Laboratory of Fermentation Physiology and Applied Microbiology

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2.3.7

Laboratory:Fermentation Physiology and Applied
Microbiology


















Member:

Professor


Ogawa, Jun, Dr. Agric. Sci.








Associate Professor Kataoka, Michihiko, Dr. Agric. Sci.







Assistant Professor Sakuradani, Eiji, Dr. Agric. Sci.








Doctor's program


1













Master's Program


20













Undergraduate

5













Post-Doctoral fellow 4













Researcher



4













































A. Research Activities (2009.4-2010.3)









A-1. Main Subjects












a) Microbial production of useful lipids
We have found that mycelia of the fungus Mortierella alpina, which was isolated from soil of
Kyoto, are rich source of a polyunsaturated fatty acid, arachidonic acid. Furthermore, we
succeeded in the selective production of various polyunsaturated fatty acids, for example,
dihomo-g-linolenic acid and EPA, by controlling of the culture conditions and breeding of the
mutant producers. These strains are now under investigation at practical level with
10,000-liter jar fermenter. We are doing enzymatic and genetic analysis of the filamentous
fungus and trying to establish novel transformation system for the fungus. We are making
further research in microorganisms for the production of novel functional lipids, and found
that lactic acid bacteria produce conjugated fatty acids. Further development of conjugated
linoleic acid production by lactic acid bacteria is on going.
b) Microbial production of optically active compounds
Reactions catalyzed by enzymes display for greater specificities than more conventional
forms of organic reactions. Among these specificities, stereospecificity is one of the most
excellent properties. To overcome the disadvantage of a conventional synthetic process of
optically active compounds (amino acids, vitamins and so on), i.e., the troublesome resolution
of a racemic mixture, microbial transformations with enzymes possessing stereospecificities
(carbonyl reductases, lactonase, aldolase, etc.) have been applied to the asymmetric synthesis
of them. Studies on enzyme and protein chemistry of the enzymes involved in these
reactions are also carried out.
c) Functional analysis and application of novel microbial enzymes
Microbial transformations of nucleic acid-related compounds are studied. The enzymes
involved in these transformations are applied for followings: 1) dihydropyrimidinase, which
functions in nucleic acid-base degradation, is applied for D-amino acids production from
DL-5-monosubstituted hydantoins, 2) the enzymes involved in creatinine metabolism are
applied to clinical diagnosis of renal dysfunction. A variety of microbial oxidases, such as
peroxidases and laccases, are screened and its applicabilities are evaluated. Peroxidases
from filamentous fungi are now under development as bleaching agents in clothes washing
and as analytical tools for diagnosis. Laccases from basidiomycete are examined as potential
tools for bioremediation, novel bio-control reagents and dyeing/bleaching reagents.
d) Microbial nitrile degradation and its application
Nitriles are widely manufactured and extensively used by chemical industries. Some
microorganisms have the ability to utilize nitriles as carbon and/or nitrogen sources. The
microbial degradation of nitriles has been found to proceed through two enzymatic pathways.
Nitrilase catalyzes the direct cleavage on nitriles to the corresponding acids and ammonia. In
the second pathway, nitriles are catabolized in two stages, via conversion to the corresponding
amides by nitrile hydratase and then the acids plus ammonia by amidase. These
nitrile-converting enzymes are expected to have great potential as catalysts in organic
chemical processing, because of the mild conditions, quantitative yields, absence of
by-products and in some cases enantio- or regioselectivity. Thus, we established the process
for the industrial production of acrylamide, an important chemical commodity, from
acrylonitrile using the Rhodococcus rhodochrous J1 nitrile hydratase in 1991. The same
enzymatic reaction was applied to nicotinamide production from 3-cyanopyridine and
industrialized from 1998.
e) Collaboration with the Research Division of Microbial Sciences
The purposes are to discover and develop the latent possibilities of microorganisms and to
tune these possibilities into useful applications such as bioprocess design, bio-fuel production,
biomass utilization, bioremediation, bio-control, probiotics development, functional food
production, pharmaceutical synthesis, etc. For the purposes, the research division
encourages broad collections of various kinds of microorganisms and fundamental
understandings of microbial metabolisms. The examples of the research topics are: 1)
analysis on microbial amino acid metabolism for production of hydroxyl amino acids useful
as functional food materials, 2) analysis on microbial reductive fatty acid metabolism for the
production of conjugated fatty acids useful for prevention of lifestyle-related diseases.
Furthermore, researches on nucleic acid metabolism in lactic acid bacteria for the
development of probiotics for hyperuricemia prevention, microbial laccase as a module of
bio-battery, and extracellular production of lipids by filamentous fungi are also on going.

















