system could facilitate the discovery of functional peptide ligands of GPCRs.
Kenshi Yaginuma; Wataru Aoki; Natsuko Miura; Yuta Ohtani; Shunsuke Aburaya; Masato Kogawa; Yohei Nishikawa; Masahito Hosokawa; Haruko Takeyama; Mitsuyoshi Ueda. High-throughput identification of peptide agonists against GPCRs by co-culture of mammalian reporter cells and peptide-secreting yeast cells using droplet microfluidics. Scientific Reports 2019, 9, 1 -11.
AMA StyleKenshi Yaginuma, Wataru Aoki, Natsuko Miura, Yuta Ohtani, Shunsuke Aburaya, Masato Kogawa, Yohei Nishikawa, Masahito Hosokawa, Haruko Takeyama, Mitsuyoshi Ueda. High-throughput identification of peptide agonists against GPCRs by co-culture of mammalian reporter cells and peptide-secreting yeast cells using droplet microfluidics. Scientific Reports. 2019; 9 (1):1-11.
Chicago/Turabian StyleKenshi Yaginuma; Wataru Aoki; Natsuko Miura; Yuta Ohtani; Shunsuke Aburaya; Masato Kogawa; Yohei Nishikawa; Masahito Hosokawa; Haruko Takeyama; Mitsuyoshi Ueda. 2019. "High-throughput identification of peptide agonists against GPCRs by co-culture of mammalian reporter cells and peptide-secreting yeast cells using droplet microfluidics." Scientific Reports 9, no. 1: 1-11.
Easy preparation of chimeric nanobodies with various scaffolds is important for customizing abilities of nanobodies toward practical utilization. To accomplish high-throughput production of various nanobodies, utilization of microbes is an attractive option. In the present study, various chimeric nanobodies were prepared using the methylotrophic yeast Pichia pastoris. We designed chimeric nanobodies with complementarity-determining regions (CDRs) against green fluorescent protein (GFP) or cluster of differentiation 4 (CD4) based on the scaffold of GFP-nanobody. FLAG-tagged chimeric nanobodies were prepared by one-step cloning and produced using P. pastoris. Secreted chimeric nanobodies were purified from the culture media of P. pastoris transformants. Relative binding abilities of purified chimeric nanobodies to GFP and CD4 was tested using a BIACORE T-200. P. pastoris successfully produced a high yield of FLAG-tagged chimeric nanobodies. FLAG-tagged GFP- and CD4-nanobodies were shown to specifically bind to GFP and CD4, respectively. Chimeric nanobodies, in which the CDR2 or 3 of GFP-nanobody was replaced with CDRs of CD4-nanobody, acquired the ability to bind to CD4 without binding to GFP. These results demonstrate successful production of functional chimeric nanobodies using P. pastoris. These results also suggest that swapping of CDRs, especially CDRs 2 or 3, potentially enables a novel method of creating nanobodies.
Natsuko Miura; Kana Miyamoto; Yuta Ohtani; Kenshi Yaginuma; Shunsuke Aburaya; Yoshinori Kitagawa; Wataru Aoki; Mitsuyoshi Ueda. Domain swapping of complementarity-determining region in nanobodies produced by Pichia pastoris. AMB Express 2019, 9, 1 -8.
AMA StyleNatsuko Miura, Kana Miyamoto, Yuta Ohtani, Kenshi Yaginuma, Shunsuke Aburaya, Yoshinori Kitagawa, Wataru Aoki, Mitsuyoshi Ueda. Domain swapping of complementarity-determining region in nanobodies produced by Pichia pastoris. AMB Express. 2019; 9 (1):1-8.
Chicago/Turabian StyleNatsuko Miura; Kana Miyamoto; Yuta Ohtani; Kenshi Yaginuma; Shunsuke Aburaya; Yoshinori Kitagawa; Wataru Aoki; Mitsuyoshi Ueda. 2019. "Domain swapping of complementarity-determining region in nanobodies produced by Pichia pastoris." AMB Express 9, no. 1: 1-8.
