Researcher Profile and Settings

Affiliation

• Research Faculty of Agriculture　Fundamental AgriScience Research　Bioscience and Chemistry

Job Title

• Assistant Professor

Degree

• Ph.D.（Tokyo Institute of Technology）

J-Global ID

• 201801003191000738

Research Interests

• Antibiotic resistance   Protoplast fusion   Metabolic engineering   RNase   Escherichia coli   Corynebacterium glutamicum   Laboratory evolution   

Research Areas

• Life sciences / Bacteriology
• Life sciences / Evolutionary biology
• Life sciences / Applied microbiology
• Life sciences / Systems genomics

Educational Organization

•  Bachelor's degree program　School of Agriculture
•  Master's degree program　Graduate School of Agriculture
•  Doctoral (PhD) degree program　Graduate School of Agriculture

Academic & Professional Experience

• 2021/02 - Today Hokkaido University Graduate School of Agriculture Research Faculty of Agriculture Assistant professor
• 2021 - Today RIKEN Center for Biosystems Dynamics Research Visiting Researcher
• 2018/04 - 2021/01 RIKEN Center for Biosystems Dynamics Research Special Postdoctoral Researcher
• 2016/01 - 2017/03 RIKEN Quantitative Biology Center Postdoctoral researcher
• 2013/09 - 2015/12 Research Institute of Innovative Technology for the Earth Molecular Microbiology and Biotechnology Group Researcher
• 2012/07 - 2013/08 Forschungszentrum Jülich Institute of Bio-and Geosciences, Biotechnology IBG-1 Postdoctoral researcher
• 2012/04 - 2012/06 Tokyo Institute of Technology Productive Leader Incubation Platform (PLIP) Postdoctoral researcher

Education

• 2008/04 - 2012/03  Tokyo Institute of Technology, Graduate School  Graduate School of Bioscience and Biotechnology  Department of Bioengineering
• 2009/09 - 2010/03  University of Cologne  Institute for Biochemistry
• 2004/04 - 2008/03  Tokyo Institute of Technology  School of Bioscience and Biotechnology  Department of Bioengineering

Association Memberships

• Society of Genome Microbiology, Japan   JAPAN SOCIETY FOR BIOSCIENCE, BIOTECHNOLOGY, AND AGROCHEMISTRY   The Society for Biotechnology, Japan   

Research Activities

Published Papers

• Laboratory Evolution of Antimicrobial Resistance in Bacteria to Develop Rational Treatment Strategies

  Tomoya Maeda, Chikara Furusawa

  Antibiotics 13 (1) 94 - 94 2024/01/18 [Refereed][Invited]
   

  Laboratory evolution studies, particularly with Escherichia coli, have yielded invaluable insights into the mechanisms of antimicrobial resistance (AMR). Recent investigations have illuminated that, with repetitive antibiotic exposures, bacterial populations will adapt and eventually become tolerant and resistant to the drugs. Through intensive analyses, these inquiries have unveiled instances of convergent evolution across diverse antibiotics, the pleiotropic effects of resistance mutations, and the role played by loss-of-function mutations in the evolutionary landscape. Moreover, a quantitative analysis of multidrug combinations has shed light on collateral sensitivity, revealing specific drug combinations capable of suppressing the acquisition of resistance. This review article introduces the methodologies employed in the laboratory evolution of AMR in bacteria and presents recent discoveries concerning AMR mechanisms derived from laboratory evolution. Additionally, the review outlines the application of laboratory evolution in endeavors to formulate rational treatment strategies.
• Inference of Fitness Landscape Based on Multi-Dimensional Phenotype Measurements of <i>Escherichia coli</i>

  Chikara FURUSAWA, Junichiro IWASAWA, Tomoya MAEDA

  Seibutsu Butsuri 63 (5) 263 - 265 0582-4052 2023
• Adaptive Laboratory Evolution of Microorganisms: Methodology and Application for Bioproduction.

  Takashi Hirasawa, Tomoya Maeda

  Microorganisms 11 (1) 2022/12/29 [Refereed][Invited]
   

  Adaptive laboratory evolution (ALE) is a useful experimental methodology for fundamental scientific research and industrial applications to create microbial cell factories. By using ALE, cells are adapted to the environment that researchers set based on their objectives through the serial transfer of cell populations in batch cultivations or continuous cultures and the fitness of the cells (i.e., cell growth) under such an environment increases. Then, omics analyses of the evolved mutants, including genome sequencing, transcriptome, proteome and metabolome analyses, are performed. It is expected that researchers can understand the evolutionary adaptation processes, and for industrial applications, researchers can create useful microorganisms that exhibit increased carbon source availability, stress tolerance, and production of target compounds based on omics analysis data. In this review article, the methodologies for ALE in microorganisms are introduced. Moreover, the application of ALE for the creation of useful microorganisms as cell factories has also been introduced.
• Analysis of the evolution of resistance to multiple antibiotics enables prediction of the Escherichia coli phenotype-based fitness landscape.

  Junichiro Iwasawa, Tomoya Maeda, Atsushi Shibai, Hazuki Kotani, Masako Kawada, Chikara Furusawa

  PLoS biology 20 (12) e3001920  2022/12 [Refereed]
   

  The fitness landscape represents the complex relationship between genotype or phenotype and fitness under a given environment, the structure of which allows the explanation and prediction of evolutionary trajectories. Although previous studies have constructed fitness landscapes by comprehensively studying the mutations in specific genes, the high dimensionality of genotypic changes prevents us from developing a fitness landscape capable of predicting evolution for the whole cell. Herein, we address this problem by inferring the phenotype-based fitness landscape for antibiotic resistance evolution by quantifying the multidimensional phenotypic changes, i.e., time-series data of resistance for eight different drugs. We show that different peaks of the landscape correspond to different drug resistance mechanisms, thus supporting the validity of the inferred phenotype-fitness landscape. We further discuss how inferred phenotype-fitness landscapes could contribute to the prediction and control of evolution. This approach bridges the gap between phenotypic/genotypic changes and fitness while contributing to a better understanding of drug resistance evolution.
• A manually curated compendium of expression profiles for the microbial cell factory Corynebacterium glutamicum.

