| Ken-ichi Honjoh | Last modified date：2022.09.09 |
Associate Professor /
Division of Food Science and Biotechnology
Department of Bioscience and Biotechnology
Faculty of Agriculture
Department of Bioscience and Biotechnology
Faculty of Agriculture
Graduate School
Undergraduate School
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Homepage
https://kyushu-u.pure.elsevier.com/en/persons/ken-ichi-honjoh
Reseacher Profiling Tool Kyushu University Pure
http://www.agr.kyushu-u.ac.jp/lab/foodhygienicchemistry/eindex.html
Phone
092-802-4758
Fax
092-802-4757
Academic Degree
Ph.D of Agriculture
Country of degree conferring institution (Overseas)
No
Field of Specialization
Food Preservation
Total Priod of education and research career in the foreign country
01years00months
Outline Activities
Some plant species develop freezing tolerance when exposed to low, non-freezing temperatures. If freezing tolerance could be provided to cold-sensitive plants, the contribution to stable production of crops would be significant. In our laboratory, we found that hardened cells of Chlorella vulgaris C-27 survive slow freezing to -196°C. We are investigating the mechanism of the development of freezing tolerance of Chlorella. Furthermore, based on the research of Chlorella, enhancement of freezing tolerance of higher plants is also carried out.
Research
Research Interests
Membership in Academic Society
- Development of a method for lowering contamination with Listeria to fresh produce during cultivation
keyword : Listeria monocytogenes, fresh produce, decontamination
2013.05. - Studies on new compatible solutes for freezing tolerance
keyword : compatible solutes, freezing tolerance, taurine, tolehalose
2009.04. - Studies on the mechanisms of the development of freezing tolerance of chlorella
keyword : chlorella, freezing tolerance, hardening
1991.04～2017.03Studies on the mechanism of the development of freezing tolerance of Chlorella. - Studies of enhancement of freezing tolerance of Saccharomyces cerevisiae
keyword : freezing tolerance, Saccharomyces cerevisiae
1996.04Enhancement of freezing tolerance of yeast. - Studies on the enhancement of freezing tolerance of higher plants
keyword : freezing tolerance, Arabidopsis thaliana, lettuce, plants
1994.04Enhancement of freezing tolerance of higher plants.
Books
Papers
| 1. | Ken-ichi Honjoh, Yin Lin, Kiyomi Jo, Yuri Iwaizako, Masayuki Maeda, Nobuyuki Kjima, Takahisa Miyamoto, Possible Contamination Routes of Listeria monocytogenes in Leaf Lettuce during Cultivation, FOOD SCIENCE AND TECHNOLOGY RESEARCH, 10.3136/fstr.24.911, 24, 5, 911-920, 2018.09, Outbreaks of foodborne illness caused by Listeria monocytogenes in or on fresh produce have been reported. Several contamination routes have been proposed and the cultivation process is one of the suspected routes. Leaf lettuce is one of the most important fresh produce products. In the present paper, we investigated the possible routes of L. monocytogenes contamination in leaf lettuce during cultivation. Leaf lettuce was cultivated in soils inoculated with cocktails of three isolates of L. monocytogenes belonging to different serotypes (1/2a, 1/2b, and 4b). The viability and injury state of L. monocytogenes in soils, and bacteria survival in or on leaf lettuce were investigated during 10 weeks of cultivation. Soils were artificially contaminated with L. monocytogenes at levels of 4, 6 or 8 log CFU/g, followed by cultivation of leaf lettuce in the contaminated soils. Populations of L. monocytogenes in the soil decreased to less than the detection limit (< 2 log CFU/g) at 8-10 weeks after the start of cultivation. L. monocytogenes was detected in some harvested leaf lettuce leaves at low levels, almost equivalent to the detection limit. As L. monocytogenes was not detected in the leaves of leaf lettuce plants cultivated in highly-contaminated soils after surface disinfection, the possibility of internalization of L. monocytogenes into leaf lettuce plants was considered low. Spraying of water contaminated with bacteria at greater than 3.2 log CFU/plant led to the survival of bacteria on the leaf lettuce leaves even after seven days. Furthermore, leaf damage prolonged the survival period of bacteria on the leaves.. |
