研究者データベース

ATSUMI SHOTA(アツミ シヨウタ)
農学研究院 食水土資源グローバルセンター
教授

基本情報

所属

  • 農学研究院 食水土資源グローバルセンター

職名

  • 教授

学位

  • 理学博士(2002年03月 京都大学)

ホームページURL

J-Global ID

研究分野

  • ライフサイエンス / 応用微生物学

学歴

  •         - 2002年03月   京都大学   理学研究科

研究活動情報

論文

  • Yohei Tashiro, Shinichi Hirano, Morgan M. Matson, Shota Atsumi, Akihiko Kondo
    Metabolic Engineering 47 211 - 218 2018年05月01日 [査読有り][通常論文]
     
    Here we have developed an electrochemical-biological hybrid system to fix CO2. Natural biological CO2 fixation processes are relatively slow. To increase the speed of fixation we applied electrocatalysts to reduce CO2 to formate. We chose a user-friendly organism, Escherichia coli, as host. Overall, the newly constructed CO2 and formate fixation pathway converts two formate and one CO2 to one pyruvate via glycine and L-serine in E. coli. First, one formate and one CO2 are converted to one glycine. Second, L-serine is produced from one glycine and one formate. Lastly, L-serine is converted to pyruvate. E. coli's genetic tractability allowed us to balance various parameters of the pathway. The carbon flux of the pathway was sufficient to compensate L-serine auxotrophy in the strain. In total, we integrated both electrocatalysis and biological systems into a single pot to support E. coli growth with CO2 and electricity. Results show promise for using this hybrid system for chemical production from CO2 and electricity.
  • Matson Morgan M, Atsumi Shota
    CURRENT OPINION IN BIOTECHNOLOGY 50 65 - 71 2018年04月 [査読有り][通常論文]
  • Nicole E. Nozzi, Anna E. Case, Austin L. Carroll, Shota Atsumi
    ACS SYNTHETIC BIOLOGY 6 11 2136 - 2144 2017年11月 [査読有り][通常論文]
     
    Cyanobacteria have attracted significant interest as a platform for renewable production of fuel and feedstock chemicals from abundant atmospheric carbon dioxide by way of photosynthesis. While great strides have been made in developing this technology in freshwater cyanobacteria, logistical issues remain in scale-up. Use of the cyanobacterium Synechococcus sp. PCC 7002 (7002) as a chemical production chassis could address a number of these issues given the higher tolerance to salt, light, and heat as well as the fast growth rate of 7002 in comparison to traditional model cyanobacteria such as Synechococcus elongatus PCC 7942 and Synechocystis sp. PCC 6803. However, despite growing interest, the development of genetic engineering tools for 7002 continues to lag behind those available for model cyanobacterial strains. In this work we demonstrate the systematic development of a 7002 production strain for the feedstock chemical 2,3-butanediol (23BD). We expand the range of tools available for use in 7002 by identifying and utilizing new integration sites for homologous recombination, demonstrating the inducibility of theophylline riboswitches, and screening a set of isopropyl beta-D-1-thiogalactopyranoside (IPTG) inducible promoters. We then demonstrate improvements of 23BD production with the systematic screening of different conditions including: operon arrangement and copy number, light strength, inducer concentration, cell density at the time of induction, and nutrient concentration. Final production tests yielded titers of 1.6 g/L 23BD after 16 days at a rate of 100 mg/L/day. This work represents great strides in the development of 7002 as an industrially relevant production host.
  • Angela Zhang, Austin L. Carroll, Shota Atsumi
    FEMS MICROBIOLOGY LETTERS 364 16 2017年08月 [査読有り][通常論文]
     
    Atmospheric CO2 levels have reached an alarming level due to industrialization and the burning of fossil fuels. In order to lower the level of atmospheric carbon, strategies to sequester excess carbon need to be implemented. The CO2-fixing mechanism in photosynthetic organisms enables integration of atmospheric CO2 into biomass. Additionally, through exogenous metabolic pathways in these photosynthetic organisms, fixed CO2 can be routed to produce various commodity chemicals that are currently produced from petroleum. This review will highlight studies and modifications to different components of cyanobacterial CO2-fixing systems, as well as the application of these systems toward CO2-derived chemical production. 2,3-Butanediol is given particular focus as one of the most thoroughly studied systems for conversion of CO2 to a bioproduct.
  • Masahiro Kanno, Austin L. Carroll, Shota Atsumi
    NATURE COMMUNICATIONS 8 2017年03月 [査読有り][通常論文]
     
    Cyanobacteria have attracted much attention as hosts to recycle CO2 into valuable chemicals. Although cyanobacteria have been engineered to produce various compounds, production efficiencies are too low for commercialization. Here we engineer the carbon metabolism of Synechococcus elongatus PCC 7942 to improve glucose utilization, enhance CO2 fixation and increase chemical production. We introduce modifications in glycolytic pathways and the Calvin Benson cycle to increase carbon flux and redirect it towards carbon fixation. The engineered strain efficiently uses both CO2 and glucose, and produces 12.6 g l(-1) of 2,3-butanediol with a rate of 1.1 g l(-1) d(-1) under continuous light conditions. Removal of native regulation enables carbon fixation and 2,3-butanediol production in the absence of light. This represents a significant step towards industrial viability and an excellent example of carbon metabolism plasticity.
  • Masahiro Kanno, Shota Atsumi
    ACS SYNTHETIC BIOLOGY 6 1 69 - 75 2017年01月 [査読有り][通常論文]
     
    Cyanobacteria have attracted much attention as a means to directly recycle carbon dioxide into valuable chemicals that are currently produced from petroleum. However, the titers and productivities achieved are still far below the level required in industry. To make a more industrially applicable production scheme, glycerol, a byproduct of biodiesel production, OH can be used as an additional carbon source for photomixotrophic chemical production. Glycerol is an ideal candidate due to its availability and low cost. In this study, we found that a heterologous glycerol respiratory pathway enabled Synechococcus elongatus PCC 7942 to utilize extracellular glycerol. The engineered strain produced 761 mg/L of 2,3-butanediol in 48 h with a 290% increase over the control strain under continuous light conditions. Glycerol supplementation also allowed for continuous cell growth and 2,3-butanediol production in diurnal light conditions. These results highlight the potential of glycerol as an additional carbon source for photomixotrophic chemical production in cyanobacteria.
  • Neal J. Oliver, Christine A. Rabinovitch-Deere, Austin L. Carroll, Nicole E. Nozzi, Anna E. Case, Shota Atsumi
    CURRENT OPINION IN CHEMICAL BIOLOGY 35 43 - 50 2016年12月 [査読有り][通常論文]
     