  Angela Kranz, Tino Polen, Christian Kotulla, Annette Arndt, Graziella Bosco, Michael Bussmann, Ava Chattopadhyay, Annette Cramer, Cedric-Farhad Davoudi, Ursula Degner, Ramon Diesveld, Raphael Freiherr von Boeselager, Kim Gärtner, Cornelia Gätgens, Tobias Georgi, Christian Geraths, Sabine Haas, Antonia Heyer, Max Hünnefeld, Takeru Ishige, Armin Kabus, Nicolai Kallscheuer, Larissa Kever, Simon Klaffl, Britta Kleine, Martina Kočan, Abigail Koch-Koerfges, Kim J Kraxner, Andreas Krug, Aileen Krüger, Andreas Küberl, Mohamed Labib, Christian Lange, Christina Mack, Tomoya Maeda, Regina Mahr, Stephan Majda, Andrea Michel, Xenia Morosov, Olga Müller, Arun M Nanda, Jens Nickel, Jennifer Pahlke, Eugen Pfeifer, Laura Platzen, Paul Ramp, Doris Rittmann, Steffen Schaffer, Sandra Scheele, Stephanie Spelberg, Julia Schulte, Jens-Eric Schweitzer, Georg Sindelar, Ulrike Sorger-Herrmann, Markus Spelberg, Corinna Stansen, Apilaasha Tharmasothirajan, Jan van Ooyen, Philana van Summeren-Wesenhagen, Michael Vogt, Sabrina Witthoff, Lingfeng Zhu, Bernhard J Eikmanns, Marco Oldiges, Georg Schaumann, Meike Baumgart, Melanie Brocker, Lothar Eggeling, Roland Freudl, Julia Frunzke, Jan Marienhagen, Volker F Wendisch, Michael Bott

  Scientific data 9 (1) 594 - 594 2022/10/01 [Refereed]
   

  Corynebacterium glutamicum is the major host for the industrial production of amino acids and has become one of the best studied model organisms in microbial biotechnology. Rational strain construction has led to an improvement of producer strains and to a variety of novel producer strains with a broad substrate and product spectrum. A key factor for the success of these approaches is detailed knowledge of transcriptional regulation in C. glutamicum. Here, we present a large compendium of 927 manually curated microarray-based transcriptional profiles for wild-type and engineered strains detecting genome-wide expression changes of the 3,047 annotated genes in response to various environmental conditions or in response to genetic modifications. The replicates within the 927 experiments were combined to 304 microarray sets ordered into six categories that were used for differential gene expression analysis. Hierarchical clustering confirmed that no outliers were present in the sets. The compendium provides a valuable resource for future fundamental and applied research with C. glutamicum and contributes to a systemic understanding of this microbial cell factory. Measurement(s) Gene Expression Analysis Technology Type(s) Two Color Microarray Factor Type(s) WT condition A vs. WT condition B • Plasmid-based gene overexpression in parental strain vs. parental strain with empty vector control • Deletion mutant vs. parental strain Sample Characteristic - Organism Corynebacterium glutamicum Sample Characteristic - Environment laboratory environment Sample Characteristic - Location Germany.
• Multi-dimensional antibiotic resistance time series reveals the phenotype-based fitness landscape for Escherichia coli

  Junichiro Iwasawa, Tomoya Maeda, Atsushi Shibai, Hazuki Kotani, Masako Kawada, Chikara Furusawa

  2022/08/24
• Acceleration of target production in co-culture by enhancing intermediate consumption through adaptive laboratory evolution.

  Ryutaro Kawai, Yoshihiro Toya, Kenta Miyoshi, Manami Murakami, Teppei Niide, Takaaki Horinouchi, Tomoya Maeda, Atsushi Shibai, Chikara Furusawa, Hiroshi Shimizu

  Biotechnology and bioengineering 119 (3) 936 - 945 2021/12/16 [Refereed]
   

  Co-culture is a promising way to alleviate metabolic burden by dividing the metabolic pathways into several modules and sharing the conversion processes with multiple strains. Since an intermediate is passed from the donor to the recipient via the extracellular environment, it is inevitably diluted. Therefore, enhancing the intermediate consumption rate is important for increasing target productivity. In the present study, we demonstrated the enhancement of mevalonate consumption in Escherichia coli by adaptive laboratory evolution and applied the evolved strain to isoprenol production in an E. coli (upstream: glucose to mevalonate)-E. coli (downstream: mevalonate to isoprenol) co-culture. An engineered mevalonate auxotroph strain was repeatedly sub-cultured in a synthetic medium supplemented with mevalonate, where the mevalonate concentration was decreased stepwise from 100 to 20 µM. In five parallel evolution experiments, all growth rates gradually increased, resulting in five evolved strains. Whole-genome re-sequencing and reverse engineering identified three mutations involved in enhancing mevalonate consumption. After introducing nudF gene for producing isoprenol, the isoprenol-producing parental and evolved strains were respectively co-cultured with a mevalonate-producing strain. At an inoculation ratio of 1:3 (upstream:downstream), isoprenol production using the evolved strain was 3.3 times higher than that using the parental strain.
• Laboratory evolution of Mycobacterium on agar plates for analysis of resistance acquisition and drug sensitivity profiles.

  Tomoya Maeda, Masako Kawada, Natsue Sakata, Hazuki Kotani, Chikara Furusawa

  Scientific reports 11 (1) 15136 - 15136 2021/07/23 [Refereed]
   

  Drug-resistant tuberculosis (TB) is a growing public health problem. There is an urgent need for information regarding cross-resistance and collateral sensitivity relationships among drugs and the genetic determinants of anti-TB drug resistance for developing strategies to suppress the emergence of drug-resistant pathogens. To identify mutations that confer resistance to anti-TB drugs in Mycobacterium species, we performed the laboratory evolution of nonpathogenic Mycobacterium smegmatis, which is closely related to Mycobacterium tuberculosis, against ten anti-TB drugs. Next, we performed whole-genome sequencing and quantified the resistance profiles of each drug-resistant strain against 24 drugs. We identified the genes with novel meropenem (MP) and linezolid (LZD) resistance-conferring mutation, which also have orthologs, in M. tuberculosis H37Rv. Among the 240 possible drug combinations, we identified 24 pairs that confer cross-resistance and 18 pairs that confer collateral sensitivity. The acquisition of bedaquiline or linezolid resistance resulted in collateral sensitivity to several drugs, while the acquisition of MP resistance led to multidrug resistance. The MP-evolved strains showed cross-resistance to rifampicin and clarithromycin owing to the acquisition of a mutation in the intergenic region of the Rv2864c ortholog, which encodes a penicillin-binding protein, at an early stage. These results provide a new insight to tackle drug-resistant TB.
• Mutational property of newly identified mutagen l-glutamic acid γ-hydrazide in Escherichia coli.