| 2. | Ken-ichi HONJOH, Hitomi OKANO, Aya KAWABATA, Masaru KUROKAWA, Taiki KIMURA, Takeshi MACHIDA, Yoshimitsu MASUDA and Takahisa MIYAMOTO, Freezing tolerance of Lactuca sativa and induction of CBF and GolS genes during cold treatment, Journal of Faculty of Agriculture, Kyushu University, 63, 2, 249-257, 2018.09. |
| 3. | Currently, injury of foodborne pathogen in agricultural environment is unclear. So, we observed survival of foodborne pathogens on common procedure for growing vegetable by cultivation on two types of agar medium that possess different selectivity. In case of Salmonella enterica serovar Infantis (SI) on the process of manure preparation, injury was estimated by cultivation on selective agar medium and resuscitation on non-selective agar medium. While in Listeria monocytogenes (LM) on cultivation environment of tomato, physiological changes were confirmed by salt content in agar medium. In manure environment, insufficient elevation of temperature in piled feedstock caused injured SI. LM survived for long term and part of these survived as injured LM in soil and on the surface of tomato fruits. It might be possible to estimate injured SI in actual condition of manure preparation by above procedure. On the contrary, there are practical difficulties for discriminating injured LM in actual agricultural environment by only salt sensitivity.. |
| 4. | Ken-ichi Honjoh, Yuri Iwazako, Yin Lin, Nobuyuki Kijima, Takahisa Miyamoto, Possibilities for Contamination of Tomato Fruit by Listeria monocytogenes during Cultivation, FOOD SCIENCE AND TECHNOLOGY RESEARCH, 10.3136/fstr.22.349, 22, 3, 349-357, 2016.05, [URL], Outbreaks of food-borne illness caused by Listeria monocytogenes in or on fresh produce are sometimes reported. Tomatoes have been considered as one of the most implicated vehicles for produce-associated outbreaks. In the present paper, using tomato plants and three isolates of L. monocytogenes showing different serotypes (1/2a, 1/2b, 4b), viability and injury of L. monocytogenes in soil and in or on tomato plants during cultivation was investigated. Soil was artificially contaminated with L. monocytogenes at levels of 2, 4, 6 or 8 log CFU/g, followed by cultivation of tomato plants in the contaminated soils. The population of L. monocytogenes in the soil decreased to less than the detection limit (< 2 log CFU/g) 6 - 8 weeks after the start of cultivation. L. monocytogenes was not detected in any harvested fruit after a 16-week cultivation, although it was detected qualitatively from soil samples. Artificial injury of the root did not induce contamination of tomato fruit by L. monocytogenes via vessels. Thus, the possibility of internalization or contamination of tomato fruit by L. monocytogenes from contaminated soil is considered quite low during cultivation. However, L. monocytogenes on the fruit surface survived up to 2 - 3 weeks. Regardless of contamination level, hygienically inappropriate handling by workers might lead to contamination of tomato fruit by bacteria such as L. monocytogenes and thus to food poisoning.. |
| 5. | Approaches of Improvement of Freezing-Stress Tolerance of Yeast. |