A-2.Publications and presentations










a) Publications














Books
















- Kataoka, M., N. Urano, S. Shimizu. Microbial production of bio-propanol by
metabolic engineering. Research and development of second-generation bio-fuels (eds.
By M. Ueda, E. Fukusaki, K. Yoshida), CMC Press, Tokyo, 189-195 (2009). (in
Japanese)

- Kataoka, M., S. Shimizu. 5-1-5-1. Enzymatic production of chiral alcohols (1).
Thechnology of Enzyme (ed. by M. Komiyama), NTS Press, 418-422 (2010). (in
Japanese)

- Ogawa, J., E. Sakuradani, S. Kishino, A. Ando, S. Shimizu. 5-1-5-2. Useful fatty
acids production. Thechnology of Enzyme (ed. by M. Komiyama), NTS Press, 430-433
(2010). (in Japanese)

- Shimizu, S., M. Kataoka. Aldehyde reductase (Carbonyl reductase). Encyclopedia of
Industrial Biotechnology: Bioprocess, Bioseparation, and Cell Technology (ed. by M.C.
Flickinger) John Wiley & Sons, in press (2010).

- Shimizu, S., M. Kataoka. Lactonohydrolase (Lactonase ). Encyclopedia of Industrial
Biotechnology: Bioprocess, Bioseparation, and Cell Technology (ed. by M.C.
Flickinger) John Wiley & Sons, in press (2010).

- Sakuradani, E., J. Ogawa, S. Kishino, A. Ando, K. Yokozeki, S. Shimizu. Oils,
Microbial Production. Encyclopedia of Industrial Biotechnology: Bioprocess,
Bioseparation, and Cell Technology (ed. by M.C. Flickinger) John Wiley & Sons, in
press (2010).

- Shimizu, S., M. Kataoka. Pantothenic Acid and Related Compounds. Encyclopedia of
Industrial Biotechnology: Bioprocess, Bioseparation, and Cell Technology (ed. by M.C.
Flickinger) John Wiley & Sons, in press (2010).

- Ogawa, J., N. Horinouchi, S. Shimizu. Retrosynthetic production of
2'-deoxyribonucleoside from glucose, acetaldehyde, and nucleobase through multistep
enzyme reactions. Biocatalysis and Agricultural Biotechnology (ed. by C.T. Hou, J.-F.
Shaw) CRC Press, 269-278 (2009).

Original Papers















- Ando, A., J. Ogawa, S. Kishino, T. Ito, N. Shirasaka, E. Sakuradani, K. Yokozeki, S.
Shimizu. Fatty acid desturation and elongation reactions of Trichoderma sp. 1-OH-2-3.
J. Am. Oil Chem. Soc., 86, 227-233 (2009).

- Ando, A., J. Ogawa, S. Sugimoto, S. Kishino, E. Sakuradani, K. Yokozeki, S.
Shimizu. Selective production of cis-9, trans-11 isomer of conjugated linoleic acidfrom
trans-vaccenic acid methyl ester by Delacroixia coronata. J. Appl. Microbiol., 106,
1697-1704 (2009).

- Ando, A., E. Sakuradani, K. Horinaka, J. Ogawa, S. Shimizu. Transformation of an
oleaginous zygomycete Mortierella alpina 1S-4 with the carboxin resistance gene
conferred by mutation of the iron-sulfur subunit of succinate dehydrogenase.
Curr.Genet., 55 (3), 349-356 (2009).

- Ando, A., Y. Sumida, H. Negoro, D.A. Suroto, J. Ogawa, E. Sakuradani, S. Shimizu.
Establishment of Agrobacterium tumefaciens-mediated transformation of an oleaginous
fungus, Mortierella alpina 1S-4, and its application for eicosapentaenoic acid producer
breeding. Appl. Environ. Microbiol., 75 (17), 5529-5535 (2009).