  Tomoya Maeda, Atsushi Shibai, Naomi Yokoi, Yumeko Tarusawa, Masako Kawada, Hazuki Kotani, Chikara Furusawa

  Mutation research 823 111759 - 111759 2021/07/21 [Refereed]
   

  We previously found that an l-glutamine analog l-glutamic acid γ-hydrazide has high mutagenic activity through the high-throughput laboratory evolution of Escherichia coli. In this study, mutagenicity and mutational property of l-glutamic acid γ-hydrazide were examined by the Ames test and mutation accumulation experiments using E. coli. The Ames test revealed that l-glutamic acid γ-hydrazide showed higher mutagenic activity without metabolic activation than known mutagens 2-aminoanthracene, and cobalt(II) acetate tetrahydrate. This result indicates that l-glutamic acid γ-hydrazide does not require metabolic activation for mutagenic activity in E. coli. Mutation accumulation experiments and whole-genome sequencing analysis revealed the number and spectrum of the accumulated mutations with or without l-glutamic acid γ-hydrazide. In the presence of l-glutamic acid γ-hydrazide, MDS42 strain accumulated 392.3 ± 116.2 point mutations during 30 passages corresponding to 777 generations, while MDS42 strain accumulated 1.5 ± 2.5 point mutations without l-glutamic acid γ-hydrazide during 50 passages corresponding to 1341 generations. The mutational spectrum of l-glutamic acid γ-hydrazide was G/C to A/T transition (82.2 ± 4.3 %) and A/T to G/C transition (17.4 ± 4.3 %). These results indicated that l-glutamic acid γ-hydrazide has a strong mutagenic activity.
• Morphological change of coiled bacterium Spirosoma linguale with acquisition of β-lactam resistance.

  Tomoya Maeda, Hazuki Kotani, Chikara Furusawa

  Scientific reports 11 (1) 13278 - 13278 2021/06/24 [Refereed]
   

  Spirosoma linguale is a gram-negative, coiled bacterium belonging to the family Cytophagaceae. Its coiled morphology is unique in contrast to closely related bacteria belonging to the genus Spirosoma, which have a short, rod-shaped morphology. The mechanisms that generate unique cell morphology are still enigmatic. In this study, using the Spirosoma linguale ATCC33905 strain, we isolated β-lactam (cefoperazone and amoxicillin)-resistant clones. These clones showed two different cell morphological changes: relatively loosely curved cells or small, horseshoe-shaped cells. Whole-genome resequencing analysis revealed the genetic determinants of β-lactam resistance and changes in cell morphology. The loose-curved clones commonly had mutations in Slin_5958 genes encoding glutamyl-tRNA amidotransferase B subunit, whereas the small, horseshoe-shaped clones commonly had mutations in either Slin_5165 or Slin_5509 encoding pyruvate dehydrogenase (PDH) components. Two clones, CFP1ESL11 and CFL5ESL4, which carried only one mutation in Slin_5958, showed almost perfectly straight, rod-shaped cells in the presence of amoxicillin. This result suggests that penicillin-binding proteins targeted by amoxicillin play an important role in the formation of a coiled morphology in this bacterium. In contrast, supplementation with acetate did not rescue the growth defect and abnormal cell size of the CFP5ESL9 strain, which carried only one mutation in Slin_5509. These results suggest that PDH is involved in cell-size maintenance in this bacterium.
• Relevance of NADH Dehydrogenase and Alternative Two-Enzyme Systems for Growth of Corynebacterium glutamicum With Glucose, Lactate, and Acetate

  Tomoya Maeda, Abigail Koch-Koerfges, Michael Bott

  Frontiers in Bioengineering and Biotechnology 8 2021/01/20 [Refereed]
   

  The oxidation of NADH with the concomitant reduction of a quinone is a crucial step in the metabolism of respiring cells. In this study, we analyzed the relevance of three different NADH oxidation systems in the actinobacterial model organism Corynebacterium glutamicum by characterizing defined mutants lacking the non-proton-pumping NADH dehydrogenase Ndh (Δndh) and/or one of the alternative NADH-oxidizing enzymes, L-lactate dehydrogenase LdhA (ΔldhA) and malate dehydrogenase Mdh (Δmdh). Together with the menaquinone-dependent L-lactate dehydrogenase LldD and malate:quinone oxidoreductase Mqo, the LdhA-LldD and Mdh-Mqo couples can functionally replace Ndh activity. In glucose minimal medium the Δndh mutant, but not the ΔldhA and Δmdh strains, showed reduced growth and a lowered NAD⁺/NADH ratio, in line with Ndh being the major enzyme for NADH oxidation. Growth of the double mutants ΔndhΔmdh and ΔndhΔldhA, but not of strain ΔmdhΔldhA, in glucose medium was stronger impaired than that of the Δndh mutant, supporting an active role of the alternative Mdh-Mqo and LdhA-LldD systems in NADH oxidation and menaquinone reduction. In L-lactate minimal medium the Δndh mutant grew better than the wild type, probably due to a higher activity of the menaquinone-dependent L-lactate dehydrogenase LldD. The ΔndhΔmdh mutant failed to grow in L-lactate medium and acetate medium. Growth with L-lactate could be restored by additional deletion of sugR, suggesting that ldhA repression by the transcriptional regulator SugR prevented growth on L-lactate medium. Attempts to construct a ΔndhΔmdhΔldhA triple mutant were not successful, suggesting that Ndh, Mdh and LdhA cannot be replaced by other NADH-oxidizing enzymes in C. glutamicum.
• High-throughput laboratory evolution reveals evolutionary constraints in Escherichia coli

  Tomoya Maeda, Junichiro Iwasawa, Hazuki Kotani, Natsue Sakata, Masako Kawada, Takaaki Horinouchi, Aki Sakai, Kumi Tanabe, Chikara Furusawa

  Nature Communications 11 (1) 5970 - 5970 2020/11 [Refereed]
   

  Understanding the constraints that shape the evolution of antibiotic resistance is critical for predicting and controlling drug resistance. Despite its importance, however, a systematic investigation of evolutionary constraints is lacking. Here, we perform a high-throughput laboratory evolution of Escherichia coli under the addition of 95 antibacterial chemicals and quantified the transcriptome, resistance, and genomic profiles for the evolved strains. Utilizing machine learning techniques, we analyze the phenotype–genotype data and identified low dimensional phenotypic states among the evolved strains. Further analysis reveals the underlying biological processes responsible for these distinct states, leading to the identification of trade-off relationships associated with drug resistance. We also report a decelerated evolution of β-lactam resistance, a phenomenon experienced by certain strains under various stresses resulting in higher acquired resistance to β-lactams compared to strains directly selected by β-lactams. These findings bridge the genotypic, gene expression, and drug resistance gap, while contributing to a better understanding of evolutionary constraints for antibiotic resistance.
• Suppression of antibiotic resistance evolution by single-gene deletion.