| 6. | Ken-ichi Honjoh, Tomoko Mishima, Nozomi Kido, Misako Shimamoto, Takahisa Miyamoto, Investigation of Routes of Salmonella Contamination Via Soils and the Use of Mulch for Contamination Control during Lettuce Cultivation, FOOD SCIENCE AND TECHNOLOGY RESEARCH, 10.3136/fstr.20.961, 20, 5, 961-969, 2014.09, [URL], Foodborne illnesses associated with the consumption of fresh produce such as raw vegetables have become a major health concern worldwide in recent years. In the present study, we investigated the possible routes of Salmonella contamination in leafy lettuce via soil during cultivation. After 10-week cultivation of lettuce plants in soils inoculated with S. Enteritidis expressing green fluorescent protein (SE-EGFP), the bacterium was detected in soil inoculated with >10(4) cfu/g and from most lettuce leaves cultivated in soils inoculated with >4.4 x 10(7) cfu/g. As Salmonella was not detected in intact lettuce leaves or lettuce leaves with root injury cultivated in highly contaminated soils and after surface disinfection, the lettuce plants were not considered to internalize the bacterium. Overhead irrigation led to the contamination of one in 10 lettuce plants; however, all sets of three leaves of the plant were contaminated (>110 MPN/g). In an effort to prevent Salmonella contamination from soils, we investigated the effects of mulch on contamination levels during cultivation. Mulch effectively reduced Salmonella contamination levels of lettuce plants cultivated in highly contaminated soils.. |
| 7. | Takeshi Machida, Akiko Ishibashi, Ai Kirino, Jun-ichi Sato, Shinji Kawasaki, Youichi Niimura, Ken-ichi Honjoh, Takahisa Miyamoto, Chloroplast NADPH-Dependent Thioredoxin Reductase from Chlorella vulgaris Alleviates Environmental Stresses in Yeast Together with 2-Cys Peroxiredoxin, PLOS ONE, 10.1371/journal.pone.0045988, 7, 9, e45988, 2012.09, Chloroplast NADPH-dependent thioredoxin reductase (NTRC) catalyzes the reduction of 2-Cys peroxiredoxin (2-Cys Prx) and, thus, probably functions as an antioxidant system. The functions of the enzyme in oxidative and salt stresses have been reported previously. We have previously identified and characterized NTRC in Chlorella vulgaris. In the present study, we isolated a full-length cDNA clone encoding 2-Cys Prx from C. vulgaris and investigated the involvement of Chlorella NTRC/2-Cys Prx system in several environmental stress tolerances by using yeast as a eukaryotic model. Deduced Chlorella 2-Cys Prx was homologous to those of chloroplast 2-Cys Prxs from plants, and two conserved cysteine residues were found in the deduced sequence. Enzyme assay showed that recombinant mature C. vulgaris NTRC (mCvNTRC) transferred electrons from NADPH to recombinant mature C. vulgaris 2-Cys Prx (mCvPrx), and mCvPrx decomposed hydrogen peroxide, tert-butyl hydroperoxide, and peroxynitrite by cooperating with mCvNTRC. Based on the results, the mCvNTRC/mCvPrx antioxidant system was identified in Chlorella. The antioxidant system genes were expressed in yeast separately or coordinately. Stress tolerances of yeast against freezing, heat, and menadione-induced oxidative stresses were significantly improved by expression of mCvNTRC, and the elevated tolerances were more significant when both mCvNTRC and mCvPrx were co-expressed. Our results reveal a novel feature of NTRC: it functions as an antioxidant system with 2-Cys Prx in freezing and heat stress tolerances.. |