    Rising levels of atmospheric CO2 are contributing to the global greenhouse effect. Large scale use of atmospheric CO2 may be a sustainable and renewable means of chemical and liquid fuel production to mitigate global climate change. Photosynthetic organisms are an ideal platform for efficient, natural CO2 conversion to a broad range of chemicals. Cyanobacteria are especially attractive for these purposes, due to their genetic malleability and relatively fast growth rate. Recent years have yielded a range of work in the metabolic engineering of cyanobacteria and have led to greater knowledge of the host metabolism. Understanding of endogenous and heterologous carbon regulation mechanisms leads to the expansion of productive capacity and chemical variety. This review discusses the recent progress in metabolic engineering of cyanobacteria for biofuel and bulk chemical production since 2014.
  • Shuchi H. Desai, Irina Koryakina, Anna E. Case, Michael D. Toney, Shota Atsumi
    METABOLIC ENGINEERING 38 98 - 104 2016年11月 [査読有り][通常論文]
     
    Industrial gas-to-liquid (GTL) technologies are well developed. They generally employ syngas, require complex infrastructure, and need high capital investment to be economically viable. Alternatively, biological conversion has the potential to be more efficient, and easily deployed to remote areas on relatively small scales for the utilization of otherwise stranded resources. The present study demonstrates a novel biological GTL process in which engineered Escherichia coli converts C2-C4 gaseous alkenes into liquid diols. Diols are versatile industrially important chemicals, used routinely as antifreeze agents, polymer precursors amongst many other applications. Heterologous co-expression of a monooxygenase and an epoxide hydrolase in E. coli allows whole cell conversion of C2-C4 alkenes for the formation of ethylene glycol, 1,2-propanediol, 1,2-butanediol, and 2,3-butanediol at ambient temperature and pressure in one pot. Increasing intracellular NADH supply via addition of formate and a formate dehydrogenase increases ethylene glycol production titers, resulting in an improved productivity of 9mg/L/h and a final titer of 250mg/L. This represents a novel biological method for GTL conversion of alkenes to industrially valuable diols. (C) 2016 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.
  • Anna E. Case, Shota Atsumi
    JOURNAL OF BIOTECHNOLOGY 231 106 - 114 2016年08月 [査読有り][通常論文]
     
    The increase in global temperatures caused by rising CO2 levels necessitates the development of alternative sources of fuel and chemicals. One appealing alternative that has been receiving increased attention in recent years is the photosynthetic conversion of atmospheric CO2 to biofuels and chemical products using genetically engineered cyanobacteria. This can help to not only provide an alternate "greener" source for some of the most popular petroleum based products but it can also help to reduce atmospheric CO2. Utilizing cyanobacteria rather than plants allows for reduced land requirements and reduces competition with food crops. This review discusses advancements in the field since 2012 with a particular emphasis on production of hydrocarbons. (C) 2016 Elsevier B.V. All rights reserved.
  • Jordan T. McEwen, Masahiro Kanno, Shota Atsumi
    METABOLIC ENGINEERING 36 28 - 36 2016年07月 [査読有り][通常論文]
     
    Cyanobacteria are under investigation as a means to utilize light energy to directly recycle CO2 into chemical compounds currently derived from petroleum. Any large-scale photosynthetic production scheme must rely on natural sunlight for energy, thereby limiting production time to only lighted hours during the day. Here, an obligate photoautotrophic cyanobacterium was engineered for enhanced production of 2,3-butanediol (23BD) in continuous light, 12 h:12 h light-dark diurnal, and continuous dark conditions via supplementation with glucose or xylose. This study achieved 23BD production under diurnal conditions comparable to production under continuous light conditions. The maximum 23BD titer was 3.0 g L-1 in 10 d. Also achieving chemical production under dark conditions, this work enhances the feasibility of using Cyanobacteria as industrial chemical-producing microbes. (C) 2016 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.
  • Austin L. Carroll, Shuchi H. Desai, Shota Atsumi
    CURRENT OPINION IN BIOTECHNOLOGY 37 8 - 15 2016年02月 [査読有り][通常論文]
     
    Scents and flavors like those of fresh oranges are no longer limited to just the natural product. Fruit, flower, and essential oil scents have found place in cosmetics, soaps, candles, and food amongst many common household products. With their increasing global demand and difficulty in extractation from the natural source, alternative methods of their production are being sought. One sustainable method is to employ microorganisms for the production of these high value compounds. With the tools of metabolic engineering, microorganisms can be modified to produce compounds such as esters, terpenoids, aldehydes, and methyl ketones. Approaches and challenges for the production of these compounds from microbial hosts are discussed in this review.
  • Nicole E. Nozzi, Shota Atsumi
    ACS SYNTHETIC BIOLOGY 4 11 1197 - 1204 2015年11月 [査読有り][通常論文]
     
    Cyanobacteria have gained popularity among the metabolic engineering community as a tractable photosynthetic host for renewable chemical production. However, though a number of successfully engineered production systems have been reported, long-term genetic stability remains an issue for cyanobacterial systems. The genetic engineering toolbox for cyanobacteria is largely lacking inducible systems for expression control. The characterization of tight regulation systems for use in cyanobacteria may help to alleviate this problem. In this work we explore the function of the IPTG inducible promoter P(L)lacO(1), in the model cyanobacterium Synechococcus elongatus PCC 7942 as well as the effect of gene order within an operon on pathway expression. According to our experiments, P(L)lacO(1) functions well as an inducible promoter in S. elongatus. Additionally, we found that gene order within an operon can strongly influence control of expression of each gene.
  • Yohei Tashiro, Shuchi H. Desai, Shota Atsumi
    NATURE COMMUNICATIONS 6 2015年06月 [査読有り][通常論文]
     