  Takaaki Horinouchi, Tomoya Maeda, Hazuki Kotani, Chikara Furusawa

  Scientific reports 10 (1) 4178 - 4178 2020/03/06 [Refereed][Not invited]
   

  Antibiotic treatment generally results in the selection of resistant bacterial strains, and the dynamics of resistance evolution is dependent on complex interactions between cellular components. To better characterize the mechanisms of antibiotic resistance and evaluate its dependence on gene regulatory networks, we performed systematic laboratory evolution of Escherichia coli strains with single-gene deletions of 173 transcription factors under three different antibiotics. This resulted in the identification of several genes whose deletion significantly suppressed resistance evolution, including arcA and gutM. Analysis of double-gene deletion strains suggested that the suppression of resistance evolution caused by arcA and gutM deletion was not caused by epistatic interactions with mutations known to confer drug resistance. These results provide a methodological basis for combinatorial drug treatments that may help to suppress the emergence of resistant pathogens by inhibiting resistance evolution.
• Complete Genome Sequences of Three Star-Shaped Bacteria, Stella humosa, Stella vacuolata, and Stella Species ATCC 35155

  Atsushi Shibai, Tomoya Maeda, Masako Kawada, Hazuki Kotani, Natsue Sakata, Chikara Furusawa

  Microbiology Resource Announcements 8 (32) 2019/08/08 [Refereed]
   

  Stella species are unique star-shaped alphaproteobacteria found in various environments. We report the complete genome sequences of three Stella strains, Stella humosa ATCC 43930, Stella vacuolata ATCC 43931, and Stella species ATCC 35155. These are the first complete genome sequences of members of the genus Stella.
• High-throughput identification of the sensitivities of an Escherichia coli ΔrecA mutant strain to various chemical compounds.

  Maeda T, Horinouchi T, Sakata N, Sakai A, Furusawa C

  The Journal of antibiotics 72 (7) 566 - 573 0021-8820 2019/02 [Refereed][Not invited]
   

  Antibiotic resistance is considered a global threat to public health. Adaptive resistance mutations and the acquisition of resistance genes by horizontal gene transfer are known to be facilitated by the RecA-dependent SOS response during antibiotic treatment, making RecA inhibitors promising agents for the prevention of antibiotic resistance. However, the impact of RecA inactivation on antibiotic sensitivities remains unclear. Therefore, in this study, we performed high-throughput screening to determine the minimum inhibitory concentrations (MICs) of 217 chemicals, including both antibiotics and toxic chemicals of unknown drug action, in the wild-type MDS42 and the ΔrecA mutant strains of Escherichia coli. The ΔrecA mutant showed increased sensitivity to DNA-damaging agents, DNA replication inhibitors, and chromate stress, as well as to other chemicals, such as S-(2-aminoethyl)-L-cysteine, L-histidine, ruthenium red, D-penicillamine, carbonyl cyanide 3-chlorophenylhydrazone (CCCP), cerulenin, and L-cysteine. Microarray analysis showed further that the ΔrecA mutant had lower expressions of glnK, nac, and glnLG, which encode nitrogen assimilation regulators, as well as amtB, which encodes an ammonium transporter, compared with the wild type. These findings suggest that the ΔrecA mutation affects not only the SOS response but also amino acid metabolism.
• RNase E/G-dependent degradation of metE mRNA, encoding methionine synthase, in Corynebacterium glutamicum

  Satoshi Endo, Tomoya Maeda, Takahiro Kawame, Noritaka Iwai, Masaaki Wachi

  The Journal of General and Applied Microbiology 65 (1) 47 - 52 0022-1260 2019 [Refereed]
  
• Toward prediction and control of antibiotic-resistance evolution

  Chikara Furusawa, Takaaki Horinouchi, Tomoya Maeda

  Current Opinion in Biotechnology 54 45 - 49 1879-0429 2018/12/01 [Refereed][Not invited]
   

  The emergence of antibiotic-resistant bacteria is a serious public concern. To deal with this problem, recent advances in technology and the use of laboratory evolution experiments have provided valuable information on the phenotypic and genotypic changes that occur during the evolution of resistance. These studies have demonstrated the existence of evolutionary constraints on the development of drug-resistance, which suggests predictability in its evolution. In this review, we focus on the possibility to predict and control the evolution of antibiotic resistance, based on quantitative analysis of phenotypic and genotypic changes observed in bacterial laboratory evolution. We emphasize the key challenges in evolutionary biology that will contribute to the development of appropriate treatment strategies for preventing resistance evolution.
• Understanding and engineering alcohol-tolerant bacteria using OMICS technology.

  Horinouchi T, Maeda T, Furusawa C

  World journal of microbiology & biotechnology 34 (11) 157 - 157 0959-3993 2018/10 [Refereed][Not invited]
   

  Microbes are capable of producing alcohols, making them an important source of alternative energy that can replace fossil fuels. However, these alcohols can be toxic to the microbes themselves, retaring or inhibiting cell growth and decreasing the production yield. One solution is improving the alcohol tolerance of such alcohol-producing organisms. Advances in omics technologies, including transcriptomic, proteomic, metabolomic, and genomic technologies, have helped us understand the complex mechanisms underlying alcohol toxicity, and such advances could assist in devising strategies for engineering alcohol-tolerant strains. This review highlights these advances and discusses strategies for improving alcohol tolerance using omics analyses.
• Glutamine-rich toxic proteins GrtA, GrtB and GrtC together with the antisense RNA AsgR constitute a toxin–antitoxin-like system in Corynebacterium glutamicum

  Tomoya Maeda, Yuya Tanaka, Masayuki Inui

  Molecular Microbiology 108 (5) 578 - 594 1365-2958 2018/06/01 [Refereed][Not invited]
   

  The Corynebacterium glutamicum R grtA (cgR_2936), grtB (cgR_2934) and grtC (cgR_2933) genes were identified as paralogs encoding glutamine-rich toxic proteins. We also identified a new antisense small RNA AsgR (antisense sRNA for grtA) that overlaps the 3′ end of the grtA gene. Single over-expressions of grtA, grtB and grtC resulted in complete inhibition of Escherichia coli cell growth. This growth was rescued by co-expression of AsgR. Similar effects were observed in C. glutamicum, although the toxicities of these proteins were moderate. Inhibition of AsgR transcription resulted in increased levels and prolonged half-lives of grtA, grtB and grtC mRNAs. We also found that the expression levels of grtA, grtB and grtC were increased in an RNase III deletion mutant. Primer extension analysis revealed the RNase III cleavage site to be in the 3′ untranslated region (3′-UTR) of the grtA mRNA. The expression levels of grtA, grtB and grtC were increased after exposure to several stresses, including heat shock, treatment with penicillin G, lysozyme or H2O2. The deletions of grtABC and asgR genes resulted in decreased survival rate under several stresses. These results indicate that GrtABC and AsgR constitute a type I toxin–antitoxin-like system in C. glutamicum.
• Polynucleotide Phosphorylase, RNase E/G, and YbeY Are Involved in the Maturation of 4.5S RNA in Corynebacterium glutamicum