| 8. | Takeshi Machida, Ken-ichi Honjoh, Ayuko Aso, Maiko Yamamoto, Masayoshi Ho, Takahisa Miyamoto, Trehalose 6-Phosphate Synthase and Trehalose 6-Phosphate Phosphatase from Nicotiana tabacum Function in Trehalose Biosynthesis and Environmental Stress Tolerance of Yeast, JOURNAL OF THE FACULTY OF AGRICULTURE KYUSHU UNIVERSITY, 55, 2, 261-268, 55, 2, 261-268, 2010.10, To investigate functions of trehalose 6 phosphate synthase (TPS) and trehalose 6 phosphate phosphatase (TPP) from a tobacco plant, Nicotiana tabacum, the corresponding cDNA clones were isolated. Those genes were designated NtTPS and NtTPP, respectively NtTPS included a N-terminal extension according to comparison of deduced amino acid sequence of NtTPS with those of TPSs from Escherichia coli and yeast. The NtTPS was genetically modified to lack a region for the N-terminal extension and the modified gene was designated Delta NNtTPS. The genes were expressed in yeast tps1 mutant as two separate proteins and as a NtTPS (or Delta NNtTPS) NtTPP fusion protein. Western blot analysis showed that the NtTPS, NtTPP, and NtTPS NtTPP were expressed abundantly in yeast, while the Delta NNtTPS and Delta NNtTPS-NtTPP were not detected. Interestingly, high levels of trehalose were accumulated in yeast expressing Delta NNtTPS and Delta NNtTPS-NtTPP in spite of their low level expressions. Furthermore, stress tolerances of yeast against osmotic, freezing thawing, and heat stresses were significantly improved by the expression of the tobacco gene, and the increased levels in tolerance were proportional to their trehalose levels. Our results showed that NtTPS and NtTPP functioned in trehalose synthesis by the removal of N-terminal extension of NtTPS and several environmental stress tolerances.. |
| 9. | Ken-ichi Honjoh, Kanae Matsuura, Takeshi Machida, Koutarou Nishi, Miki Nakao, Tomoki Yano, Takahisa Miyamoto, Masayoshi Iio, Enhancement of menadione stress tolerance in yeast by accumulation of hypotaurine and taurine: co-expression of cDNA clones, from Cyprinus carpio, for cysteine dioxygenase and cysteine sulfinate decarboxylase in Saccharomyces cerevisiae, AMINO ACIDS, 10.1007/s00726-009-0328-6, 38, 4, 1173-1183, 38(4): 1173-1183, 2010.04, Taurine is known to function as a protectant against various stresses in animal cells. In order to utilize taurine as a compatible solute for stress tolerance of yeast, isolation of cDNA clones for genes encoding enzymes involved in biosynthesis of taurine was attempted. Two types of cDNA clones corresponding to genes encoding cysteine dioxygenase (CDO1 and CDO2) and a cDNA clone for cysteine sulfinate decarboxylase (CSD) were isolated from Cyprinus carpio. Deduced amino acid sequences of the two CDOs and that of CSD showed high similarity to those of CDOs and those of CSDs from other organisms, respectively. The coding regions of CDO1, CDO2, and CSD were subcloned into an expression vector, pESC-TRP, for Saccharomyces cerevisiae. Furthermore, to enhance the efficiency of synthesis of taurine in S. cerevisiae, a CDO-CSD fusion was designed and expressed. Expression of CDO and CSD proteins, or the CDO-CSD fusion protein was confirmed by Western blot analysis. HPLC analysis showed that the expression of the proteins led to enhancement of the accumulation level of hypotaurine, a precursor of taurine, rather than taurine. The yeast cells expressing corresponding genes showed tolerance to oxidative stress induced by menadione, but not to freezing-thawing stress.. |
| 10. | Takeshi Machida, Naoto Ohashi, Ayano Mimura, Ken-ichi Honjoh, Masayoshi Ho, Takahisa Miyamoto, Chloroplastic Glucose 6-Phosphate Dehydrogenase from Chlorella vulgaris Alleviates Freezing and Menadione-Induced Oxidative Stresses in Saccharomyces cerevisiae, JOURNAL OF THE FACULTY OF AGRICULTURE KYUSHU UNIVERSITY, 55, 1, 29-38, 55, 1, 29-38, 2010.02, Enhanced glucose 6-phosphate dehydrogenase (E C 1 1 1 49, G6PDH) activity has been identified as a hardening-induced intracellular change of Chlorella vulgaris, which acquires freezing tolerance during hardening. In the present study, a full-length cDNA clone corresponding to a gene encoding a chloroplastic isoform of G6PDH, designated CvchG6PDH, was isolated from C vulgarus C-27. By comparing the deduced amino acid sequence of CvchG6PDH with the N-terminal aminoacid