Updated on 2024/03/12

写真b

 
MUKAI Takahito
 
*Items subject to periodic update by Rikkyo University (The rest are reprinted from information registered on researchmap.)
Affiliation*
College of Science
Title*
Assistant Professor
Research Interests
  • tRNA

  • Bioinformatics

  • non-canonical amino acids

  • The genetic code

  • Synthetic biology

  • Campus Career*
    • 4 2023 - Present 
      College of Science   Assistant Professor
    • 4 2018 - 3 2023 
      College of Science   Department of Life Science   Assistant Professor
    Profile
    研究テーマは,遺伝暗号の人為的改変と,自然界における特殊な遺伝暗号の探索です.生命の起源と可能性を追い求めています.
     

    Research Areas

    • Life Science / Genome biology

    Research History

    • 4 2018 - Present 
      Rikkyo University   Department of Life Science, College of Science

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    • 4 2016 - 3 2018 
      Yale大学   Department of Molecular Biophysics and Biochemistry   日本学術振興会 海外特別研究員 (Yale大学においては、平成28年度中はPostdoctoral Fellowの身分で、平成29年度からはAssociate Research Scientistの身分)

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    • 6 2014 - 3 2016 
      Yale大学   Department of Molecular Biophysics and Biochemistry   ポスドク

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    • 4 2012 - 5 2014 
      理化学研究所   生命分子システム基盤研究領域(平成25年度から改組してライフサイエンス技術基盤研究センター)   基礎科学特別研究員

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    • 4 2011 - 3 2012 
      理化学研究所   生命分子システム基盤研究領域   特別研究員

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    • 4 2008 - 3 2011 
      東京大学大学院   理学系研究科生物化学専攻   日本学術振興会特別研究員DC1

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    Education

    • 4 2006 - 3 2011 
      東京大学大学院   理学系研究科   生物化学専攻

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    • 4 2002 - 3 2006 
      The University of Tokyo

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    Papers

    • Recoding UAG to selenocysteine in Saccharomyces cerevisiae Peer-reviewed

      Kyle S Hoffman, Christina Z Chung, Takahito Mukai, Natalie Krahn, Han-Kai Jang, Nileeka Balasuriya, Patrick O'Donoghue, Dieter Söll

      RNA   rna.079658.123 - rna.079658.123   6 6 2023

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      Publishing type:Research paper (scientific journal)   Publisher:Cold Spring Harbor Laboratory  

      Unique chemical and physical properties are introduced by inserting selenocysteine (Sec) at specific sites within proteins. Recombinant and facile production of eukaryotic selenoproteins would benefit from a yeast expression system, however, the selenoprotein biosynthetic pathway was lost in the evolution of the kingdom Fungi as it diverged from its eukaryotic relatives. Based on our previous development of efficient selenoprotein production in bacteria, we designed a novel selenocysteine biosynthesis pathway in Saccharomyces cerevisiae using Aeromonas salmonicida translation components. S. cerevisiae tRNASer was mutated to resemble A. salmonicida tRNASec to allow recognition by S. cerevisiae seryl-tRNA synthetase as well as A. salmonicida selenocysteine synthase (SelA) and selenophosphate synthetase (SelD). Expression of these selenocysteine pathway components was then combined with metabolic engineering of yeast to enable the production of active methionine sulfate reductase enzyme containing genetically encoded selenocysteine. Our report is the first demonstration that yeast is capable of selenoprotein production by site-specific incorporation of selenocysteine.

      DOI: 10.1261/rna.079658.123

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    • Ancestral Archaea Expanded the Genetic Code with Pyrrolysine Peer-reviewed

      Li-Tao Guo, Kazuaki Amikura, Han-Kai Jiang, Takahito Mukai, Xian Fu, Yane-Shih Wang, Patrick O’Donoghue, Dieter Söll, Jeffery M. Tharp

      Journal of Biological Chemistry   102521 - 102521   9 2022

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

      DOI: 10.1016/j.jbc.2022.102521

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    • Enzymatic Supercoiling of Bacterial Chromosomes Facilitates Genome Manipulation Peer-reviewed

      Hironobu Fujita, Ayane Osaku, Yuto Sakane, Koki Yoshida, Kayoko Yamada, Seia Nara, Takahito Mukai, Masayuki Su’etsugu

      ACS Synthetic Biology   23 8 2022

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      Authorship:Corresponding author   Language:English   Publishing type:Research paper (scientific journal)   Publisher:American Chemical Society (ACS)  

      DOI: 10.1021/acssynbio.2c00353

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    • The tRNA discriminator base defines the mutual orthogonality of two distinct pyrrolysyl-tRNA synthetase/tRNAPyl pairs in the same organism Peer-reviewed

      Haolin Zhang, Xuemei Gong, Qianqian Zhao, Takahito Mukai, Oscar Vargas-Rodriguez, Huiming Zhang, Yuxing Zhang, Paul Wassel, Kazuaki Amikura, Julie Maupin-Furlow, Yan Ren, Xun Xu, Yuri I Wolf, Kira S Makarova, Eugene V Koonin, Yue Shen, Dieter Söll, Xian Fu

      Nucleic Acids Research   25 4 2022

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      Publishing type:Research paper (scientific journal)   Publisher:Oxford University Press (OUP)  

      Abstract

      Site-specific incorporation of distinct non-canonical amino acids into proteins via genetic code expansion requires mutually orthogonal aminoacyl-tRNA synthetase/tRNA pairs. Pyrrolysyl-tRNA synthetase (PylRS)/tRNAPyl pairs are ideal for genetic code expansion and have been extensively engineered for developing mutually orthogonal pairs. Here, we identify two novel wild-type PylRS/tRNAPyl pairs simultaneously present in the deep-rooted extremely halophilic euryarchaeal methanogen Candidatus Methanohalarchaeum thermophilum HMET1, and show that both pairs are functional in the model halophilic archaeon Haloferax volcanii. These pairs consist of two different PylRS enzymes and two distinct tRNAs with dissimilar discriminator bases. Surprisingly, these two PylRS/tRNAPyl pairs display mutual orthogonality enabled by two unique features, the A73 discriminator base of tRNAPyl2 and a shorter motif 2 loop in PylRS2. In vivo translation experiments show that tRNAPyl2 charging by PylRS2 is defined by the enzyme's shortened motif 2 loop. Finally, we demonstrate that the two HMET1 PylRS/tRNAPyl pairs can simultaneously decode UAG and UAA codons for incorporation of two distinct noncanonical amino acids into protein. This example of a single base change in a tRNA leading to additional coding capacity suggests that the growth of the genetic code is not yet limited by the number of identity elements fitting into the tRNA structure.