- Horinouchi, N., T. Kawano, T. Sakai, S. Matsumoto, M. Sasaki, Y. Mikami, J .Ogawa,
S. Shimizu. Screening and characterization of a phosphopentomutase useful for
enzymatic production of 2'-deoxyribonucleoside. N. Biotechnol., 26(1-2), 75-82 (2009).


- Isobe, K., T. Takahashi, J. Ogawa, M. Kataoka, S. Shimizu. Production and
characterization of alcohol oxidase from Penicillium purpurescens AIU 063.. J. Biosci.
Bioeng., 107 (2), 108-112 (2009).

- Kishino, S., J. Ogawa, K. Yokozeki, S. Shimizu. Metabolic diversity in
biohydrogenation of polyunsaturated fatty acids by lactic acid bacteria involving
conjugated fatty acid production. Appl. Microbiol. Biotechnol., 84, 87-97 (2009).

- Kishino, S., J. Ogawa, K. Yokozeki, S. Shimizu. Microbial production of conjugated
fatty acids. Lipid Technology, 21(8-9), 177-181 (2009).

- Kodera, T., SV. Smirnov, NN. Samsonova, YI. Kozlov, R. Koyama, M. Hibi, J.
Ogawa, K. Yokozeki, S. Shimizu. A novel l-isoleucine hydroxylating enzyme,
l-isoleucine dioxygenase from Bacillus thuringiensis, produces
(2S,3R,4S)-4-hydroxyisoleucine.. Biochem. Biophys. Res. Commun., 390(3), 506-510
(2009).

- Matsushita, N., M. Miyashita, Y. Ichiki, T. Ogura, E. Sakuradani, Y. Nakagawa, S.
Shimizu, H. Miyagawa. Purification and cDNA cloning of LaIT2, a novel insecticidal
toxin from venom of the scorpion Liocheles australasiae. Biosci. Biotechnol. Biochem.,
73(12), 2769-2772 (2009).

- Muramatsu, M., C. Ohto, S. Obata, E. Sakuradani, S. Shimizu. Alkaline pH enhances
farnesol production by Saccharomyces cerevisiae. J. Biosci. Bioeng., 108 (1),52-55
(2009).

- Ogawa, J., J. Mano, T. Hagishita, S. Shimizu. Enantioselective ester hydrolase from
Sphingobacterium sp. 238C5 useful for chiral resolution of β-phenylalanine and for its
β-peptide synthesis. Journal of Molecular Catalysis B: Enzymatic, 60(3-4), 138-144
(2009).

- Ohto, C., M. Muramatsu, S. Obata, E. Sakuradani, S. Shimizu. Overexpression of the
gene encoding HMG-CoA reductase in Saccharomyces cerevisiae for production of
prenyl alcohols. Appl. Microbiol. Biotechnol., 82, 837-845 (2009).

- Ohto, C., M. Muramatsu, S. Obata, E. Sakuradani, S. Shimizu. Prenyl alcohol
production by expression of exogenous isopentenyl diphosphate isomerase and farnesyl
diphosphate synthase genes in Escherichia coli. Biosci. Biotechnol. Biochem., 73 (1),
186-188 (2009).

- Sakuradani, E., T. Abe, S. Shimizu. Identification of mutation sites on w3 desaturase
genes from Mortierella alpina 1S-4 mutants. J. Biosci. Bioeng., 107 (1), 7-9 (2009).

- Sakuradani, E., M. Nojiri, H. Suzuki, S. Shimizu. Identification of a novel fatty acid
elongase with a wide substrate specificity from arachidonic acid-producing fungus
Mortierella alpina 1S-4. Appl. Microbiol. Biotechnol., 84(4), 709-716 (2009).

- Takeshita, D., M. Kataoka, T. Miyakawa, K. Miyazono, A. Uzura, K. Nagata, S.
Shimizu, M. Tanokura. Crystallization and preliminary X-ray analysis of the
NADPH-dependent3-quinuclidinone reductase from Rhodotorula rubra. Acta Cryst.,
F65, 645-647 (2009).