  Tomoya Maeda, Yuya Tanaka, Masaaki Wachi, Masayuki Inui

  JOURNAL OF BACTERIOLOGY 199 (5) 0021-9193 2017/03 [Refereed][Not invited]
   

  Corynebacterium glutamicum has been applied for the industrial production of various metabolites, such as amino acids. To understand the biosynthesis of the membrane protein in this bacterium, we investigated the process of signal recognition particle (SRP) assembly. SRP is found in all three domains of life and plays an important role in the membrane insertion of proteins. SRP RNA is initially transcribed as precursor molecules; however, relatively little is known about its maturation. In C. glutamicum, SRP consists of the Ffh protein and 4.5S RNA lacking an Alu domain. In this study, we found that 3'-to-5' exoribonuclease, polynucleotide phosphorylase (PNPase), and two endo-type RNases, RNase E/G and YbeY, are involved in the 3' maturation of 4.5S RNA in C. glutamicum. The mature form of 4.5S RNA was inefficiently formed Delta rneG Delta pnp mutant cells, suggesting the existence of an alternative pathway for the 3' maturation of 4.5S RNA. Primer extension analysis also revealed that the 5' mature end of 4.5S RNA corresponds to that of the transcriptional start site. Immunoprecipitated Ffh protein contained immature 4.5S RNA Delta pnp, Delta rneG, and Delta ybeY mutants, suggesting that 4.5S RNA precursors can interact with Ffh. These results imply that the maturation of 4.5S RNA can be performed in the 4.5S RNA-Ffh complex. IMPORTANCE Overproduction of a membrane protein, such as a transporter, is useful for engineering of strains of Corynebacterium glutamicum, which is a workhorse of amino acid production. To understand membrane protein biogenesis in this bacterium, we investigated the process of signal recognition particle (SRP) assembly. SRP contains the Ffh protein and SRP RNA and plays an important role in the membrane insertion of proteins. Although SRP RNA is highly conserved among the three domains of life, relatively little is known about its maturation. We show that PNPase, RNase E/G, and YbeY are involved in the 3' maturation of the SRP RNA (4.5S RNA) in this bacterium. This indicates that 3' end processing in this organism is different from that in other bacteria, such as Escherichia coli.
• RNase III mediated cleavage of the coding region of mraZ mRNA is required for efficient cell division in Corynebacterium glutamicum

  Tomoya Maeda, Yuya Tanaka, Norihiko Takemoto, Nagisa Hamamoto, Masayuki Inui

  MOLECULAR MICROBIOLOGY 99 (6) 1149 - 1166 0950-382X 2016/03 [Refereed][Not invited]
   

  The Corynebacterium glutamicum R cgR_1959 gene encodes an endoribonuclease of the RNase III family. Deletion mutant of cgR_1959 (rnc mutant) showed an elongated cell shape, and presence of several lines on the cell surface, indicating a required of RNase III for maintaining normal cell morphology in C. glutamicum. The level of mraZ mRNA was increased, whereas cgR_1596 mRNA encoding a putative cell wall hydrolase and ftsEX mRNA were decreased in the rnc mutant. The half-life of mraZ mRNA was significantly prolonged in the rnc and the pnp mutant strains. This indicated that the degradation of mraZ mRNA was performed by RNase III and the 3-to-5 exoribonuclease, PNPase. Northern hybridization and primer extension analysis revealed that the cleavage site for mraZ mRNA by RNase III is in the coding region. Overproduction of MraZ resulted in an elongated cell shape. The expression of ftsEX decreased while that of cgR_1596 unchanged in an MraZ-overexpressing strain. An electrophoretic mobility shift assay and a transcriptional reporter assay indicate that MraZ is a transcriptional repressor of ftsEX in C. glutamicum. These results indicate that RNase III is required for efficient expression of MraZ-dependent ftsEX and MraZ-independent cgR_1596.
• 細菌におけるRNaseを介した代謝制御 産業微生物コリネ型細菌を例に

  前田智也, 和地正明

  化学と生物 53 (2) 0453-073X 2015
• Engineering of Corynebacterium glutamicum for growth and L-lysine and lycopene production from N-acetyl-glucosamine

  Christian Matano, Andreas Uhde, Jung-Won Youn, Tomoya Maeda, Lina Clermont, Kay Marin, Reinhard Kraemer, Volker F. Wendisch, Gerd M. Seibold

  APPLIED MICROBIOLOGY AND BIOTECHNOLOGY 98 (12) 5633 - 5643 0175-7598 2014/06 [Refereed][Not invited]
   

  Sustainable supply of feedstock has become a key issue in process development in microbial biotechnology. The workhorse of industrial amino acid production Corynebacterium glutamicum has been engineered towards utilization of alternative carbon sources. Utilization of the chitin-derived aminosugar N-acetyl-glucosamine (GlcNAc) for both cultivation and production with C. glutamicum has hitherto not been investigated. Albeit this organism harbors the enzymes N-acetylglucosamine-6-phosphatedeacetylase and glucosamine-6P deaminase of GlcNAc metabolism (encoded by nagA and nagB, respectively) growth of C. glutamicum with GlcNAc as substrate was not observed. This was attributed to the lack of a functional system for GlcNAc uptake. Of the 17 type strains of the genus Corynebacterium tested here for their ability to grow with GlcNAc, only Corynebacterium glycinophilum DSM45794 was able to utilize this substrate. Complementation studies with a GlcNAc-uptake deficient Escherichia coli strain revealed that C. glycinophilum possesses a nagE-encoded EII permease for GlcNAc uptake. Heterologous expression of the C. glycinophilum nagE in C. glutamicum indeed enabled uptake of GlcNAc. For efficient GlcNac utilization in C. glutamicum, improved expression of nagE with concurrent overexpression of the endogenous nagA and nagB genes was found to be necessary. Based on this strategy, C. glutamicum strains for the efficient production of the amino acid l-lysine as well as the carotenoid lycopene from GlcNAc as sole substrate were constructed.
• L-Glutamate Secretion by the N-Terminal Domain of the Corynebacterium glutamicum NCgl1221 Mechanosensitive Channel