      DOI: 10.1093/nar/gkac271

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    • Indirect Routes to Aminoacyl-tRNA: The Diversity of Prokaryotic Cysteine Encoding Systems Peer-reviewed

      Takahito Mukai, Kazuaki Amikura, Xian Fu, Dieter Söll, Ana Crnković

      Frontiers in Genetics12   3 1 2022

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      Authorship:Lead author   Publishing type:Research paper (scientific journal)   Publisher:Frontiers Media SA  

      Universally present aminoacyl-tRNA synthetases (aaRSs) stringently recognize their cognate tRNAs and acylate them with one of the proteinogenic amino acids. However, some organisms possess aaRSs that deviate from the accurate translation of the genetic code and exhibit relaxed specificity toward their tRNA and/or amino acid substrates. Typically, these aaRSs are part of an indirect pathway in which multiple enzymes participate in the formation of the correct aminoacyl-tRNA product. The indirect cysteine (Cys)-tRNA pathway, originally thought to be restricted to methanogenic archaea, uses the unique <italic>O</italic>-phosphoseryl-tRNA synthetase (SepRS), which acylates the non-proteinogenic amino acid <italic>O</italic>-phosphoserine (Sep) onto tRNA<sup>Cys</sup>. Together with Sep-tRNA:Cys-tRNA synthase (SepCysS) and the adapter protein SepCysE, SepRS forms a transsulfursome complex responsible for shuttling Sep-tRNA<sup>Cys</sup> to SepCysS for conversion of the tRNA-bound Sep to Cys. Here, we report a comprehensive bioinformatic analysis of the diversity of indirect Cys encoding systems. These systems are present in more diverse groups of bacteria and archaea than previously known. Given the occurrence and distribution of some genes consistently flanking SepRS, it is likely that this gene was part of an ancient operon that suffered a gradual loss of its original components. Newly identified bacterial SepRS sequences strengthen the suggestion that this lineage of enzymes may not rely on the m<sup>1</sup>G37 identity determinant in tRNA. Some bacterial SepRSs possess an N-terminal fusion resembling a threonyl-tRNA synthetase editing domain, which interestingly is frequently observed in the vicinity of archaeal SepCysS genes. We also found several highly degenerate SepRS genes that likely have altered amino acid specificity. Cross-analysis of selenocysteine (Sec)-utilizing traits confirmed the co-occurrence of SepCysE and the Sec-utilizing machinery in archaea, but also identified an unusual <italic>O</italic>-phosphoseryl-tRNA<sup>Sec</sup> kinase fusion with an archaeal Sec elongation factor in some lineages, where it may serve in place of SepCysE to prevent crosstalk between the two minor aminoacylation systems. These results shed new light on the variations in SepRS and SepCysS enzymes that may reflect adaptation to lifestyle and habitat, and provide new information on the evolution of the genetic code.

      DOI: 10.3389/fgene.2021.794509

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    • Grand scale genome manipulation via chromosome swapping in Escherichia coli programmed by three one megabase chromosomes Peer-reviewed International journal

      Tatsuya Yoneji, Hironobu Fujita, Takahito Mukai, Masayuki Su’etsugu

      Nucleic Acids Research   4 2021

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      Authorship:Corresponding author   Language:English   Publishing type:Research paper (scientific journal)   Publisher:Oxford University Press (OUP)  

      In bacterial synthetic biology, whole genome transplantation has been achieved only in mycoplasmas that contain a small genome and are competent for foreign genome uptake. In this study, we developed Escherichia coli strains programmed by three 1-megabase (Mb) chromosomes by splitting the 3-Mb chromosome of a genome-reduced strain. The first split-chromosome retains the original replication origin (oriC) and partitioning (par) system. The second one has an oriC and the par locus from the F plasmid, while the third one has the ori and par locus of the Vibrio tubiashii secondary chromosome. The tripartite-genome cells maintained the rod-shaped form and grew only twice as slowly as their parent, allowing their further genetic engineering. A proportion of these 1-Mb chromosomes were purified as covalently closed supercoiled molecules with a conventional alkaline lysis method and anion exchange columns. Furthermore, the second and third chromosomes could be individually electroporated into competent cells. In contrast, the first split-chromosome was not able to coexist with another chromosome carrying the same origin region. However, it was exchangeable via conjugation between tripartite-genome strains by using different selection markers. We believe that this E. coli-based technology has the potential to greatly accelerate synthetic biology and synthetic genomics.

      DOI: 10.1093/nar/gkab298

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    • Bioinformatic Prediction of an tRNASec Gene Nested inside an Elongation Factor SelB Gene in Alphaproteobacteria Invited Peer-reviewed

      Takahito Mukai

      IJMS22 ( 9 ) 4605   4 2021

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

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    • Rational Design of Aptamer-Tagged tRNAs Invited Peer-reviewed

      Takahito Mukai

      IJMS21 ( 20 ) 7793   10 2020

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

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    • Overcoming the Challenges of Megabase-Sized Plasmid Construction in Escherichia coli Peer-reviewed

      Takahito Mukai, Tatsuya Yoneji, Kayoko Yamada, Hironobu Fujita, Seia Nara, Masayuki Su’etsugu

      ACS Synthetic Biology   27 5 2020

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      Authorship:Lead author, Corresponding author   Language:English   Publishing type:Research paper (scientific journal)   Publisher:American Chemical Society (ACS)  

      DOI: 10.1021/acssynbio.0c00008

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    • A cysteinyl-tRNA synthetase variant confers resistance against selenite toxicity and decreases selenocysteine misincorporation. Peer-reviewed

      Hoffman KS, Vargas-Rodriguez O, Bak DW, Mukai T, Woodward LK, Weerapana E, Söll D, Reynolds NM

      The Journal of biological chemistry   7 2019

    • Cell-Free Protein Synthesis Using S30 Extracts from <i>Escherichia coli</i> RFzero Strains for Efficient Incorporation of Non-Natural Amino Acids into Proteins. Invited Peer-reviewed International journal

      Adachi J, Katsura K, Seki E, Takemoto C, Shirouzu M, Terada T, Mukai T, Sakamoto K, Yokoyama S