Clostridium cellulovorans is a mesophilic, cellulosome-producing bacterium containing 57 genomic cellulosomal enzyme-encoding genes. In addition to cellulosomal proteins, C. cellulovorans also secretes non-cellulosomal proteins to degrade plant cell wall polysaccharides. Unlike other cellulosome-producing Clostridium species, C. cellulovorans can metabolize all major plant cell wall polysaccharides (cellulose, hemicelluloses, and pectins). In this study, we performed a temporal proteome analysis of C. cellulovorans to reveal strategies underlying plant cell wall polysaccharide degradation. We cultured C. cellulovorans with five different carbon sources (glucose, cellulose, xylan, galactomannan, and pectin) and performed proteome analysis on cellular and secreted proteins. In total, we identified 1895 cellular proteins and 875 secreted proteins. The identified unique carbohydrate-degrading enzymes corresponding to each carbon source were annotated to have specific activity against each carbon source. However, we identified pectate lyase as a unique enzyme in C. cellulovorans cultivated on xylan, which was not previously associated with xylan degradation. We performed k-means clustering analysis for elucidation of temporal changes of the cellular and secreted proteins in each carbon sources. We found that cellular proteins in most of the k-means clusters are involved in carbohydrate metabolism, amino acid metabolism, translation, or membrane transport. When xylan and pectin were used as the carbon sources, the most increasing k-means cluster contained proteins involved in the metabolism of cofactors and vitamins. In case of secreted proteins of C. cellulovorans cultured either on cellulose or xylan, galactomannan, and pectin, the clusters with the most increasing trend contained either 25 cellulosomal proteins and five non-cellulosomal proteins or 8–19 cellulosomal proteins and 9–16 non-cellulosomal proteins, respectively. These differences might reflect mechanisms for degrading cellulose of other carbon source. Co-abundance analysis of the secreted proteins revealed that proteases and protease inhibitors accumulated coordinately. This observation implies that the secreted protease inhibitors and proteases protect carbohydrate-degrading enzymes from an attack from the plant. In this study, we clarified, for the first time, the temporal proteome dynamics of cellular and secreted proteins in C. cellulovorans. This data will be valuable in understanding strategies employed by C. cellulovorans for degrading major plant cell wall polysaccharides. The online version of this article (10.1186/s12866-019-1480-0) contains supplementary material, which is available to authorized users.
Shunsuke Aburaya; Wataru Aoki; Kouichi Kuroda; Hiroshi Minakuchi; Mitsuyoshi Ueda. Temporal proteome dynamics of Clostridium cellulovorans cultured with major plant cell wall polysaccharides. BMC Microbiology 2019, 19, 118 .
AMA StyleShunsuke Aburaya, Wataru Aoki, Kouichi Kuroda, Hiroshi Minakuchi, Mitsuyoshi Ueda. Temporal proteome dynamics of Clostridium cellulovorans cultured with major plant cell wall polysaccharides. BMC Microbiology. 2019; 19 (1):118.
Chicago/Turabian StyleShunsuke Aburaya; Wataru Aoki; Kouichi Kuroda; Hiroshi Minakuchi; Mitsuyoshi Ueda. 2019. "Temporal proteome dynamics of Clostridium cellulovorans cultured with major plant cell wall polysaccharides." BMC Microbiology 19, no. 1: 118.
Yeast surface display is one of the most successful in vitro protein engineering platforms. In vitro selection enables antibodies screening with specific functions in the primary screening process, which is difficult when using the classical hybridoma systems. Moreover, eukaryotic quality control systems assist in displaying the complex proteins, and the applicability of flow cytometry facilitates the quantitative evaluation of protein libraries. Based on these characteristics, yeast surface display has broad applications from antibody screening and maturation to the high-throughput evaluation of de novo designer proteins. In this chapter, we review yeast surface display technologies, highlighting their roles in protein engineering and in therapeutic uses.
Wataru Aoki. Engineering Antibodies and Alternative Binders for Therapeutic Uses. Yeast Cell Surface Engineering 2019, 123 -147.
AMA StyleWataru Aoki. Engineering Antibodies and Alternative Binders for Therapeutic Uses. Yeast Cell Surface Engineering. 2019; ():123-147.
Chicago/Turabian StyleWataru Aoki. 2019. "Engineering Antibodies and Alternative Binders for Therapeutic Uses." Yeast Cell Surface Engineering , no. : 123-147.