  Chikako Yamashita, Ken-ichi Hashimoto, Kosuke Kumagai, Tomoya Maeda, Ayako Takada, Isamu Yabe, Hisashi Kawasaki, Masaaki Wachi

  BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 77 (5) 1008 - 1013 0916-8451 2013/05 [Refereed][Not invited]
   

  The Corynebacterium glutamicum NCgl1221 mechanosensitive channel mediates L-glutamate secretion by sensing changes in membrane tension caused by treatments such as biotin limitation and penicillin. The NCgl1221 protein has an N-terminal domain (1-286 a.a.) homologous to the Escherichia coli MscS and a long C-terminal domain (287-533 a.a.) of unknown function. In order to investigate the role of the C-terminal domain in L-glutamate secretion, we constructed a series of C-terminally truncated mutants of NCgl1221. We found that the N-terminal domain, homologous to E. coli MscS, retained the ability to cause L-glutamate secretion in response to the treatment. Electrophysiological analysis confirmed that the N-terminal domain mediated L-glutamate secretion. 3D homology modeling has suggested that the N-terminal domain of NCgl1221 has an extra loop structure (221-232 a.a.) that is not found in most other MscS proteins. The mutant NCgl1221, deleted for this loop structure, lost the ability to secrete L-glutamate. In addition, we found that mutant NCgl1221 lacking the C-terminal extracytoplasmic domain (420-533 a.a.) produced L-glutamate without any inducing treatment. These results suggest that the N-terminal domain is necessary and sufficient for the excretion of L-glutamate in response to inducing treatment, and that the C-terminal extracytoplasmic domain has a negative regulatory role in L-glutamate production.
• Glucosamine as carbon source for amino acid-producing Corynebacterium glutamicum

  Andreas Uhde, Jung-Won Youn, Tomoya Maeda, Lina Clermont, Christian Matano, Reinhard Kraemer, Volker F. Wendisch, Gerd M. Seibold, Kay Marin

  APPLIED MICROBIOLOGY AND BIOTECHNOLOGY 97 (4) 1679 - 1687 0175-7598 2013/02 [Refereed][Not invited]
   

  Corynebacterium glutamicum grows with a variety of carbohydrates and carbohydrate derivatives as sole carbon sources; however, growth with glucosamine has not yet been reported. We isolated a spontaneous mutant (M4) which is able to grow as fast with glucosamine as with glucose as sole carbon source. Glucosamine also served as a combined source of carbon, energy and nitrogen for the mutant strain. Characterisation of the M4 mutant revealed a significantly increased expression of the nagB gene encoding the glucosamine-6P deaminase NagB involved in degradation of glucosamine, as a consequence of a single mutation in the promoter region of the nagAB-scrB operon. Ectopic nagB overexpression verified that the activity of the NagB enzyme is in fact the growth limiting factor under these conditions. In addition, glucosamine uptake was studied, which proved to be unchanged in the wild-type and M4 mutant strains. Using specific deletion strains, we identified the PTSGlc transport system to be responsible for glucosamine uptake in C. glutamicum. The affinity of this uptake system for glucosamine was about 40-fold lower than that for its major substrate glucose. Because of this difference in affinity, glucosamine is efficiently taken up only if external glucose is absent or present at low concentrations. C. glutamicum was also examined for its suitability to use glucosamine as substrate for biotechnological purposes. Upon overexpression of the nagB gene in suitable C. glutamicum producer strains, efficient production of both the amino acid l-lysine and the diamine putrescine from glucosamine was demonstrated.
• A role of the transcriptional regulator LldR (NCgl2814) in glutamate metabolism under biotin-limited conditions in Corynebacterium glutamicum

  Tanyanut Supkulsutra, Tomoya Maeda, Kosuke Kumagai, Masaaki Wachi

  JOURNAL OF GENERAL AND APPLIED MICROBIOLOGY 59 (3) 207 - 214 0022-1260 2013 [Refereed][Not invited]
   

  Corynebacterium glutamicum is a Gram-positive, rod-shaped, aerobic bacterium used for the fermentative production of L-glutamate. LldR (NCgl2814) is known as a repressor for ldhA and lldD encoding lactate dehydrogenases. LdhA is responsible for production of L-lactate, while LldD is for its assimilation. Since L-lactate production was observed as a by-product of glutamate production under biotin-limited conditions, LldR might play a regulatory role in the glutamate metabolism. Here for the first time, we investigated effects of overproduction or deletion of LldR on the glutamate metabolism under biotin-limited conditions in C. glutamicum. It was found that glutamate production under biotin-limited conditions was decreased by overproduction of LldR. In the wild-type cells, L-lactate was produced in the first 24 h and it was re-consumed thereafter. On the other hand, in the overproduced cells, L-lactate was produced like the wild type, but it was not re-consumed. This means that L-lactate assimilation, which is catalyzed by LldD, was suppressed by the overproduction of LldR, but L-lactate production, which is catalyzed by LdhA, was not affected, indicating that LldR mainly controls the expression of lldD but not of ldhA under biotin-limited conditions. This was confirmed by quantitative real-time RT-PCR. From these results, it is suggested that L-lactate metabolism, which is controlled by LldR, has a buffering function of the pyruvate pool for glutamate production.
• 3 ' Untranslated Region-Dependent Degradation of the aceA mRNA, Encoding the Glyoxylate Cycle Enzyme Isocitrate Lyase, by RNase E/G in Corynebacterium glutamicum

  Tomoya Maeda, Masaaki Wachi

  APPLIED AND ENVIRONMENTAL MICROBIOLOGY 78 (24) 8753 - 8761 0099-2240 2012/12 [Refereed][Not invited]
   

  We previously reported that the Corynebacterium glutamicum RNase E/G encoded by the rneG gene (NCgl2281) is required for the 5' maturation of 5S rRNA. In the search for the intracellular target RNAs of RNase E/G other than the 5S rRNA precursor, we detected that the amount of isocitrate lyase, an enzyme of the glyoxylate cycle, increased in rneG knockout mutant cells grown on sodium acetate as the sole carbon source. Rifampin chase experiments showed that the half-life of the aceA mRNA was about 4 times longer in the rneG knockout mutant than in the wild type. Quantitative real-time PCR analysis also confirmed that the level of aceA mRNA was approximately 3-fold higher in the rneG knockout mutant strain than in the wild type. Such differences were not observed in other mRNAs encoding enzymes involved in acetate metabolism. Analysis by 3' rapid amplification of cDNA ends suggested that RNase E/G cleaves the aceA mRNA at a single-stranded AU-rich region in the 3' untranslated region (3'-UTR). The lacZ fusion assay showed that the 3'-UTR rendered lacZ mRNA RNase E/G dependent. These findings indicate that RNase E/G is a novel regulator of the glyoxylate cycle in C. glutamicum.
• Corynebacterium glutamicum RNase E/G-type endoribonuclease encoded by NCgl2281 is involved in the 5 ' maturation of 5S rRNA