      International journal of molecular sciences20 ( 3 )   1 2019

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

      Cell-free protein synthesis is useful for synthesizing difficult targets. The site-specific incorporation of non-natural amino acids into proteins is a powerful protein engineering method. In this study, we optimized the protocol for cell extract preparation from the Escherichia coli strain RFzero-iy, which is engineered to lack release factor 1 (RF-1). The BL21(DE3)-based RFzero-iy strain exhibited quite high cell-free protein productivity, and thus we established the protocols for its cell culture and extract preparation. In the presence of 3-iodo-l-tyrosine (IY), cell-free protein synthesis using the RFzero-iy-based S30 extract translated the UAG codon to IY at various sites with a high translation efficiency of >90%. In the absence of IY, the RFzero-iy-based cell-free system did not translate UAG to any amino acid, leaving UAG unassigned. Actually, UAG was readily reassigned to various non-natural amino acids, by supplementing them with their specific aminoacyl-tRNA synthetase variants (and their specific tRNAs) into the system. The high incorporation rate of our RFzero-iy-based cell-free system enables the incorporation of a variety of non-natural amino acids into multiple sites of proteins. The present strategy to create the RFzero strain is rapid, and thus promising for RF-1 deletions of various E. coli strains genomically engineered for specific requirements.

      DOI: 10.3390/ijms20030492

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    • Engineering an auto-maturing transglutaminase with enhanced thermostability by genetic code expansion with two codon reassignments Peer-reviewed

      Kazumasa Ohtake, Takahito Mukai, Fumie Iraha, Mihoko Takahashi, Ken-ichi Haruna, Masayo Date, Keiichi Yokoyama, Kensaku Sakamoto

      ACS Synthetic Biology   7 2018

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      Language:English  

      DOI: 10.1021/acssynbio.8b00157

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    • Recoding of the selenocysteine UGA codon by cysteine in the presence of a non-canonical tRNACys and elongation factor SelB Invited Peer-reviewed

      Oscar Vargas-Rodriguez, Markus Englert, Anna Merkuryev, Takahito Mukai, Dieter Söll

      RNA Biology   6 2018

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

      DOI: 10.1080/15476286.2018.1474074

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    • Eine einfache Methode zur Produktion von Selenoproteinen Invited Peer-reviewed

      Mukai T, Sevostyanova A, Suzuki T, Fu X, Söll D

      Angewandte Chemie   5 2018

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

      DOI: 10.1002/ange.201713215

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    • A facile method for producing selenocysteine‐containing proteins. Peer-reviewed

      Mukai T, Sevostyanova A, Suzuki T, Fu X, Söll D

      Angewandte Chemie International Editiondoi: 10.1002/anie.201713215   4 2018

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      DOI: 10.1002/anie.201713215

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    • RNA-Dependent Cysteine Biosynthesis in Bacteria and Archaea Peer-reviewed

      Takahito Mukai, Ana Crnkovic, Takuya Umehara, Natalia N. Ivanova, Nikos C. Kyrpides, Dieter Soll

      MBIO8 ( 3 ) doi: 10.1128/mBio.00561-17   5 2017

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      Language:English   Publishing type:Research paper (scientific journal)   Publisher:AMER SOC MICROBIOLOGY  

      The diversity of the genetic code systems used by microbes on earth is yet to be elucidated. It is known that certain methanogenic archaea employ an alternative system for cysteine (Cys) biosynthesis and encoding; tRNACys is first acylated with phosphoserine (Sep) by O-phosphoseryl-tRNA synthetase (SepRS) and then converted to Cys-tRNACys by Sep-tRNA:Cys-tRNA synthase (SepCysS). In this study, we searched all genomic and metagenomic protein sequence data in the Integrated Microbial Genomes (IMG) system and at the NCBI to reveal new clades of SepRS and SepCysS proteins belonging to diverse archaea in the four major groups (DPANN, Euryarchaeota, TACK, and Asgard) and two groups of bacteria ("Candidatus Parcubacteria" and Chloroflexi). Bacterial SepRS and SepCysS charged bacterial tRNACys species with cysteine in vitro. Homologs of SepCysE, a scaffold protein facilitating SepRS.SepCysS complex assembly in Euryarchaeota class I methanogens, are found in a few groups of TACK and Asgard archaea, whereas the C-terminally truncated homologs exist fused or genetically coupled with diverse SepCysS species. Investigation of the selenocysteine (Sec)-and pyrrolysine (Pyl)-utilizing traits in SepRSutilizing archaea and bacteria revealed that the archaea carrying full-length SepCysE employ Sec and that SepRS is often found in Pyl-utilizing archaea and Chloroflexi bacteria. We discuss possible contributions of the SepRS-SepCysS system for sulfur assimilation, methanogenesis, and other metabolic processes requiring large amounts of iron-sulfur enzymes or Pyl-containing enzymes.
      IMPORTANCE Comprehensive analyses of all genomic and metagenomic protein sequence data in public databases revealed the distribution and evolution of an alternative cysteine-encoding system in diverse archaea and bacteria. The finding that the SepRS-SepCysS-SepCysE-and the selenocysteine-encoding systems are shared by the Euryarchaeota class I methanogens, the Crenarchaeota AK8/W8A-19 group, and an Asgard archaeon suggests that ancient archaea may have used both systems. In contrast, bacteria may have obtained the SepRS-SepCysS system from archaea. The SepRS-SepCysS system sometimes coexists with a pyrrolysine-encoding system in both archaea and bacteria. Our results provide additional bioinformatic evidence for the contribution of the SepRS-SepCysS system for sulfur assimilation and diverse metabolisms which require vast amounts of iron-sulfur enzymes and proteins. Among these biological activities, methanogenesis, methylamine metabolism, and organohalide respiration may have local and global effects on earth. Taken together, uncultured bacteria and archaea provide an expanded record of the evolution of the genetic code.