    For an economically competitive biological process, achieving high carbon yield of a target chemical is crucial. In biochemical production, pyruvate and acetyl-CoA are primary building blocks. When sugar is used as the sole biosynthetic substrate, acetyl-CoA is commonly generated by pyruvate decarboxylation. However, pyruvate decarboxylation during acetyl-CoA formation limits the theoretical maximum carbon yield (TMCY) by releasing carbon, and in some cases also leads to redox imbalance. To avoid these problems, we describe here the construction of a metabolic pathway that simultaneously utilizes glucose and acetate. Acetate is utilized to produce acetyl-CoA without carbon loss or redox imbalance. We demonstrate the utility of this approach for isobutyl acetate (IBA) production, wherein IBA production with glucose and acetate achieves a higher carbon yield than with either sole carbon source. These results highlight the potential for this multiple carbon source approach to improve the TMCY and balance redox in biosynthetic pathways.
  • John W. K. Oliver, Shota Atsumi
    METABOLIC ENGINEERING 29 106 - 112 2015年05月 [査読有り][通常論文]
     
    The burning of fossil reserves, and subsequent release of carbon into the atmosphere is depleting the supply of carbon-based molecules used for synthetic materials including plastics, oils, medicines, and glues. To provide for future society, innovations are needed for the conversion of waste carbon (CO2) into organic carbon useful for materials. Chemical production directly from photosynthesis is a nascent technology, with great promise for capture of CO2 using sunlight. To improve low yields, it has been proposed that photosynthetic capacity can be increased by a relaxation of bottlenecks inherent to growth. The limits of carbon partitioning away from growth within the cell and the effect of partitioning on carbon fixation are not well known. Here we show that expressing genes in a pathway between carbon fixation and pyruvate increases partitioning to 2,3-butanediol (23BD) and leads to a 1.8-fold increase in total carbon yield in the cyanobacterium Synechococcus elongatus PCC 7942, Specific 2,3-butanediol production increases 2.4-fold. As partitioning increases beyond 30%, it leads to a steep decline in total carbon yield. The data suggests a local maximum for carbon partitioning from the Calvin Benson cycle that is scalable with light intensity. (C) 2015 international Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.
  • Shuchi H. Desai, Christine A. Rabinovitch-Deere, Zhiliang Fan, Shota Atsumi
    MICROBIAL CELL FACTORIES 14 2015年04月 [査読有り][通常論文]
     
    Background: Liquid fuels needed for the global transportation industry can be produced from sugars derived from plant-based lignocellulosics. Lignocellulosics contain a range of sugars, only some of which (such as cellulose) have been shown to be utilizable by microorganisms capable of producing biofuels. Cellobionic acid makes up a small but significant portion of lignocellulosic degradation products, and had not previously been investigated as an utilizable substrate. However, aldonic acids such as cellobionic acid are the primary products of a promising new group of lignocellulosic-degrading enzymes, which makes this compound group worthy of study. Cellobionic acid doesn't inhibit cellulose degradation enzymes and so its inclusion would increase lignocellulosic degradation efficiency. Also, its use would increase overall product yield from lignocellulose substrate. For these reasons, cellobionic acid has gained increased attention for cellulosic biofuel production. Results: This study describes the discovery that Escherichia coli are naturally able to utilize cellobionic acid as a sole carbon source with efficiency comparable to that of glucose and the construction of an E. coli strain able to produce the drop-in biofuel candidate isobutanol from cellobionic acid. The gene primarily responsible for growth of E. coli on cellobionic acid is ascB, a gene previously thought to be cryptic (expressed only after incurring specific mutations in nearby regulatory genes). In addition to AscB, the ascB knockout strain can be complemented by the cellobionic acid phosphorylase from the fungus Neurospora crassa. An E. coli strain engineered to express the isobutanol production pathway was successfully able to convert cellobionic acid into isobutanol. Furthermore, to demonstrate potential application of this strain in a sequential two-step bioprocessing system, E. coli was grown on hydrolysate (that was degraded by a fungus) and was successfully able to produce isobutanol. Conclusions: These results demonstrate that cellobionic acid is a viable carbon source for biofuel production. This work suggests that with further optimization, a bacteria-fungus co-culture could be used in decreased-cost biomass-based biofuel production systems.
  • Yohei Tashiro, Gabriel M. Rodriguez, Shota Atsumi
    JOURNAL OF INDUSTRIAL MICROBIOLOGY & BIOTECHNOLOGY 42 3 361 - 373 2015年03月 [査読有り][通常論文]
     
    Global energy and environmental concerns have driven the development of biological chemical production from renewable sources. Biological processes using microorganisms are efficient and have been traditionally utilized to convert biomass (i. e., glucose) to useful chemicals such as amino acids. To produce desired fuels and chemicals with high yield and rate, metabolic pathways have been enhanced and expanded with metabolic engineering and synthetic biology approaches. 2-Keto acids, which are key intermediates in amino acid biosynthesis, can be converted to a wide range of chemicals. 2-Keto acid pathways were engineered in previous research efforts and these studies demonstrated that 2-keto acid pathways have high potential for novel metabolic routes with high productivity. In this review, we discuss recently developed 2-keto acid-based pathways.
  • Nicole E. Nozzi, Shuchi H. Desai, Annae E. Case, Shota Atsumi
    METABOLIC ENGINEERING 25 174 - 182 2014年09月 [査読有り][通常論文]
     
    Engineering microbial hosts for the production of higher alcohols looks to combine the benefits of renewable biological production with the useful chemical properties of larger alcohols. In this review we outline the array of metabolic engineering strategies employed for the efficient diversion of carbon flux from native biosynthetic pathways to the overproduction of a target alcohol. Strategies for pathway design from amino acid biosynthesis through 2-keto acids, from isoprenoid biosynthesis through pyrophosphate intermediates, from fatty acid biosynthesis and degradation by tailoring chain length specificity, and the use and expansion of natural solvent production pathways will be covered. (C) 2014 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.
  • Gabriel M. Rodriguez, Shota Atsumi
    METABOLIC ENGINEERING 25 227 - 237 2014年09月 [査読有り][通常論文]
     