  Tomoya Maeda, Masaaki Wachi

  ARCHIVES OF MICROBIOLOGY 194 (2) 65 - 73 0302-8933 2012/02 [Refereed][Not invited]
   

  Corynebacterium glutamicum has one RNase E/G ortholog and one RNase J ortholog but no RNase Y. We previously reported that the C. glutamicum NCgl2281 gene encoding the RNase E/G ortholog complemented the rng::cat mutation in Escherichia coli but not the rne-1 mutation. In this study, we constructed an NCgl2281 knockout mutant and found that the mutant cells accumulated 5S rRNA precursor molecules. The processing of 16S and 23S rRNA, tRNA, and tmRNA was normal. Primer extension analysis revealed that the RNase E/G ortholog cleaved at the -1 site of the 5' end of 5S rRNA. However, 3' maturation was essentially unaffected. These findings showed that C. glutamicum NCgl2281 endoribonuclease is involved in the 5' maturation of 5S rRNA. This is the first report showing the physiological function of the RNase E/G ortholog in bacteria having one RNase E/G and one RNase J but no RNase Y.
• The Corynebacterium glutamicum NCgl2281 Gene Encoding an RNase E/G Family Endoribonuclease Can Complement the Escherichia coli rng::cat Mutation but Not the rne-1 Mutation

  Tomoya Maeda, Taro Sakai, Masaaki Wachi

  BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 73 (10) 2281 - 2286 0916-8451 2009/10 [Refereed][Not invited]
   

  The Corynebacterium glutamicum NCgl2281. gene encodes an RNase E/G family endoribonuclease having an additional N-terminal domain of unknown function. In this study, we constructed plasmids expressing the full length (FL) and the N-terminally truncated form (Delta N) of NCgl2281 and examined their complementation ability as to Escherichia coli rng::cat and rne-1 mutations. Both FL- and Delta N-NCgl2281 rescued the defects caused by the rng::cat mutation, i.e., accumulation of 16S rRNA precursor, overproduction of the AdhE protein, and growth inhibition on M9 glucose medium. On the other hand, they did not complement the rne-1 mutation. These results indicate that the C. glutamicum NCgl2281 endoribonuclease is functionally more closely related to the E. coli RNase G than to RNase E.

MISC

• Isoprenol production by co-culture with two Escherichia coli strains with consuming different carbon sources

  村上茉奈美, 川井隆太郎, 三吉健太, 芝井厚, 前田智也, 堀之内貴明, 古澤力, 古澤力, 戸谷吉博, 清水浩  日本生物工学会大会講演要旨集  74th-  2022
• Genomic mutation analysis of an evolved strain with enhanced mevalonate consumption

  村上茉奈美, 川井隆太郎, 前田智也, 堀之内貴明, 古澤力, 古澤力, 戸谷吉博, 清水浩  日本生物工学会大会講演要旨集  73rd-  2021
• 代謝シミュレーションと指向性進化に基づく有用物質生産微生物の開発

  戸谷吉博, 川井隆太郎, 三吉健太, 村上茉奈美, 一色衣香, 堀之内貴明, 前田智也, 古澤力, 清水浩  化学工学会秋季大会研究発表講演要旨集(CD-ROM)  52nd-  2021
• Directed evolution for enhancing mevalonate uptake ability in Escherichia coli under a continuous culture

  村上茉奈美, 川井隆太郎, 前田智也, 堀之内貴明, 古澤力, 戸谷吉博, 清水浩  日本農芸化学会大会講演要旨集(Web)  2021-  2021
• 機械学習と大腸菌実験室進化データを用いた進化拘束の解析

  岩澤諄一郎, 前田智也, 堀之内貴明, 古澤力, 古澤力  日本ゲノム微生物学会年会要旨集  14th-  2020
• Phenotypic and genotypic analysis of laboratory evolution of Escherichia coli: Toward prediction and control of ecosystem evolution

  古澤力, 前田智也, 芝井厚  実験医学  38-  (18)  2020
• 全自動培養システムを用いた多種ストレス環境下における大腸菌の大規模実験室進化

  前田智也, 岩澤諄一郎, 堀之内貴明, 阪田奈津枝, 小谷葉月, 川田正子, 田邊久美, 古澤力, 古澤力  日本ゲノム微生物学会年会要旨集  13th-  2019
• 全自動培養システムを用いた多種ストレス環境下における大腸菌の大規模実験室進化

  前田智也, 堀之内貴明, 阪田奈津江, 小谷葉月, 酒井亜希, 田邊久美, 古澤力, 古澤力  日本ゲノム微生物学会年会要旨集  12th-  2018
• 大規模実験室進化を用いた転写因子の破壊による大腸菌の薬剤耐性化の制御

  堀之内貴明, 前田智也, 小谷葉月, 酒井亜希, 古澤力, 古澤力  日本生物工学会大会講演要旨集  70th-  2018
• 全自動培養システムを用いた多種ストレス環境化における大腸菌進化ダイナミクスの解析

  前田智也, 堀之内貴明, 阪田奈津枝, 小谷葉月, 酒井亜希, 田邊久美, 古澤力, 古澤力  日本ゲノム微生物学会年会要旨集  11th-  2017
• 超大規模実験室進化とオミックス解析による大腸菌のストレス耐性機構の解析

  堀之内貴明, 前田智也, 古澤力, 古澤力  日本生物工学会大会講演要旨集  69th-  2017
• 大腸菌進化実験による進化的制約の解析

  古澤力, 古澤力, 前田智也, 堀之内貴明  日本進化学会大会プログラム・講演要旨集(Web)  19th-  2017
• 1P-046 Analysis of mRNA recognition mechanism of Corynebacterium glutamicum RNase E/G

  Endo Satoshi, Maeda Tomoya, Wachi Masaaki  日本生物工学会大会講演要旨集  66-  28  -28  2014
• 2P-031 5'-UTR dependent degradation of mRNAs by RNase E/G in Corynebacterium glutamicum