      DOI: 10.1128/mBio.00561-17

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    • Transfer RNAs with novel cloverleaf structures Peer-reviewed

      Takahito Mukai, Oscar Vargas-Rodriguez, Markus Englert, H. James Tripp, Natalia N. Ivanova, Edward M. Rubin, Nikos C. Kyrpides, Dieter Soell

      NUCLEIC ACIDS RESEARCH45 ( 5 ) 2776 - 2785   3 2017

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      Language:English   Publishing type:Research paper (scientific journal)   Publisher:OXFORD UNIV PRESS  

      We report the identification of novel tRNA species with 12-base pair amino-acid acceptor branches composed of longer acceptor stem and shorter T-stem. While canonical tRNAs have a 7/5 configuration of the branch, the novel tRNAs have either 8/4 or 9/3 structure. They were found during the search for selenocysteine tRNAs in terabytes of genome, metagenome and metatranscriptome sequences. Certain bacteria and their phages employ the 8/4 structure for serine and histidine tRNAs, while minor cysteine and selenocysteine tRNA species may have a modified 8/4 structure with one bulge nucleotide. In Acidobacteria, tRNAs with 8/4 and 9/3 structures may function as missense and nonsense suppressor tRNAs and/or regulatory non-coding RNAs. In delta-proteobacteria, an additional cysteine tRNA with an 8/4 structure mimics selenocysteine tRNA and may function as opal suppressor. We examined the potential translation function of suppressor tRNA species in Escherichia coli; tRNAs with 8/4 or 9/3 structures efficiently inserted serine, alanine and cysteine in response to stop and sense codons, depending on the identity element and anticodon sequence of the tRNA. These findings expand our view of how tRNA, and possibly the genetic code, is diversified in nature.

      DOI: 10.1093/nar/gkw898

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    • Bioinformatic Analysis Reveals Archaeal tRNA<sup>Tyr</sup> and tRNA<sup>Trp</sup> Identities in Bacteria. Invited Peer-reviewed

      Mukai T, Reynolds NM, Crnković A, Söll D

      Life (Basel, Switzerland)7 ( 1 )   2 2017

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

      DOI: 10.3390/life7010008

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    • Rewriting the Genetic Code Invited Peer-reviewed

      Takahito Mukai, Marc J. Lajoie, Markus Englert, Dieter Soll

      ANNUAL REVIEW OF MICROBIOLOGY, VOL 7171 ( 1 ) 557 - 577   2017

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      Language:English   Publishing type:Part of collection (book)   Publisher:ANNUAL REVIEWS  

      The genetic code-the language used by cells to translate their genomes into proteins that perform many cellular functions-is highly conserved throughout natural life. Rewriting the genetic code could lead to new biological functions such as expanding protein chemistries with noncanonical amino acids (ncAAs) and genetically isolating synthetic organisms from natural organisms and viruses. It has long been possible to transiently produce proteins bearing ncAAs, but stabilizing an expanded genetic code for sustained function in vivo requires an integrated approach: creating recoded genomes and introducing new translation machinery that function together without compromising viability or clashing with endogenous pathways. In this review, we discuss design considerations and technologies for expanding the genetic code. The knowledge obtained by rewriting the genetic code will deepen our understanding of how genomes are designed and how the canonical genetic code evolved.

      DOI: 10.1146/annurev-micro-090816-093247

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    • Facile Recoding of Selenocysteine in Nature Peer-reviewed

      Takahito Mukai, Markus Englert, H. James Tripp, Corwin Miller, Natalia N. Ivanova, Edward M. Rubin, Nikos C. Kyrpides, Dieter Soell

      ANGEWANDTE CHEMIE-INTERNATIONAL EDITION55 ( 17 ) 5337 - 5341   4 2016

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      Language:English   Publishing type:Research paper (scientific journal)   Publisher:WILEY-V C H VERLAG GMBH  

      Selenocysteine (Sec or U) is encoded by UGA, a stop codon reassigned by a Sec-specific elongation factor and a distinctive RNA structure. To discover possible code variations in extant organisms we analyzed 6.4trillion base pairs of metagenomic sequences and 24903 microbial genomes for tRNA(Sec) species. As expected, UGA is the predominant Sec codon in use. We also found tRNA(Sec) species that recognize the stop codons UAG and UAA, and ten sense codons. Selenoprotein synthesis programmed by UAG in Geodermatophilus and Blastococcus, and by the Cys codon UGU in Aeromonas salmonicida was confirmed by metabolic labeling with Se-75 or mass spectrometry. Other tRNA(Sec) species with different anticodons enabled E.coli to synthesize active formate dehydrogenaseH, a selenoenzyme. This illustrates the ease by which the genetic code may evolve new coding schemes, possibly aiding organisms to adapt to changing environments, and show the genetic code is much more flexible than previously thought.

      DOI: 10.1002/anie.201511657

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    • Leichte Neucodierung von Selenocystein in der Natur. Invited Peer-reviewed

      Mukai T, Englert M, Tripp HJ, Miller C, Ivanova NN, Rubin EM, Kyrpides NC, Söll D

      Angewandte Chemie (Weinheim an der Bergstrasse, Germany)128 ( 17 ) 5423 - 5427   4 2016

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

      DOI: 10.1002/ange.201511657

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    • Reassignment of a rare sense codon to a non-canonical amino acid in Escherichia coli Peer-reviewed

      Takahito Mukai, Atsushi Yamaguchi, Kazumasa Ohtake, Mihoko Takahashi, Akiko Hayashi, Fumie Iraha, Satoshi Kira, Tatsuo Yanagisawa, Shigeyuki Yokoyama, Hiroko Hoshi, Takatsugu Kobayashi, Kensaku Sakamoto

      Nucleic Acids Research43 ( 16 ) 8111 - 8122   18 9 2015

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      Authorship:Lead author, Corresponding author   Publishing type:Research paper (scientific journal)   Publisher:Oxford University Press (OUP)  

      DOI: 10.1093/nar/gkv787

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    • Highly reproductive Escherichia coli cells with no specific assignment to the UAG codon Peer-reviewed

      Takahito Mukai, Hiroko Hoshi, Kazumasa Ohtake, Mihoko Takahashi, Atsushi Yamaguchi, Akiko Hayashi, Shigeyuki Yokoyama, Kensaku Sakamoto

      SCIENTIFIC REPORTS5   9699   5 2015

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      Language:English   Publishing type:Research paper (scientific journal)   Publisher:NATURE PUBLISHING GROUP  

      Escherichia coli is a widely used host organism for recombinant technology, and the bacterial incorporation of non-natural amino acids promises the efficient synthesis of proteins with novel structures and properties. In the present study, we developed E. coli strains in which the UAG codon was reserved for non-natural amino acids, without compromising the reproductive strength of the host cells. Ninety-five of the 273 UAG stop codons were replaced synonymously in the genome of E. coli BL21(DE3), by exploiting the oligonucleotide-mediated base-mismatch-repair mechanism. This genomic modification allowed the safe elimination of the UAG-recognizing cellular component (RF-1), thus leaving the remaining 178 UAG codons with no specific molecule recognizing them. The resulting strain B-95.Delta A grew as vigorously as BL21(DE3) in rich medium at 25-42 degrees C, and its derivative B-95.Delta A Delta fabR was better adapted to low temperatures and minimal media than B-95.Delta A. UAG was reassigned to synthetic amino acids by expressing the specific pairs of UAG-reading tRNA and aminoacyl-tRNA synthetase. Due to the preserved growth vigor, the B-95.Delta A strains showed superior productivities for hirudin molecules sulfonated on a particular tyrosine residue, and the Fab fragments of Herceptin containing multiple azido groups.