    Advances in synthetic biology and metabolic engineering have enabled the construction of novel biological routes to valuable chemicals using suitable microbial hosts. Aldehydes serve as chemical feedstocks in the synthesis of rubbers, plastics, and other larger molecules. Microbial production of alkanes is dependent on the formation of a fatty aldehyde intermediate which is converted to an alkane by an aldehyde deformylating oxygenase (ADO). However, microbial hosts such as Escherichia coli are plagued by many highly active endogenous aldehyde reductases (ALRs) that convert aldehydes to alcohols, which greatly complicates strain engineering for aldehyde and alkane production. It has been shown that the endogenous ALR activity outcompetes the ADO enzyme for fatty aldehyde substrate. The large degree of ALR redundancy coupled with an incomplete database of ALRs represents a significant obstacle in engineering E. cob for either aldehyde or alkane production. In this study, we identified 44 ALR candidates encoded in the E. coli genome using bioinformatics tools, and undertook a comprehensive screening by measuring the ability of these enzymes to produce isobutanol. From the pool of 44 candidates, we found five new ALRs using this screening method (YahK, DkgA, GldA, YbbO, and YghA). Combined deletions of all 13 known ALRs resulted in a 90-99% reduction in endogenous ALR activity for a wide range of aldehyde substrates (C2-C12). Elucidation of the ALRs found in E. coli could guide one in reducing competing alcohol formation during alkane or aldehyde production. (C) 2014 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved
  • John W. K. Oliver, Shota Atsumi
    PHOTOSYNTHESIS RESEARCH 120 3 249 - 261 2014年06月 [査読有り][通常論文]
     
    Photosynthetic chemical production in cyanobacteria is a promising technology for renewable energy, CO2 mitigation, and fossil fuel replacement. Metabolic engineering has enabled a direct biosynthetic process from CO2 fixation to chemical feedstocks and biofuels, without requiring costly production, storage, and breakdown of cellulose or sugars. However, direct production technology is challenged by a need to achieve high-carbon partitioning to products in order to be competitive. This review discusses principles for the design of biosynthetic pathways in cyanobacteria and describes recent advances in relevant genetic tools. This field is at a critical juncture in assessing the strength and feasibility of carbon partitioning. To address this, we have included the stoichiometry of reducing equivalents and carbon conservation for heterologous pathways, and a method for calculating product yields against a theoretical maximum.
  • Gabriel M. Rodriguez, Yohei Tashiro, Shota Atsumi
    NATURE CHEMICAL BIOLOGY 10 4 259 - + 2014年04月 [査読有り][通常論文]
     
    To expand the capabilities of whole-cell biocatalysis, we have engineered Escherichia coli to produce various esters. The alcohol O-acyltransferase (ATF) class of enzyme uses acyl-CoA units for ester formation. The release of free CoA upon esterification with an alcohol provides the free energy to facilitate ester formation. The diversity of CoA molecules found in nature in combination with various alcohol biosynthetic pathways allows for the biosynthesis of a multitude of esters. Small to medium volatile esters have extensive applications in the flavor, fragrance, cosmetic, solvent, paint and coating industries. The present work enables the production of these compounds by designing several ester pathways in E. coli. The engineered pathways generated acetate esters of ethyl, propyl, isobutyl, 2-methyl-1-butyl, 3-methyl-1-butyl and 2-phenylethyl alcohols. In particular, we achieved high-level production of isobutyl acetate from glucose (17.2 g l(-1)). This strategy was expanded to realize pathways for tetradecyl acetate and several isobutyrate esters.
  • Shuchi H. Desai, Christine A. Rabinovitch-Deere, Yohei Tashiro, Shota Atsumi
    APPLIED MICROBIOLOGY AND BIOTECHNOLOGY 98 8 3727 - 3736 2014年04月 [査読有り][通常論文]
     
    Converting lignocellulosics into biofuels remains a promising route for biofuel production. To facilitate strain development for specificity and productivity of cellulosic biofuel production, a user friendly Escherichia coli host was engineered to produce isobutanol, a drop-in biofuel candidate, from cellobiose. A beta-glucosidase was expressed extracellularly by either excretion into the media, or anchoring to the cell membrane. The excretion system allowed for E. coli to grow with cellobiose as a sole carbon source at rates comparable to those with glucose. The system was then combined with isobutanol production genes in three different configurations to determine whether gene arrangement affected isobutanol production. The most productive strain converted cellobiose to isobutanol in titers of 7.64 +/- 0.19 g/L with a productivity of 0.16 g/L/h. These results demonstrate that efficient cellobiose degradation and isobutanol production can be achieved by a single organism, and provide insight for optimization of strains for future use in a consolidated bioprocessing system for renewable production of isobutanol.
  • John W. K. Oliver, Iara M. P. Machado, Hisanari Yoneda, Shota Atsumi
    METABOLIC ENGINEERING 22 76 - 82 2014年03月 [査読有り][通常論文]
     
    A vital goal of renewable technology is the capture and re-energizing of exhausted CO2 into usable carbon products. Cyanobacteria fix CO2 more efficiently than plants, and can be engineered to produce carbon feedstocks useful for making plastics, solvents, and medicines. However, fitness of this technology in the economy is threatened by low yields in engineered strains. Robust engineering of photosynthetic microorganisms is lagging behind model microorganisms that rely on energetic carbon, such as Escherichia coli, due in part to slower growth rates and increased metabolic complexity. In this work we show that protein expression from characterized parts is unpredictable in Synechococcus elongatus sp. strain PCC 7942, and may contribute to slow development. To overcome this, we apply a combinatorial approach and show that modulation of the 5'-untranslated region (UTR) can produce a range of protein expression sufficient to optimize chemical feedstock production from CO2. (C) 2014 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.
  • Hisanari Yoneda, Dean J. Tantillo, Shota Atsumi
    CHEMSUSCHEM 7 1 92 - 95 2014年01月 [査読有り][通常論文]
     
    An Escherichia coli (E. coli) strain was engineered to synthesize 2-butanone from glucose by extending the 2,3-butanediol synthesis reaction sequence catalyzed by exogenous enzymes. To convert 2,3-butanediol to 2-butanone, B12-dependent glycerol dehydratase from Klebsiella pneumoniae was introduced into E. coli. It has been proposed that the enzyme has a weak activity toward 2,3-butanediol. The activity in E. coli is confirmed in this study. Furthermore, co-expressing coenzyme B12 reactivators increased the 2-butanone titer. This demonstration of 2-butanone production by extending the 2,3-butanediol biosynthetic pathway provides the possibility to produce this valuable chemical renewably.
  • Shuchi H. Desai, Shota Atsumi
    CURRENT OPINION IN BIOTECHNOLOGY 24 6 1031 - 1036 2013年12月 [査読有り][通常論文]
     