Measuring binding properties of binders (e.g., antibodies) is essential for developing useful experimental reagents, diagnostics, and pharmaceuticals. Display technologies can evaluate a large number of binders in a high-throughput manner, but the immobilization effect and the avidity effect prohibit the precise evaluation of binding properties. In this paper, we propose a novel methodology, peptide barcoding, to quantitatively measure the binding properties of multiple binders without immobilization. In the experimental scheme, unique peptide barcodes are fused with each binder, and they represent genotype information. These peptide barcodes are designed to have high detectability for mass spectrometry, leading to low identification bias and a high identification rate. A mixture of different peptide-barcoded nanobodies is reacted with antigen-coated magnetic beads in one pot. Peptide barcodes of functional nanobodies are cleaved on beads by a specific protease, and identified by selected reaction monitoring using triple quadrupole mass spectrometry. To demonstrate proof-of-principle for peptide barcoding, we generated peptide-barcoded anti-CD4 nanobody and anti-GFP nanobody, and determined whether we could simultaneously quantify their binding activities. We showed that peptide barcoding did not affect the properties of the nanobodies, and succeeded in measuring the binding activities of these nanobodies in one shot. The results demonstrate the advantages of peptide barcoding, new types of genotype–phenotype linkages.
Kana Miyamoto; Wataru Aoki; Yuta Ohtani; Natsuko Miura; Shunsuke Aburaya; Yusei Matsuzaki; Kaho Kajiwara; Yoshinori Kitagawa; Mitsuyoshi Ueda. Peptide barcoding for establishment of new types of genotype–phenotype linkages. PLOS ONE 2019, 14, e0215993 .
AMA StyleKana Miyamoto, Wataru Aoki, Yuta Ohtani, Natsuko Miura, Shunsuke Aburaya, Yusei Matsuzaki, Kaho Kajiwara, Yoshinori Kitagawa, Mitsuyoshi Ueda. Peptide barcoding for establishment of new types of genotype–phenotype linkages. PLOS ONE. 2019; 14 (4):e0215993.
Chicago/Turabian StyleKana Miyamoto; Wataru Aoki; Yuta Ohtani; Natsuko Miura; Shunsuke Aburaya; Yusei Matsuzaki; Kaho Kajiwara; Yoshinori Kitagawa; Mitsuyoshi Ueda. 2019. "Peptide barcoding for establishment of new types of genotype–phenotype linkages." PLOS ONE 14, no. 4: e0215993.
Proteome is extremely complex as many proteins with a large dynamic range are involved. Nano-liquid chromatography/mass spectrometry-based proteomics has made it possible to separate and identify thousands of proteins in one shot. Although the number of identified proteins in proteomics has significantly improved, it is necessary to increase detection sensitivity to clearly identify low-abundant proteins. In this study, we developed meter-long monolithic columns with a small inner diameter and applied them to selected reaction monitoring-based proteomics for improving proteomic detection sensitivity. Bovine serum albumin tryptic digests were analyzed with optimized selected reaction monitoring methods, and separation efficiency and detection sensitivity in each monolithic column were evaluated. As a result, peak capacity increased by about 1.8-fold and peak area of peptide levels increased by about 2.3-fold. Although flow rate was reduced during analysis with columns of a smaller inner diameter, the peak area reproducibility was maintained. These data displayed the value of meter-long monolithic columns with small inner diameter for selected reaction monitoring-based proteomics.
Yuta Ohtani; Shunsuke Aburaya; Hiroyoshi Minakuchi; Natsuko Miura; Wataru Aoki; Mitsuyoshi Ueda. Evaluation of meter-long monolithic columns for selected reaction monitoring mass spectrometry. Journal of Bioscience and Bioengineering 2019, 128, 379 -383.
AMA StyleYuta Ohtani, Shunsuke Aburaya, Hiroyoshi Minakuchi, Natsuko Miura, Wataru Aoki, Mitsuyoshi Ueda. Evaluation of meter-long monolithic columns for selected reaction monitoring mass spectrometry. Journal of Bioscience and Bioengineering. 2019; 128 (3):379-383.
Chicago/Turabian StyleYuta Ohtani; Shunsuke Aburaya; Hiroyoshi Minakuchi; Natsuko Miura; Wataru Aoki; Mitsuyoshi Ueda. 2019. "Evaluation of meter-long monolithic columns for selected reaction monitoring mass spectrometry." Journal of Bioscience and Bioengineering 128, no. 3: 379-383.