      DOI: 10.1038/srep09699

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    • Protein stabilization utilizing a redefined codon Peer-reviewed

      Kazumasa Ohtake, Atsushi Yamaguchi, Takahito Mukai, Hiroki Kashimura, Nobutaka Hirano, Mitsuru Haruki, Sosuke Kohashi, Kenji Yamagishi, Kazutaka Murayama, Yuri Tomabechi, Takashi Itagaki, Ryogo Akasaka, Masahito Kawazoe, Chie Takemoto, Mikako Shirouzu, Shigeyuki Yokoyama, Kensaku Sakamoto

      SCIENTIFIC REPORTS5   9762   5 2015

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      Language:English   Publishing type:Research paper (scientific journal)   Publisher:NATURE PUBLISHING GROUP  

      Recent advances have fundamentally changed the ways in which synthetic amino acids are incorporated into proteins, enabling their efficient and multiple-site incorporation, in addition to the 20 canonical amino acids. This development provides opportunities for fresh approaches toward addressing fundamental problems in bioengineering. In the present study, we showed that the structural stability of proteins can be enhanced by integrating bulky halogenated amino acids at multiple selected sites. Glutathione S-transferase was thus stabilized significantly (by 5.2 and 5.6 kcal/mol) with 3-chloro- and 3-bromo-L-tyrosines, respectively, incorporated at seven selected sites. X-ray crystallographic analyses revealed that the bulky halogen moieties filled internal spaces within the molecules, and formed non-canonical stabilizing interactions with the neighboring residues. This new mechanism for protein stabilization is quite simple and applicable to a wide range of proteins, as demonstrated by the rapid stabilization of the industrially relevant azoreductase.

      DOI: 10.1038/srep09762

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    • Multiple Site-Specific Installations of N-epsilon-Monomethyl-L-Lysine into Histone Proteins by Cell-Based and Cell-Free Protein Synthesis Invited Peer-reviewed

      Tatsuo Yanagisawa, Mihoko Takahashi, Takahito Mukai, Shin Sato, Masatoshi Wakamori, Mikako Shirouzu, Kensaku Sakamoto, Takashi Umehara, Shigeyuki Yokoyama

      CHEMBIOCHEM15 ( 12 ) 1830 - 1838   8 2014

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      Language:English   Publishing type:Research paper (scientific journal)   Publisher:WILEY-V C H VERLAG GMBH  

      Lysine methylation is one of the important post-translational modifications of histones, and produces an N-epsilon-mono-, di-, or trimethyllysine residues. Multiple and site-specific lysine methylations of histones are essential to define epigenetic statuses and control heterochromatin formation, DNA repair, and transcription regulation. A method was previously developed to build an analogue of N-epsilon-monomethyllysine, with cysteine substituting for lysine. Here, we have developed a new method of preparing histones bearing multiple N-epsilon-monomethyllysine residues at specified positions. Release factor 1-knockout (RFzero) Escherichia coli cells or a cell-free system based on the RFzero cell lysate was used for protein synthesis, as in RFzero cells UAG is redefined as a sense codon for non-canonical amino acids. During protein synthesis, a tert-butyloxycarbonyl- protected N-epsilon-monomethyllysine analogue is ligated to Methanosarcina mazei pyrrolysine tRNA (tRNAPyl) by M. mazei pyrrolysyl-tRNA synthetase mutants, and is translationally incorporated into one or more positions specified by the UAG codon. Protecting groups on the protein are then removed with trifluoroacetic acid to generate N-epsilon-monomethyllysine residues. We installed N-epsilon-monomethyllysine residues at positions 4, 9, 27, 36, and/or 79 of human histone H3. Each of the N-epsilon-monomethyllysine residues within the produced histone H3 was recognized by its specific antibody. Furthermore, the antibody recognized the authentic N-epsilon-monomethyllysine residue at position 27 better than the N-epsilon-monomethyllysine analogue built with cysteine. Mass spectrometry analyses also confirmed the lysine modifications on the produced histone H3. Thus, our method enables the installation of authentic N-epsilon-monomethyllysines at multiple positions within a protein for largescale production.

      DOI: 10.1002/cbic.201402291

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    • Efficient Decoding of the UAG Triplet as a Full-Fledged Sense Codon Enhances the Growth of a prfA-Deficient Strain of Escherichia coli Peer-reviewed

      Kazumasa Ohtake, Aya Sato, Takahito Mukai, Nobumasa Hino, Shigeyuki Yokoyama, Kensaku Sakamoto

      JOURNAL OF BACTERIOLOGY194 ( 10 ) 2606 - 2613   5 2012

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      Language:English   Publishing type:Research paper (scientific journal)   Publisher:AMER SOC MICROBIOLOGY  

      We previously reassigned the amber UAG stop triplet as a sense codon in Escherichia coli by expressing a UAG-decoding tRNA and knocking out the prfA gene, encoding release factor 1. UAG triplets were left at the ends of about 300 genes in the genome. In the present study, we showed that the detrimental effect of UAG reassignment could be alleviated by increasing the efficiency of UAG translation instead of reducing the number of UAGs in the genome. We isolated an amber suppressor tRNA(Gln) variant displaying enhanced suppression activity, and we introduced it into the prfA knockout strain, RFzero-q, in place of the original suppressor tRNA(Gln). The resulting strain, RFzero-q(3), translated UAG to glutamine almost as efficiently as the glutamine codons, and it proliferated faster than the parent RFzero-q strain. We identified two major factors in this growth enhancement. First, the sucB gene, which is involved in energy regeneration and has two successive UAG triplets at the end, was expressed at a higher level in RFzero-q3 than RFzero-q. Second, the ribosome stalling that occurred at UAG in RFzero-q was resolved in RFzero-q3. The results revealed the importance of "backup" stop triplets, UAA or UGA downstream of UAG, to avoid the deleterious impact of UAG reassignment on the proteome.