    National interest and environmental advocates encourage alternatives to petroleum-based products. Besides biofuels, many other valuable chemicals used in every-day life are petroleum derivatives or require petroleum for their production. A plausible alternative to production using petroleum for chemical production is to harvest the abundant carbon dioxide resources in the environment to produce valuable hydrocarbons. Currently, efforts are being made to utilize a natural biological system, photosynthetic microorganisms, to perform this task. Photosynthetic microorganisms are attractive to use for biochemical production because they utilize economical resources for survival: sunlight and carbon dioxide. This review examines the various compounds produced by photosynthetic microorganisms.
  • Tamami Kusakabe, Tsuneyuki Tatsuke, Keigo Tsuruno, Yasutaka Hirokawa, Shota Atsumi, James C. Liao, Taizo Hanai
    METABOLIC ENGINEERING 20 101 - 108 2013年11月 [査読有り][通常論文]
     
    Production of alternate fuels or chemicals directly from solar energy and carbon dioxide using engineered cyanobacteria is an attractive method to reduce petroleum dependency and minimize carbon emissions. Here, we constructed a synthetic pathway composed of acetyl-CoA acetyl transferase (encoded by thl), acetoacetyl-CoA transferase (encoded by atoAD), acetoacetate decarboxylase (encoded by ado) and secondary alcohol dehydrogenase (encoded by adh) in Synechococcus elongatus strain PCC 7942 to produce isopropanol. The enzyme-coding genes, heterogeneously originating from Clostridium acetobutylicum ATCC 824 (thl and adc), Escherichia coli K-12 MG1655 (atoAD) and Clostridium beijerinckii (adh), were integrated into the S. elongatus genome. Under the optimized production conditions, the engineered cyanobacteria produced 26.5 mg/L of isopropanol after 9 days. (C) 2013 Elsevier Inc. All rights reserved.
  • Rabinovitch-Deere Christine A, Oliver John W. K, Rodriguez Gabriel M, Atsumi Shota
    CHEMICAL REVIEWS 113 7 4611 - 4632 2013年07月 [査読有り][通常論文]
  • McEwen Jordan T, Machado Iara M. P, Connor Michael R, Atsumi Shota
    APPLIED AND ENVIRONMENTAL MICROBIOLOGY 79 5 1668 - 1675 2013年03月 [査読有り][通常論文]
  • John W. K. Oliver, Iara M. P. Machado, Hisanari Yoneda, Shota Atsumi
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 110 4 1249 - 1254 2013年01月 [査読有り][通常論文]
     
    Conversion of CO2 for the synthesis of chemicals by photosynthetic organisms is an attractive target for establishing independence from fossil reserves. However, synthetic pathway construction in cyanobacteria is still in its infancy compared with model fermentative organisms. Here we systematically developed the 2,3-butanediol (23BD) biosynthetic pathway in Synechococcus elongatus PCC7942 as a model system to establish design methods for efficient exogenous chemical production in cyanobacteria. We identified 23BD as a target chemical with low host toxicity, and designed an oxygen-insensitive, cofactor-matched biosynthetic pathway coupled with irreversible enzymatic steps to create a driving force toward the target. Production of 23BD from CO2 reached 2.38 g/L, which is a significant increase for chemical production from exogenous pathways in cyanobacteria. This work demonstrates that developing strong design methods can continue to increase chemical production in cyanobacteria.
  • Iara M. P. Machado, Shota Atsumi
    JOURNAL OF BIOTECHNOLOGY 162 1 50 - 56 2012年11月 [査読有り][通常論文]
     
    The development of new technologies for production of alternative fuel became necessary to circumvent finite petroleum resources, associate rising costs, and environmental concerns due to rising fossil fuel CO2 emissions. Several alternatives have been proposed to develop a sustainable industrial society and reduce greenhouse emissions. The idea of biological conversion of CO2 to fuel and chemicals is receiving increased attention. In particular, the direct conversion of CO2 with solar energy to biofuel by photosynthetic microorganisms such as microalgae and cyanobacteria has several advantages compared to traditional biofuel production from plant biomass. Photosynthetic microorganisms have higher growth rates compared with plants, and the production systems can be based on non-arable land. The advancement of synthetic biology and genetic manipulation has permitted engineering of cyanobacteria to produce non-natural chemicals typically not produced by these organisms in nature. This review addresses recent publications that utilize different approaches involving engineering cyanobacteria for production of high value chemicals including biofuels. Published by Elsevier B.V.
  • Jordan T. McEwen, Shota Atsumi
    CURRENT OPINION IN BIOTECHNOLOGY 23 5 744 - 750 2012年10月 [査読有り][通常論文]
     
    Global energy and environmental concerns have stimulated increased efforts in synthesizing petroleum-derived products from renewable resources. Biological production of metabolites for fuel is increasingly becoming a feasible, renewable, environmentally sound alternative. However, many of these chemicals are not highly produced in any known native organism. Here we review the current progress of modifying microorganisms with heterogeneous elements for the production of biofuels. This strategy has been extensively employed in a variety of hosts for the development of production of various alcohols, fatty acids, alkenes and alkanes.
  • Gabriel M. Rodriguez, Shota Atsumi
    MICROBIAL CELL FACTORIES 11 2012年06月 [査読有り][通常論文]
     