sequence of mature G6PDH(2) protein isolated previously, a DNA region encoding mature CvchG6PDH was determined and designated mCvchG6PDH. The deduced amino acid sequence of CvchG6PDH showed higher homology to those of plant plastidic G6PDH genes than those of cytosolic ones. A recombinant mCvchG6PDH protein expressed in Escherichia coli showed similar enzymatic properties to previously isolated Chlorella G6PDH(2), suggesting that the gene encoded plastidic G6PDH(2) protein. Expression of CvchG6PDH was induced transcriptionally throughout 24-h hardening, while the translation was induced up to 9-h hardening and then decreased, and the change did not reflect the enhanced G6PDH activity during hardening. Furthermore, the mCvchG6PDH alleviated both freezing and menadione-induced oxidative stresses in yeast. We showed the contribution of CvchG6PDH in menadione stress tolerance as one of its functions in the acquisition of freezing tolerance of Chlorella.. |
| 11. | Takeshi Machida, Ken-ichi Honjoh, Hideyuki Shimizu, Maiko Yamamoto, Masayoshi Iio, Takahisa Miyamoto, Expression of A Gene Encoding A Functional Glycosyl Hydrolase, Trehalase, from Nicotiana Tabacum in Saccharomyces Cerevisiae, JOURNAL OF THE FACULTY OF AGRICULTURE KYUSHU UNIVERSITY, 54, 2, 297-303, 54, 2, 297-304, 2009.10, Trehalases (TREs) function in trehalose hydrolysis and commonly found in most organisms. It is said that most fungi have two types of TREs, acid trehalase and neutral trehalase, but other organisms including plants have only one type. To investigate the function of TRE from plants, a full-length cDNA clone encoding TRE was isolated and designated NtTRE. A conserved region of common trehalase can be found in deduced amino acid sequence of NtTRE, thus the gene was recognized to encode NtTRE enzyme. The NITRE was expressed in Escherichia coli as a glutathione-S-transferase (GST)-fusion protein to investigate the function of the expressed protein as trehalase. SDS-PAGE profile of the protein extract of E. coli showed that the expressed GST-NtTRE protein appeared to be cleaved into two polypeptides, which were approximately 56 and 28 kDa in size, and to form an inclusion body. Based on the results of N-terminal amino acid sequencing of the 56-kDa protein, it contained almost all parts of NtTRE protein. Thus, the protein expressed in E. coli was used only for production of anti-NtTRE antibodies. Function of NtTRE was investigated using yeast expressing NtTRE. The NtTRE protein was expressed as a soluble protein. Trehalase activity of protein extract of the transformed yeast was significantly higher than that of control yeast carrying an empty vector. In addition, intracellular trehalose content in yeast cells was significantly reduced by expression of NtTRE. Those data provided a possibility to construct a modified tobacco plant that can accumulate trehalose in the cells by suppression of the expression and/or the activity of NtTRE.. |
| 12. | Takeshi Machida, Eri Kato, Akiko Ishibashi, Jun-ichi Sato, Shinji Kawasaki, Youichi Niimura, Ken-ichi Honjoh, Takahisa Miyamoto, Expression Pattern of a Chloroplast NADPH-Dependent Thioredoxin Reductase in Chlorella vulgaris during Hardening and Its Interaction with 2-Cys Peroxiredoxin, BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 10.1271/bbb.80761, 73, 3, 695-701, 73(3), 695-701, 2009.03, A chloroplastic NADPH-dependent thioredoxin reductase gene was identified from Chlorella vulgaris and designated CvNTRC. Mature CvNTRC protein (mCvNTRC) was expressed in Escherichia coli, and it showed both NADPH-dependent thioredoxin reductase (NTR) and thioredoxin (Trx)-like dithiol-disulfide oxidoreductase activities. The transcript of CvNTRC increased throughout 24-h hardening, whereas the encoded protein amount and total NTR activity decreased once and then increased during hardening. By in vitro pull-down assay, a 21.2-kDa protein bound to mCvNTRC was isolated and identified as a 2-Cys peroxiredoxin (2-Cys Prx) based on the N-terminal sequence. These data suggest that CvNTRC is maintained at a constant level during hardening and functions as an antioxidant with 2-Cys Prx in the acquisition of freezing tolerance of Chlorella.. |