      DOI: 10.1128/JB.00195-12

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    • Wide-range protein photo-crosslinking achieved by a genetically encoded N-epsilon-(benzyloxycarbonyl)lysine derivative with a diazirinyl moiety Peer-reviewed

      Tatsuo Yanagisawa, Nobumasa Hino, Fumie Iraha, Takahito Mukai, Kensaku Sakamoto, Shigeyuki Yokoyama

      MOLECULAR BIOSYSTEMS8 ( 4 ) 1131 - 1135   2012

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      Language:English   Publishing type:Research paper (scientific journal)   Publisher:ROYAL SOC CHEMISTRY  

      A derivative of N-benzyloxycarbonyl-L-lysine with a photo-reactive diazirinyl group, N-epsilon-[((4-(3-( trifluoromethyl)-3H-diazirin-3-yl)-benzyl)oxy)carbonyl]-L-lysine, was site-specifically incorporated into target proteins in mammalian cells. The incorporated photo-crosslinker is able to react not only with residues as distant as about 15 A but also with those in closer proximity, thus enabling "wide-range'' photo-crosslinking of proteins.

      DOI: 10.1039/c2mb05321g

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    • Genetic-code evolution for protein synthesis with non-natural amino acids Peer-reviewed

      Takahito Mukai, Tatsuo Yanagisawa, Kazumasa Ohtake, Masatoshi Wakamori, Jiro Adachi, Nobumasa Hino, Aya Sato, Takatsugu Kobayashi, Akiko Hayashi, Mikako Shirouzu, Takashi Umehara, Shigeyuki Yokoyama, Kensaku Sakamoto

      BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS411 ( 4 ) 757 - 761   8 2011

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      Language:English   Publishing type:Research paper (scientific journal)   Publisher:ACADEMIC PRESS INC ELSEVIER SCIENCE  

      The genetic encoding of synthetic or "non-natural" amino acids promises to diversify the functions and structures of proteins. We applied rapid codon-reassignment for creating Escherichia coli strains unable to terminate translation at the UAG "stop" triplet, but efficiently decoding it as various tyrosine and lysine derivatives. This complete change in the UAG meaning enabled protein synthesis with these non-natural molecules at multiple defined sites, in addition to the 20 canonical amino acids. UAG was also redefined in the E. coli BL21 strain, suitable for the large-scale production of recombinant proteins, and its cell extract served the cell-free synthesis of an epigenetic protein, histone H4, fully acetylated at four specific lysine sites. (C) 2011 Elsevier Inc. All rights reserved.

      DOI: 10.1016/j.bbrc.2011.07.020

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    • (doctoral thesis) A study on the genetic-code flexibility by manipulation of the UAG codon-decoding molecules. Peer-reviewed

      Takahito Mukai

      The University of Tokyo   3 2011

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    • Genetic Incorporation of a Photo-Crosslinkable Amino Acid Reveals Novel Protein Complexes with GRB2 in Mammalian Cells Peer-reviewed

      Nobumasa Hino, Masaaki Oyama, Aya Sato, Takahito Mukai, Fumie Iraha, Akiko Hayashi, Hiroko Kozuka-Hata, Tadashi Yamamoto, Shigeyuki Yokoyama, Kensaku Sakamoto

      JOURNAL OF MOLECULAR BIOLOGY406 ( 2 ) 343 - 353   2 2011

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      Language:English   Publishing type:Research paper (scientific journal)   Publisher:ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD  

      Cell signaling pathways are essentially organized through the distribution of various types of binding domains in signaling proteins, with each domain binding to specific target molecules. Although identification of these targets is crucial for mapping the pathways, affinity-based or copurification methods are insufficient to distinguish between direct and indirect interactions in a cellular context. In the present study, we developed another approach involving the genetic encoding of a photo-crosslinkable amino acid. p-Trifluoromethyl-diazirinyl-L-phenylalanine was thus incorporated at a defined site in the Src homology 2 (SH2) domain of the adaptor protein GRB2 in human embryonic kidney cells. These cells were exposed to 365-nm light after an epidermal growth factor stimulus, and the crosslinkable GRB2-SH2 domain exclusively formed covalent bonds with directly interacting proteins. Proteomic mass spectrometry analysis identified these direct binders of GRB2-SH2 separately from the proteins noncovalently bound to the Src homology 3 domains of GRB2. In addition to two signaling-associated proteins (GIT1 and AF6), the heterogeneous nuclear ribonucleoproteins F, H1, and H2 were thus identified as novel direct binders. The results revealed a connection between the cell signaling protein and the nuclear machinery involved in mRNA processing, and demonstrated the usefulness of genetically encoded photo-crosslinkers for mapping protein protein interactions in cells. (C) 2010 Elsevier Ltd. All rights reserved.

      DOI: 10.1016/j.jmb.2010.12.022

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    • Codon reassignment in the Escherichia coli genetic code Peer-reviewed

      Takahito Mukai, Akiko Hayashi, Fumie Iraha, Aya Sato, Kazumasa Ohtake, Shigeyuki Yokoyama, Kensaku Sakamoto

      NUCLEIC ACIDS RESEARCH38 ( 22 ) 8188 - 8195   12 2010

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      Language:English   Publishing type:Research paper (scientific journal)   Publisher:OXFORD UNIV PRESS  

      Most organisms, from Escherichia coli to humans, use the &apos;universal&apos; genetic code, which have been unchanged or &apos;frozen&apos; for billions of years. It has been argued that codon reassignment causes mistranslation of genetic information, and must be lethal. In this study, we successfully reassigned the UAG triplet from a stop to a sense codon in the E. coli genome, by eliminating the UAG-recognizing release factor, an essential cellular component, from the bacterium. Only a few genetic modifications of E. coli were needed to circumvent the lethality of codon reassignment; erasing all UAG triplets from the genome was unnecessary. Thus, UAG was assigned unambiguously to a natural or non-natural amino acid, according to the specificity of the UAG-decoding tRNA. The result reveals the unexpected flexibility of the genetic code.