    Background: Increasing global demand and reliance on petroleum-derived chemicals will necessitate alternative sources for chemical feedstocks. Currently, 99% of chemical feedstocks are derived from petroleum and natural gas. Renewable methods for producing important chemical feedstocks largely remain unaddressed. Synthetic biology enables the renewable production of various chemicals from microorganisms by constructing unique metabolic pathways. Here, we engineer Escherichia coli for the production of isobutyraldehyde, which can be readily converted to various hydrocarbons currently derived from petroleum such as isobutyric acid, acetal, oxime and imine using existing chemical catalysis. Isobutyraldehyde can be readily stripped from cultures during production, which reduces toxic effects of isobutyraldehyde. Results: We adopted the isobutanol pathway previously constructed in E. coli, neglecting the last step in the pathway where isobutyraldehyde is converted to isobutanol. However, this strain still overwhelmingly produced isobutanol (1.5 g/L/OD600 (isobutanol) vs 0.14 g/L/OD600 (isobutyraldehyde)). Next, we deleted yqhD which encodes a broad-substrate range aldehyde reductase known to be active toward isobutyraldehyde. This strain produced isobutanol and isobutyraldehyde at a near 1:1 ratio, indicating further native isobutyraldehyde reductase (IBR) activity in E. coli. To further eliminate isobutanol formation, we set out to identify and remove the remaining IBRs from the E. coli genome. We identified 7 annotated genes coding for IBRs that could be active toward isobutyraldehyde: adhP, eutG, yiaY, yjgB, betA, fucO, eutE. Individual deletions of the genes yielded only marginal improvements. Therefore, we sequentially deleted all seven of the genes and assessed production. The combined deletions greatly increased isobutyraldehyde production (1.5 g/L/OD600) and decreased isobutanol production (0.4 g/L/OD600). By assessing production by overexpression of each candidate IBR, we reveal that AdhP, EutG, YjgB, and FucO are active toward isobutyraldehyde. Finally, we assessed long-term isobutyraldehyde production of our best strain containing a total of 15 gene deletions using a gas stripping system with in situ product removal, resulting in a final titer of 35 g/L after 5 days. Conclusions: In this work, we optimized E. coli for the production of the important chemical feedstock isobutyraldehyde by the removal of IBRs. Long-term production yielded industrially relevant titers of isobutyraldehyde with in situ product removal. The mutational load imparted on E. coli in this work demonstrates the versatility of metabolic engineering for strain improvements.
  • Edna N. Lamsen, Shota Atsumi
    FRONTIERS IN MICROBIOLOGY 3 2012年 [査読有り][通常論文]
     
    The growing need to address current energy and environmental problems has sparked an interest in developing improved biological methods to produce liquid fuels from renewable sources. While microbial ethanol production is well established, higher-chain alcohols possess chemical properties that are more similar to gasoline. Unfortunately, these alcohols (except 1-butanol) are not produced efficiently in natural microorganisms, and thus economical production in industrial volumes remains a challenge. Synthetic biology, however, offers additional tools to engineer synthetic pathways in user-friendly hosts to help increase titers and productivity of these advanced biofuels. This review concentrates on recent developments in synthetic biology to produce higher-chain alcohols as viable renewable replacements for traditional fuel.
  • Shota Atsumi, Tung-Yun Wu, Iara M. P. Machado, Wei-Chih Huang, Pao-Yang Chen, Matteo Pellegrini, James C. Liao
    MOLECULAR SYSTEMS BIOLOGY 6 2010年12月 [査読有り][通常論文]
     
    Escherichia coli has been engineered to produce isobutanol, with titers reaching greater than the toxicity level. However, the specific effects of isobutanol on the cell have never been fully understood. Here, we aim to identify genotype-phenotype relationships in isobutanol response. An isobutanol-tolerant mutant was isolated with serial transfers. Using whole-genome sequencing followed by gene repair and knockout, we identified five mutations (acrA, gatY, tnaA, yhbJ, and marCRAB) that were primarily responsible for the increased isobutanol tolerance. We successfully reconstructed the tolerance phenotype by combining deletions of these five loci, and identified glucosamine-6-phosphate as an important metabolite for isobutanol tolerance, which presumably enhanced membrane synthesis. The isobutanol-tolerant mutants also show increased tolerance to n-butanol and 2-methyl-1-butanol, but showed no improvement in ethanol tolerance and higher sensitivity to hexane and chloramphenicol than the parental strain. These results suggest that C4, C5 alcohol stress impacts the cell differently compared with the general solvent or antibiotic stresses. Interestingly, improved isobutanol tolerance did not increase the final titer of isobutanol production. Molecular Systems Biology 6: 449; published online 21 December 2010; doi:10.1038/msb.2010.98
  • Shota Atsumi, Tung-Yun Wu, Eva-Maria Eckl, Sarah D. Hawkins, Thomas Buelter, James C. Liao
    APPLIED MICROBIOLOGY AND BIOTECHNOLOGY 85 3 651 - 657 2010年01月 [査読有り][通常論文]
     
    Biofuels synthesized from renewable resources are of increasing interest because of global energy and environmental problems. We have previously demonstrated production of higher alcohols from Escherichia coli using a 2-keto acid-based pathway. Here, we have compared the effect of various alcohol dehydrogenases (ADH) for the last step of the isobutanol production. E. coli has the yqhD gene which encodes a broad-range ADH. Isobutanol production significantly decreased with the deletion of yqhD, suggesting that the yqhD gene on the genome contributed to isobutanol production. The adh genes of two bacteria and one yeast were also compared in E. coli harboring the isobutanol synthesis pathway. Overexpression of yqhD or adhA in E. coli showed better production than ADH2, a result confirmed by activity measurements with isobutyraldehyde.
  • Ieong Wong, Shota Atsumi, Wei-Chih Huang, Tung-Yun Wu, Taizo Hanai, Miu-Ling Lam, Ping Tang, Jian Yang, James C. Liao, Chih-Ming Ho
    LAB ON A CHIP 10 20 2710 - 2719 2010年 [査読有り][通常論文]
     
    Significance of single cell measurements stems from the substantial temporal fluctuations and cell-cell variability possessed by individual cells. A major difficulty in monitoring surface non-adherent cells such as bacteria and yeast is that these cells tend to aggregate into clumps during growth, obstructing the tracking or identification of single-cells over long time periods. Here, we developed a microfluidic platform for long term single-cell tracking and cultivation with continuous media refreshing and dynamic chemical perturbation capability. The design highlights a simple device-assembly process between PDMS microchannel and agar membrane through conformal contact, and can be easily adapted by microbiologists for their routine laboratory use. The device confines cell growth in monolayer between an agar membrane and a glass surface. Efficient nutrient diffusion through the membrane and reliable temperature maintenance provide optimal growth condition for the cells, which exhibited fast exponential growth and constant distribution of cell sizes. More than 24 h of single-cell tracking was demonstrated on a transcription-metabolism integrated synthetic biological model, the gene-metabolic oscillator. Single cell morphology study under alcohol toxicity allowed us to discover and characterize cell filamentation exhibited by different E. coli isobutanol tolerant strains. We believe this novel device will bring new capabilities to quantitative microbiology, providing a versatile platform for single cell dynamic studies.
  • Michael R. Connor, Shota Atsumi
    JOURNAL OF BIOMEDICINE AND BIOTECHNOLOGY 2010年 [査読有り][通常論文]
     