- Tokuhiro, K., M. Muramatsu, C. Ohto, T. Kawaguchi, S. Obata, N. Muramoto, M.
Hirai, H. Takahashi, A. Kondo, E. Sakuradani, S. Shimizu. Overproduction of
geranylgeraniol by metabolically engineered Saccharomyces cerevisiae. Appl. Environ.
Microbiol., 75 (17), 5536-5543 (2009).

- Uzura, A., F. Nomoto, A. Sakoda, Y. Nishimoto, M. Kataoka, S. Shimizu.
Stereoselective synthesis of (R)-3-quinuclidinol through asymmetric reduction of
3-quinuclidinone with NADPH-dependent 3-quinuclidinone reductase from
Rhodotorula rubra. Appl. Microbiol. Biotechnol., 83 (4), 617-626 (2009).

- Yamamura, A., S. Maruoka, J. Ohtsuka, T. Miyakawa, K. Nagata, M. Kataoka, N.
Kitamura, S. Shimizu, M. Tanokura. Expression, purification, crystallization and
preliminary X-ray analysis of conjugated polyketone reductase C2 (CPR-C2) from
Candida parapsilosis IFO 0708. Acta Cryst., F65, 1145-1148 (2009).

- Murai, M., T. Yamashita, M. Senoh, Y. Mashimo, M. Kataoka, H. Kosaka, A.
Matsuno-Yagi, T. Yagi, H. Miyoshi. Characterization of the ubiquinone binding site in
alternative NADH-quinone oxidoreductase of Saccharomyces cerevisiae by
photoaffinity labeling. Biochemistry., 49, 2973-2980 (2010).

Reviews
















- Sakuradani, E., A. Ando, J. Ogawa, S. Shimizu. Improved production of various
polyunsaturated fatty acids through filamentous fungus Mortierella alpina breeding.
Appl. Microbiol. Biotechnol., 84, 1-10 (2009).

- Sakuradani, E., A. Ando, J. Ogawa, S. Shimizu. Production of functional lipids by
microorganisms: arachidonic acid and related polyunsaturated fatty acids.
Tanpakushitsu Kakusan Koso., 54(6), 725-734 (2009). (in Japanese)

- Sakuradani, E., S. Shimizu. Single cell oil production by Mortierella alpina. J.
Biotechnol., 144(1), 31-36 (2009).

Reports
















- Katayama, T., J. Ogawa. The 11th Swiss-Japanese Joint Meeting on Biotechnology
and Bioprocess Debelopment. Bioscience and Industry, 67(2), 86-87 (2009). (in
Japanese)

- Ogawa, J., S. Shimizu. A view from the front line of industrial uses of microbial
enzymes. Kagaku to Seibutsu, 47(6), 412-418(2009). (in Japanese)


















b) Conference and seminar papers presented










- Annual Meeting of Japan Society for Bioscience, Biotechnology, and Agrochemistry
2009: 18 reports

- 9th Annual Meeting of Protein Science of Japan: 2 reports

- 61st Annual Meeting of the Vitamin Society of Japan: 1 report

- 3rd Meeting of Target Protein Program: 1 report

- 18th Annual Meeting of Bio-imaging Society of Japan: 1 report

- Annual Meeting of the Society for Fermentation and Bioengineering, Japan 2009: 1
report

- Enzyme Engineering XX: 3 reprots

- 82nd Annual Scientific Meeting of The Japanese Biochemical Society: 1 report

- The Joint Symposium of Branches of Japan Society for Bioscience, Biotechnology,
and Agrochemistry 2009: 9 reports

- 144th Meeting of North Japan Branch of Japan Society for Bioscience, Biotechnology,
and Agrochemistry 2009: 1 report

- 62nd Meeting of Japanese Enzyme Engineering Society: 1 report

- 157th Young Scientist Synposium of Tyubu Branch of Japan Society for Bioscience,
Biotechnology, and Agrochemistry 2009: 1 report