      DOI: 10.1093/nar/gkq707

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    • Genetic encoding of non-natural amino acids in Drosophila melanogaster Schneider 2 cells Peer-reviewed

      Takahito Mukai, Motoaki Wakiyama, Kensaku Sakamoto, Shigeyuki Yokoyama

      PROTEIN SCIENCE19 ( 3 ) 440 - 448   3 2010

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      Language:English   Publishing type:Research paper (scientific journal)   Publisher:JOHN WILEY & SONS INC  

      Insect cells are useful for the high-yield production of recombinant proteins including chemokines and membrane proteins. In this study, we developed an insect cell-based system for incorporating non-natural amino acids into proteins at specific sites. Three types of promoter systems were constructed, and their efficiencies were compared for the expression of the prokaryotic amber suppressor tRNA(Tyr) in Drosophila melanogaster Schneider 2 cells. When paired with a variant of Escherichia coli tyrosyl-tRNA synthetase specific for 3-iodo-L-tyrosine, the suppressor tRNA transcribed from the U6 promoter most efficiently incorporated the amino acid into proteins in the cells. The transient and stable introductions of these prokaryotic molecules into the insect cells were then compared in terms of the yield of proteins containing non-natural amino acids, and the "transient" method generated a sevenfold higher yield. By this method, 4-azido-L-phenylalanine was incorporated into human interleukin-8 at a specific site. The yield of the azido-containing IL-8 was 1 mu g/1 mL cell culture, and the recombinant protein was successfully labeled with a fluorescent probe by the Staudinger-Bertozzi reaction.

      DOI: 10.1002/pro.322

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    • Adding L-lysine derivatives to the genetic code of mammalian cells with engineered pyrrolysyl-tRNA synthetases Peer-reviewed

      Takahito Mukai, Takatsugu Kobayashi, Nobumasa Hino, Tatsuo Yanagisawa, Kensaku Sakamoto, Shigeyuki Yokoyama

      BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS371 ( 4 ) 818 - 822   7 2008

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      Language:English   Publishing type:Research paper (scientific journal)   Publisher:ACADEMIC PRESS INC ELSEVIER SCIENCE  

      We report a method for site-specifically incorporating L-lysine derivatives into proteins in mammalian cells, based on the expression of the pyrrolysyl-tRNA synthetase (PyIRS)-tRNA(PYl) pair from Methanosarcino mazei. Different types of external promoters were tested for the expression of tRNA(PYl) in Chinese hamster ovary cells. When tRNA(PYl) was expressed from a gene cluster under the control of the U6 promoter, the wild-type PylRS-tRNA(PYl) pair facilitated the most efficient incorporation of a pyrrolysine analog, N-epsilon-tert-butyloxycarbonyl-L-lysine (Boc-lysine), into proteins at the amber position. This PyIRS-tRNA(PYl) system yielded the Boc-lysine-containing protein in an amount accounting for 1% of the total protein in human embryonic kidney (HEK) 293 cells. We also created a PyIRS variant specific to N-epsilon-benzyloxycarbonyl-L-lysine, to incorporate this long, bulky, non-natural lysine derivative into proteins in HEK293. The recently reported variant specific to N-epsilon-acetyllysine was also expressed, resulting in the genetic encoding of this naturally-occurring lysine modification in mammalian cells. (c) 2008 Elsevier Inc. All rights reserved.

      DOI: 10.1016/j.bbrc.2008.04.164

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    Presentations

    • シンプル ゲノム合成 Invited

      向井崇人

      第17回日本ゲノム微生物学会年会  9 3 2023 

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    • Designing the central dogma system of zombie cells Invited

      Mukai Takahito

      2 12 2022 

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      Language:English   Presentation type:Symposium, workshop panel (nominated)  

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    • Chromosome shuttling between cell-free systems and E. coli cells Invited

      Takahito Mukai

      The 3rd International BioDesign Research Conference  27 11 2022 

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    • ゲノムのミライ 作れる細胞の設計

      向井 崇人

      第16回日本ゲノム微生物学会年会  2 3 2022 

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      Presentation type:Oral presentation (general)  

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    • 大腸菌ゲノムの分断化とポータブル化

      向井崇人, 末次正幸

      第17回21世紀大腸菌研究会  20 8 2021 

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    • 大腸菌を用いた1 Mbプラスミドの構築と接合伝達

      向井崇人, 米司達哉, 末次正幸, 藤田裕寛

      第14回日本ゲノム微生物学会年会  8 3 2020  日本ゲノム微生物学会

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      Language:Japanese   Presentation type:Oral presentation (general)  

      Venue:名古屋  

      新型コロナウイルス感染症対策の基本方針に基づき、年会を中止し、発表が行われたと認定

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    • 地球規模メタゲノムデータの解析 SepRS、どこ行った? Invited

      向井 崇人

      第18回微生物研究会「微生物研究の新しい潮流」  9 11 2019 

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      Language:Japanese  

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    • Megabase cloning by utilizing E. coli chromosome vectors Invited

      Takahito Mukai

      ICG-14  26 10 2019 

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      Language:English   Presentation type:Oral presentation (invited, special)  

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    • Finding new genetic code deviations in the metagenomic era Invited

      Takahito Mukai

      mbsj2018  28 11 2018 

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      Language:Japanese   Presentation type:Oral presentation (invited, special)  

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    • A new chassis tRNA for genetic code expansion? International conference

      Takahito Mukai, Dieter Söll

      AARS2017  30 10 2017 

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      Language:English   Presentation type:Oral presentation (general)  

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    • A rare codon can be captured by a non-canonical amino acid. International conference

      Takahito Mukai, Kensaku Sakamoto

      The 25th tRNA Conference  9 2014 

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      Language:English   Presentation type:Oral presentation (general)  

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    • 細胞の遺伝暗号改変と、これからの課題 Invited

      向井崇人, 大竹和正, 坂本健作

      「細胞を創る」研究会5.0  11 2012 

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    Teaching Experience

    • 5 2020 - 7 2020 
      seimerigakuseminar2 ( Rikkyo University )

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      生物化学2 ( 立教大学 )

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    Professional Memberships

    • 1 2020 - Present 
      日本ゲノム微生物学会

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      「細胞を創る」研究会

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    Research Projects

    • ゾンビ細胞を用いたゲノム入れ替え法の開発

      日本学術振興会  科学研究費助成事業 基盤研究(C) 

      向井 崇人

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      4 2023 - 3 2026

      Grant number:23K05743

      Grant amount:\4680000 ( Direct Cost: \3600000 、 Indirect Cost:\1080000 )

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    • 立体構造タグを用いたtRNA分子群の操作法の開発

      科研費  研究活動スタート支援 

      向井崇人

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      8 2018 - 3 2020

      Authorship:Principal investigator  Grant type:Competitive

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    • セレノシステイン/システイン遺伝暗号の全様解明

      日本学術振興会  海外特別研究員 

      向井崇人

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      4 2016 - 3 2018

      Authorship:Principal investigator  Grant type:Competitive

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    • 非天然型アミノ酸を用いて酵素活性をデザインするための基幹技術開発