    The advancement of microbial processes for the production of renewable liquid fuels has increased with concerns about the current fuel economy. The development of advanced biofuels in particular has risen to address some of the shortcomings of ethanol. These advanced fuels have chemical properties similar to petroleum-based liquid fuels, thus removing the need for engine modification or infrastructure redesign. While the productivity and titers of each of these processes remains to be improved, progress in synthetic biology has provided tools to guide the engineering of these processes through present and future challenges.
  • Shota Atsumi, Wendy Higashide, James C. Liao
    NATURE BIOTECHNOLOGY 27 12 1177 - U142 2009年12月 [査読有り][通常論文]
     
    Global climate change has stimulated efforts to reduce CO(2) emissions. One approach to addressing this problem is to recycle CO(2) directly into fuels or chemicals using photosynthesis. Here we genetically engineered Synechococcus elongatus PCC7942 to produce isobutyraldehyde and isobutanol directly from CO(2) and increased productivity by overexpression of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco). Isobutyraldehyde is a precursor for the synthesis of other chemicals, and isobutanol can be used as a gasoline substitute. The high vapor pressure of isobutyraldehyde allows in situ product recovery and reduces product toxicity. The engineered strain remained active for 8 d and produced isobutyraldehyde at a higher rate than those reported for ethanol(1), hydrogen(2) or lipid(3) production by cyanobacteria or algae. These results underscore the promise of direct bioconversion of CO(2) into fuels and chemicals, which bypasses the need for deconstruction of biomass.
  • Shota Atsumi, Zhen Li, James C. Liao
    APPLIED AND ENVIRONMENTAL MICROBIOLOGY 75 19 6306 - 6311 2009年10月 [査読有り][通常論文]
     
    A pathway toward isobutanol production previously constructed in Escherichia coli involves 2-ketoacid decarboxylase (Kdc) from Lactococcus lactis that decarboxylates 2-ketoisovalerate (KIV) to isobutyraldehyde. Here, we showed that a strain lacking Kdc is still capable of producing isobutanol. We found that acetolactate synthase from Bacillus subtilis (AlsS), which originally catalyzes the condensation of two molecules of pyruvate to form 2-acetolactate, is able to catalyze the decarboxylation of KIV like Kdc both in vivo and in vitro. Mutational studies revealed that the replacement of Q487 with amino acids with small side chains (Ala, Ser, and Gly) diminished only the decarboxylase activity but maintained the synthase activity.
  • Shota Atsumi, James C. Liao
    APPLIED AND ENVIRONMENTAL MICROBIOLOGY 74 24 7802 - 7808 2008年12月 [査読有り][通常論文]
     
    Biofuels synthesized from renewable resources are of increasing interest because of global energy and environmental problems. We have previously demonstrated production of higher alcohols from Escherichia coli using a 2-keto acid-based pathway. Here, we took advantage of the growth phenotype associated with 2-keto acid deficiency to construct a hyperproducer of 1-propanol and 1-butanol by evolving citramalate synthase (CimA) from Methanococcus jannaschii. This new pathway, which directly converts pyruvate to 2-ketobutyrate, bypasses threonine biosynthesis and represents the shortest keto acid-mediated pathway for producing 1-propanol and 1-butanol from glucose. Directed evolution of CimA enhanced the specific activity over a wide temperature range (30 to 70 C). The best CimA variant was found to be insensitive to feedback inhibition by isoleucine in addition to the improved activity. This CimA variant enabled 9- and 22-fold higher production levels of 1-propanol and 1-butanol, respectively, compared to the strain expressing the wild-type CimA. This work demonstrates (i) the first production of 1-propanol and 1-butanol using the citramalate pathway and (ii) the benefit of the 2-keto acid pathway that enables a growth-based evolutionary strategy to improve the production of non-growth-related products.
  • Shota Atsumi, Anthony F. Cann, Michael R. Connor, Claire R. Shen, Kevin M. Smith, Mark P. Brynildsen, Katherine J. Y. Chou, Taizo Hanai, James C. Liao
    METABOLIC ENGINEERING 10 6 305 - 311 2008年11月 [査読有り][通常論文]
     
    Compared to ethanol, butanol offers many advantages as a substitute for gasoline because of higher energy content and higher hydrophobicity. Typically, 1-butanol is produced by Clostridium in a mixed-product fermentation. To facilitate strain improvement for specificity and productivity, we engineered a synthetic pathway in Escherichia coli and demonstrated the production of 1-butanol from this non-native user-friendly host. Alternative genes and competing pathway deletions were evaluated for 1-butanol production. Results show promise for using E. coli for 1-butanol production. (C) 2007 Elsevier Inc. All rights reserved.
  • Shota Atsumi, James C. Liao
    CURRENT OPINION IN BIOTECHNOLOGY 19 5 414 - 419 2008年10月 [査読有り][通常論文]
     
    Global energy and environmental problems have stimulated increasing efforts toward synthesizing liquid biofuels as transportation energy. Compared to the traditional biofuel, ethanol, advanced biofuels should offer advantages such as higher energy density, lower hygroscopicity, lower vapor pressure, and compatibility with existing transportation infrastructure. However, these fuels are not synthesized economically using native organisms. Metabolic engineering offers an alternative approach in which synthetic pathways are engineered into user-friendly hosts for the production of these fuel molecules. These hosts could be readily manipulated to improve the production efficiency. This review summarizes recent progress in the engineering of Escherichia coli to produce advanced biofuels.
  • Shota Atsumi, Taizo Hanai, James C. Liao
    NATURE 451 7174 86 - U13 2008年01月 [査読有り][通常論文]
     
    Global energy and environmental problems have stimulated increased efforts towards synthesizing biofuels from renewable resources(1-3). Compared to the traditional biofuel, ethanol, higher alcohols offer advantages as gasoline substitutes because of their higher energy density and lower hygroscopicity. In addition, branched- chain alcohols have higher octane numbers compared with their straight- chain counterparts. However, these alcohols cannot be synthesized economically using native organisms. Here we present a metabolic engineering approach using Escherichia coli to produce higher alcohols including isobutanol, 1-butanol, 2- methyl- 1- butanol, 3- methyl- 1- butanol and 2- phenylethanol from glucose, a renewable carbon source. This strategy uses the host's highly active amino acid biosynthetic pathway and diverts its 2- keto acid intermediates for alcohol synthesis. In particular, we have achieved high- yield, high- specificity production of isobutanol from glucose. The strategy enables the exploration of biofuels beyond those naturally accumulated to high quantities in microbial fermentation.
  • Shota Atsumi, John W. Little
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 103 50 19045 - 19050 2006年12月 [査読有り][通常論文]
     