- BioMicroWorld2009: 1 report

- 13th Annual Meeting of Biocatalysis Science: 1 report

- 32nd Annual Meeting of The Molecular Biology Society of Japan: 1 report

- Symposium of Target Protein Program 2009: 1 report

- 8th Lipid Reserch Seminar: 4 reports

- 2009 bioactive lipid molecules and transportes: 2 repoets

- The 1st Seminar of Electron-oriented Biotechnology (e-Bio conference 1st open
seminar): 1 report

- The 11thJapanese CLA forum: 2 reports

- The 5th Takeda Science Foundation Symposium on PharmaSciences: 1 report

- Japanese Oil Chemist's Society 2009 Oil Industry Technology Seminar "Application
of Useful Fat - Fat and Health -": 1 report

- The 14th Marine Oil technology Forum: 1 report

- 5th International Symposiu on Biocatalysis and Biotechnology: 1 repoet

- Italy-Japan Symposium, New Trends in Enzyme Science and technology: 1 report

- 15th German-Japanese Workshop on Enzyme Technology: 1 report

















A-3.Off-campus activities











Membership in academic societies











- Ogawa, Jun, D.Agric.Sci : Japanese Society for Bioscience, Biotechnolgy, and
Agrochemistry (member of Industry-Government-Academy young person interchange
society, Secretory of Kansai branch), The Society for Fermentaton and Bioengineering, Japan
(chairman of lipid biotechnology group), Japan Bioindustry Asociation (secretary of the
soceity of Fermentation and Metabolism), Japanese Society of Brewing (secretary of young
person's society), Industry Research Association, Osaka (secretary of new frontier material
group)
- Kataoka, Michihiko, D.Agric.Sci. : The Society of Enzyme Engineering (secretary), The
Vitamin Society of Japan (topics editor)
Research grants














1. Grants-in-aid for Scientific Research(KAKENHI)








- Scientific Research (B) : Ogawa, Jun : Screening and development of microbial function
of reductive fatty acid and organic acid conversion
- Scientific Research (B) : Kataoka, Michihiko : Development of production process of
useful compounds using bioreduction system
- Young Scientist Research (A) : Sakuradani, Eiji : Production of useful compounds by
molecular breeding of oleaginous microbes
2.Other Research Grants












- Target Protein Research program:Kataoka, Michihiko:Structural analysis and modification
of enzymes useful for chiral compound production
- Research project funded by New Energy and Industrial Technology Development
Organization:Ogawa, Jun:The Project for Development of a Technological Infrastructure for
Industrial Bioprocesses

















A-4.International cooperation and overseas activities







Membership in academic societies











- Ogawa, Jun: Applied Microbiology and Biotechnology (editor), International Society of
Biocatalysis and Biotechnology (editor)
- Kataoka, Michihiko: Journal of Bioscience and Bioengineering (editor), Recent Patents on
Biotechnology (editor)
International meetings(country,roles)











- Ogawa, Jun:15th German-Japanese Workshop on Enzyme Technology (member of
organizing committee), 5th International Symposium on Biocatalysis and Biotechnology
(member of organizing committee)
Visiting Research Scholars













- Researcher 1(Korea)


































B.Educational Activities(2009.4-2010.3)









B-1.On-campus teaching











a) Courses given













- Undergraduate level:

Biochemistry I (Kataoka), Applied microbiology III (Ogawa,
Kataoka), Laboratory course in applied microbiology (Kataoka,
Sakuradani, Ando)
- Graduate level: Fermentation physiology and applied microbiology seminar (Ogawa,
Kataoka, Sakuradani, Ando), Experimental course of fermentation
physiology and applied microbiology (Ogawa, Kataoka, Sakuradani,
Ando)

















B-2.Off-campus teaching etc.











Part-time lecturer














- Ogawa, Jun: Faculty of Agricuture, Tokyo University of Agriculture and Technology
(Applied biochemistry), Graduate School of Agriculture and Life Sciences, The University of
Tokyo (Microbial Function Development)
- Kataoka, Michihiko: Faculty of Engineering, Gifu University (Bioengineering 3)

















B-3.Overseas teaching












International students













- International students : Doctral 1 (China)

















C.Other Remarks













- Ogawa, Jun:NEDO 2010 Biotechnology load map revision "production" (committee
member)