      理化学研究所  基礎科学特別研究員 

      向井崇人

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      4 2012 - 5 2014

      Authorship:Principal investigator  Grant type:Competitive

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    • Sense Codon Reassignment

      JSPS  Grants-in-Aid for Young Scientists (B) 

      Takahito Mukai

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      4 2012 - 3 2014

      Authorship:Principal investigator  Grant type:Competitive

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    • 哺乳類細胞の遺伝暗号拡張によるタンパク質翻訳後修飾の細胞機能解明

      特別研究員奨励費(DC1) 

      向井崇人

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      4 2008 - 3 2011

      Authorship:Principal investigator  Grant type:Competitive

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    Industrial property rights

    • COMPOSITIONS AND METHODS FOR MAKING SELENOCYSTEINE CONTAINING POLYPEPTIDES

      Dieter Söll, MUKAI, Takahito, HOFFMAN, Kyle

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      Application no:PCT/US2018/054437  Date applied:4 10 2018

      Announcement no:特開WO/2019/071023  Date announced:11 4 2019

      Patent/Registration no:特許US11788111B2  Date registered:17 10 2023 

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    • 機能性DNAカセット及びプラスミド

      末次 正幸, 向井 崇人

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      Application no:特願2022-006523  Date applied:19 1 2022

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    • COMPOSITIONS AND METHODS FOR MAKING SELENOCYSTEINE CONTAINING POLYPEPTIDES

      Dieter Söll, Caroline Aldag, Michael Hohn, Takahito Mukai

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      Application no:特願15/724678  Date applied:4 10 2017

      Announcement no:特開20180105854  Date announced:19 4 2018

      Patent/Registration no:特許US-10876142-B2  Date registered:29 12 2020 

      Rights holder:Yale University

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    • 非天然タンパク質製造用の組換え細菌の作製方法、及びその利用

      横山 茂之, 向井 崇人, 坂本 健作, 松元 明子

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      Applicant:国立研究開発法人理化学研究所

      Application no:特願2012-520488  Date applied:16 6 2011

      Patent/Registration no:特許第5858543号  Date issued:25 12 2015

      J-GLOBAL

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    • アミノアシルtRNA合成酵素活性を有するポリペプチド及びその利用

      横山 茂之, 坂本 健作, 大木 健二, 向井 崇人

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      Applicant:独立行政法人理化学研究所

      Application no:特願2009-024136  Date applied:4 2 2009

      Announcement no:特開2009-207490  Date announced:17 9 2009

      Patent/Registration no:特許第5585904号  Date issued:1 8 2014

      J-GLOBAL

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    • エステル結合を含む非天然タンパク質の製造方法

      横山 茂之, 坂本 健作, 柳沢 達男, 向井 崇人, 小林 隆嗣

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      Applicant:独立行政法人理化学研究所

      Application no:特願2009-542600  Date applied:21 11 2008

      Patent/Registration no:特許第5419220号  Date issued:29 11 2013

      J-GLOBAL

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    • サプレッサーtRNAの合成方法、DNA構築物及びそれを用いた非天然型アミノ酸組み込みタンパク質の製造

      横山 茂之, 坂本 健作, 樋野 展正, 向井 崇人, 小林 隆嗣

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      Applicant:独立行政法人理化学研究所

      Application no:特願2008-502740  Date applied:22 2 2007

      Patent/Registration no:特許第5196378号  Date issued:15 2 2013

      J-GLOBAL

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    • METHOD FOR SYNTHESIS OF SUPPRESSOR tRNA, DNA CONSTRUCT, AND PRODUCTION OF PROTEIN HAVING NON-NATURAL AMINO ACID INTEGRATED THEREIN BY USING THE DNA CONSTRUCT

      Shigeyuki Yokoyama, Kensaku Sakamoto, Nobumasa Hino, Takahito Mukai, Takatsugu Kobayashi

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      Patent/Registration no:特許EP1992698B1  Date issued:5 9 2018

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    • Process for production of non-natural protein having ester bond therein

      Shigeyuki Yokoyama, Kensaku Sakamoto, Tatsuo Yanagisawa, Takahito Mukai, Takatsugu Kobayashi

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      Patent/Registration no:特許US8785152B2  Date issued:22 7 2014

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    • Process for production of non-natural protein having ester bond therein

      Shigeyuki Yokoyama, Kensaku Sakamoto, Tatsuo Yanagisawa, Takahito Mukai, Takatsugu Kobayashi

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      Patent/Registration no:特許EP2221370B1  Date issued:16 4 2014

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    • Polypeptide having activity of aminoacyl-tRNA synthetase and use thereof

      Shigeyuki Yokoyama, Kensaku Sakamoto, Kenji Oki, Takahito Mukai

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      Patent/Registration no:特許US8293512B2  Date issued:23 10 2012

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    • Method for constructing recombinant bacterium for producing non-native protein, and utilization of same

      Shigeyuki Yokoyama, Takahito Mukai, Kensaku Sakamoto, Akiko Matsumoto

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      Patent/Registration no:特許US9340790B2  Date issued:17 5 2016

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    • Method for constructing recombinant bacterium for producing non-native protein, and utilization of same

      Shigeyuki Yokoyama, Takahito Mukai, Kensaku Sakamoto, Akiko Matsumoto

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      Patent/Registration no:特許EP2584037B1  Date issued:10 8 2016

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    • Method of synthesizing a suppressor tRNA, DNA construct and use thereof for producing a non-natural amino acid-incorporated protein

      Shigeyuki Yokoyama, Kensaku Sakamoto, Nobumasa Hino, Takahito Mukai, Takatsugu Kobayashi

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      Patent/Registration no:特許US8822180B2  Date issued:2 9 2014

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    • Method of synthesizing a suppressor TRNA, DNA construct and use thereof for producing a protein including a non-natural amino acid

      Shigeyuki Yokoyama, Kensaku Sakamoto, Nobumasa Hino, Takahito Mukai, Takatsugu Kobayashi

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      Patent/Registration no:特許US8168407B2  Date issued:1 5 2012

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    Media Coverage

    • 3分割したゲノムからなる大腸菌を作製、自由なゲノム出し入れを実現 ~モデル生物でのゲノムインストール技術~

      JST & 立教大学  4 2021

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    • Proving the Genetic Code’s Flexibility

      3 2016

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    • DNA情報の変換ルールを人為的に改変 -多様なアミノ酸を高い効率でタンパク質に導入することが可能に-

      理化学研究所  5 2015

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