    Analysis of synthetic gene regulatory circuits can provide insight into circuit behavior and evolution. An alternative approach is to modify a naturally occurring circuit, by using genetic methods to select functional circuits and evolve their properties. We have applied this approach to the circuitry of phage lambda. This phage grows lytically, forms stable lysogens, and can switch from this regulatory state to lytic growth. Genetic selections are available for each behavior. We previously replaced lambda Cro in the intact phage with a module including Lac repressor, whose function is tunable with small molecules, and several cis-acting sites. Here, we have in addition replaced lambda Cl repressor with another tunable module, Tet repressor and several cis-acting sites. Tet repressor lacks several important properties of Cl, including positive autoregulation and cooperative DNA binding. Using a combinatorial approach, we isolated phage variants with behavior similar to that of WT lambda. These variants grew lytically and formed stable lysogens. Lysogens underwent prophage induction upon addition of a ligand that weakens binding by the Tet repressor. Strikingly, however, addition of a ligand that weakens binding by Lac repressor also induced lysogens. This finding indicates that Lac repressor was present in the lysogens and was necessary for stable lysogeny. Therefore, these isolates had an altered wiring diagram from that of lambda. We speculate that this complexity is needed to compensate for the missing features. Our method is generally useful for making customized gene regulatory circuits whose activity is regulated by small molecules or protein cofactors.
  • S Atsumi, JW Little
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 103 12 4558 - 4563 2006年03月 [査読有り][通常論文]
     
    Using a module exchange approach, we have tested a long-standing model for the role of Cro repressor in lambda prophage induction. This epigenetic switch from lysogeny to the lytic state occurs on activation of the host SOS system, which leads to specific cleavage of CI repressor. it has been proposed that Cro repressor, which operates during lytic growth and which we shall term the lytic repressor, is crucial to prophage induction. In this view, Cro binds to the O(R)3 operator, thereby repressing the cl gene and making the switch irreversible. Here we tested this model by replacing lambda Cro with a dimeric form of Lac repressor and adding several lac operators. This approach allowed us to regulate the function of the lytic repressor at will and to prevent it from repressing cl, because lac repressor could not repress P-RM in our constructs. Repression of cl by the lytic repressor was not required for prophage induction to occur. However, our evidence suggests that this binding can make induction more efficient, particularly at intermediate levels of DNA damage that otherwise cause induction of only a fraction of the population. These results indicate that this strategy of module exchange will have broad applications for analysis of gene regulatory circuits.
  • S Atsumi, JW Little
    GENES & DEVELOPMENT 18 17 2086 - 2094 2004年09月 [査読有り][通常論文]
     
    Bistable gene regulatory circuits can adopt more than one stable epigenetic state. To understand how natural circuits have this and other systems properties, several groups have designed regulatory circuits de novo. Here we describe an alternative approach. We have modified an existing bistable circuit, that of phage lambda. With this approach, we used powerful genetic selections to identify functional circuits and selected for variants with altered behavior. The lambda circuit involves two antagonistic repressors, CI and Cro. We replaced lambda Cro with a module that included Lac repressor and several lac operators. Using a combinatorial approach, we isolated variants with different types of regulatory behavior. Several resembled wild-type lambda-they could grow lytically, could form highly stable lysogens, and carried out prophage induction. Another variant could form stable lysogens in the presence of a ligand for Lac repressor but switched to the lytic state when the ligand was removed. Several isolates evolved toward a desired behavior under selective pressure. These results strongly support the idea that complex circuits can arise during the course of evolution by a combination of simpler regulatory modules. They also underscore the advantages of modifying a natural circuit as an approach to understanding circuit design, systems behavior, and circuit evolution.
  • Y Ikawa, K Tsuda, S Matsumura, S Atsumi, T Inoue
    NUCLEIC ACIDS RESEARCH 31 5 1488 - 1496 2003年03月 [査読有り][通常論文]
     
    A hypothetical evolutionary pathway from a ribozyme to a catalytic RNA-protein complex (RNP) is proposed and examined. In this hypothesis for an early phase of molecular evolution, one RNA-RNA interaction in the starting ribozyme is replaced with an RNA-protein interaction via two intermediary stages. At each stage, the original RNA-RNA interaction and a newly introduced RNA-protein interaction are designed to coexist. The catalytic RNPs corresponding to the intermediary stages were constructed by employing the Tetrahymena ribozyme together with molecular modeling. Analyses of the RNPs indicate that the protein can fully replace the original role of the RNA-RNA interaction in the starting ribozyme and that the association of a protein with a ribozyme might be beneficial for improving the ribozymatic activity.
  • S Atsumi, Y Ikawa, H Shiraishi, T Inoue
    NUCLEIC ACIDS RESEARCH 31 2 661 - 669 2003年01月 [査読有り][通常論文]
     
    In vitro and in vivo selection techniques are developed to constitute new RNA-peptide interactions. The selection strategy is designed by employing a catalytic RNP consisting of a derivative of the Tetrahymena ribozyme and an artificial RNA-binding protein. An arginine-rich RNA-binding motif and its target RNA motif in the RNP are substituted with randomized sequences and used for the selection experiments. Previously unknown binding motifs are obtained and the newly established interactions have been indispensable for assembling a catalytically active RNP. The method employed in this study is useful for making customized self-splicing intron RNAs whose activity is regulated by protein cofactors.
  • Ikawa Y, Tsuda K, Matsumura S, Atsumi S, Inoue T
    Nucleic acids research. Supplement (2001) 2 119 - 120 2002年 [査読有り][通常論文]
  • Ikawa Y, Nohmi K, Atsumi S, Shiraishi H, Inoue T, Jou
    130 2 251 - 255 2001年08月 [査読有り][通常論文]
     
    Ikawa Y, Nohmi K, Atsumi S, Shiraishi H, Inoue T, Journal of biochemistry, 2001, vol. 130, no. 2, pp. 251-255
  • Atsumi S, Ikawa Y, Shiraishi H, Inoue T
    EMBO Journal 20 19 5453 - 5460 2001年 [査読有り][通常論文]

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