  Endo Satoshi, Maeda Tomoya, Wachi Masaaki  日本生物工学会大会講演要旨集  65-  111  -111  2013

Awards & Honors

• 2021/01 理化学研究所 Presentation award Biology Prize
  
• 2012/03 生物工学研究会 鎌田泉博士論文賞
   

  受賞者: 前田 智也

Research Grants & Projects

• 非病原性ウシ型結核菌BCG株を用いた進化実験による多剤耐性進化可能性の検証

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 2024/04 -2028/03 

  Author : 前田 智也
• Analysis of evolutionary dynamics of ecosystem using automated culture system

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2023/04 -2026/03 

  Author : 古澤 力, 前田 智也
• 腸内マイクロバイオームの大規模継代培養による栄養共生関係の解明

  日本学術振興会：科学研究費助成事業 新学術領域研究(研究領域提案型)

  Date (from‐to) : 2022/04 -2024/03 

  Author : 前田 智也
• 適応的実験室進化による産業微生物のエネルギー欠乏への潜在的な適応能力の解明

  日本学術振興会：科学研究費助成事業 基盤研究(C)

  Date (from‐to) : 2021/04 -2024/03 

  Author : 前田 智也

   

  本研究では、発酵産業に重要な大腸菌やコリネ型細菌の発酵生産効率の向上を目的として、中枢代謝が活性化している様々な呼吸鎖酵素欠損株の適応的実験室進化を行うことで、産業微生物の細胞増殖と目的物質生産のバランスを最適化させる方法を明らかにすることを最終目的としている。 大腸菌やコリネ型細菌において、酸化的リン酸化の阻害によるエネルギー欠乏の誘導が糖代謝などの中枢代謝を活性化させる一方、著しい増殖悪化を引き起こすことが先行研究から明らかにされている。申請者は、本研究において、エネルギー欠乏株の多くが酢酸を単一炭素源として生育できない、または著しく生育が阻害されることを見出した。酢酸を単一炭素源とした場合、取り込んだ酢酸をまず活性化してアセチルリン酸へ変換する過程で1分子のATPを消費し、その後基質レベルのリン酸化では1分子のATPしか合成できないため、ATP合成は酸化的リン酸化に依存していると考えられる。そのため、酢酸を単一炭素源とする選択圧をかける実験室進化を行うことで、酸化的リン酸化が阻害されているエネルギー欠乏株において、エネルギー欠乏への潜在的な適応能力が活性化された進化株が出現し得るのではないかと考えた。そこで今年度は、まず大腸菌の様々な呼吸鎖酵素を欠損したエネルギー欠乏株6株を親株として、酢酸最少培地を用いた適応的実験室進化を行った。適応的実験室進化は、コントロールとして呼吸鎖を欠損していない野生株を用い、また同一培養条件における反復数4として合計28系列行った。このような適応的実験室進化により、酢酸最少培地における生育が回復または、増殖速度が向上した進化株を取得することに成功した。
• Metabolic analysis of an Escherichia coli mutant defective in respiratory chain enzymes and its application to the production of 1,3-butanediol and gamma-aminobutyric acid.

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2019/04 -2022/03 

  Author : Atsushi Yokota

   

  The respiratory chain of Escherichia coli includes NADH dehydrogenase (NDH) and terminal oxidase. We previously knocked out the highest proton-motive force-generating capacity components (NADH dehydrogenase-1 and cytochrome bo3 oxidase) using the E. coli wild-type W1485 strain. In this study, we found that the double knockout mutant strain (ΔΔ strain) showed an increase in the glucose consumption rate, respiration rate, acetate production, NADH/NAD+ ratio, and glutamate production. A metabolome analysis and RNA-seq analysis revealed that genes encoding for pentose phosphate pathway, acetate metabolism, and gadAB encoding glutamate decarboxylase were significantly increased. We also demonstrated that the ΔΔ strain can be applied for the production of 1,3-butanediol and γ-aminobutyric acid (GABA).
• Identification of interaction network of environmental microorganisms by high-throughput laboratory evolution.

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed research area)

  Date (from‐to) : 2020/04 -2022/03 

  Author : 前田 智也

   

  自然界では、数多くの微生物種が共存しており、栄養や生息地の競合だけでなく、基質の分解や、代謝物の交換等、複雑な微生物相互作用を基とした共生関係が築かれている。そのため、このような微生物相互作用を明らかにすることは、マイクロバイオームにおける各微生物の役割や、存在割合の動態が何よって影響を受けているのかを理解する上で大変重要である。そこで本研究では、様々な環境下においてマイクロバイオームを継代培養し、共存状態を維持し続ける微生物の組合せを解析することで、自然界に存在し得る共生関係を大規模に同定することを目的としている。 今年度は、まず昨年度に実施した大規模継代培養実験によって得られた継代培養後の微生物集団におけるメタ16S解析を実施した。安定した微生物集団が得られた13種類の炭素源由来の継代培養後集団および、それに対応する継代前の初期集団を合わせた合計623サンプルについて、16S rRNAのV4領域をターゲットとしたアンプリコンシーケンス解析を行い、試料中に含まれていた微生物の種類をAmplicon sequence variant (ASV) として算出した。続いて継代培養後の集団が、同一炭素源と同一初期条件のどちらと類似性が高いか調べたところ、炭素源が同じ集団の方が、初期状態が同じ集団よりも菌叢構造の類似性が有意に高かった。このことから、菌叢構造は初期状態よりも、利用できる栄養による影響を強く受けることが明らかになった。また、継代培養後の集団から単離した異種を組み合わせたペアワイズな共培養試験を行ったところ、継代培養後の安定した集団には、栄養共生関係が広く見られたことから、同一環境下で安定して共存している種同士には栄養共生関係が成立していることが示唆された。
• Construction and analysis of bacterial strains carrying heterologous bacterial genome by heterologous protoplast fusion.

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Early-Career Scientists

  Date (from‐to) : 2018/04 -2020/03 

  Author : Maeda Tomoya

   

  In this project, I aimed to develop a new technology to construct heterologous genome fusant bacterial strain. First, protoplast fusion experiments that cause cell fusion were performed using various bacterial pairs. However, none of them produced a heterologous genome fusant. Next, to replicate and maintain heterologous genome in E. coli, the sequence of a single copy plasmid derived from E. coli was introduced to the Corynebacterium glutamicum genome. Then, the recombinant C. glutamicum strain was fused with E. coli by protoplast fusion. It was confirmed that the obtained fusant strains carry both E. coli and C. glutamicum genome sequences.
• Development of interspecies genome recombination method using cell fusion and CRISPR/Cas9 technique

  Noda Institute for Scientific Research：NISR Young Investigator Research Grants

  Date (from‐to) : 2019/04 -2020/03

Educational Activities

Teaching Experience