| 13. | Machida, T., Kato, E., Ishibashi, A., Ohashi, N., Honjoh, K., Miyamoto, T., Molecular characterization of low-temperaure-inducible NTR-C in Chlorella vulgaris, Nucleic Acids Symposium Series, No. 51, 463-464, 2007.11. |
| 14. | Ken-ichi Honjoh, Takeshi Machida, Koutarou Nishi, Kanae Matsuura, Kevin Webby Soli, Takatoshi Sakai, Hiroya Ishikawa, Kiyoshi Matsumoto, Takahisa Miyamoto, Masayoshi Iio, Improvement of freezing and oxidative stress tolerance in Saccharomyces cerevisiae by taurine, FOOD SCIENCE AND TECHNOLOGY RESEARCH, 10.3136/fstr.13.145, 13, 2, 145-154, 13,2, 145-154, 2007.05, The effect of taurine on the survival of Saccharomyces cereuisiae after freezing and oxidative stress was investigated. Proline and NaCl were used in comparison. The accumulation of taurine in yeast cells seemed to lead to the enhancement of tolerance to both freezing and oxidative stress in yeast. Although taurine appeared to be less effective than proline in the development of freezing tolerance, when based on intracellular amounts taurine protected cells more effectively than proline. In order to clarify the effect of taurine on stress tolerance, the expression patterns of stress-responsive genes were observed using RT-PCR. In addition, the contents of glycerol and trehalose as well as the redox states of glutathione in the yeast cells were investigated. Our results suggest that taurine, as well as proline, may function as a cryo-protectant and/or an antioxidant in yeast.. |
| 15. | Kenichi Honjoh, Takeshi Machida, Takahiro Hagisako, Koushirou Suga, Madoka Yonekura, Hideyuki Shimizu, Naoto Ohashi, Takahisa Miyamoto, Shoji Hatano, Masayoshi Iio, Molecular cloning and characterization of a cDNA for low-temperature inducible cytosolic glucose 6-phosphate dehydrogenase gene from Chlorella vulgaris and expression of the gene in Saccharomyces cerevisiae, Plant Science, 10.1016/j.plantsci.2006.12.004, 172, 3, 649-658, 172, 649-658, 2007.03, A cDNA clone corresponding to the gene for glucose 6-phosphate dehydrogenase (E.C.1.1.1.49) was isolated from a cDNA library constructed from poly(A)+RNA from Chlorella vulgaris C-27, a frost hardy strain. The cDNA clone was designated as Cvcg6pdh. The length of Cvcg6pdh was 1845 bp and the clone coded for 521 amino acids. The deduced amino acid sequence of Cvcg6pdh showed sequence homology to cytosolic G6PDHs from other higher plants rather than chloroplastic ones. Northern blot analysis of a transcript of Cvcg6pdh gene showed that the gene was down-regulated once and then induced after 12-h hardening. Activity staining also showed that the expression pattern of one G6PDH isoform was similar to that of the transcript of the gene. Southern blot analysis of genomic DNA showed that Cvcg6pdh seems to hybridize with, at least, one copy of g6pdh genes. The coding region of the clone was amplified by PCR and the product was introduced into Saccharomyces cerevisiae by using a pTG887 expression vector. The activity of G6PDH in transformed yeast was enhanced up to 8.7 times that of the control strain. Furthermore, after freezing-thawing, the viability of the yeast transformed with pTG887/Cvcg6pdh was significantly higher than that of the control yeast cells carrying pTG887. © 2006 Elsevier Ireland Ltd. All rights reserved.. |
| 16. | Ken Ichi Honjoh, Ayano Mimura, Eiko Kuroiwa, Takahiro Hagisako, Koushirou Suga, Hideyuki Shimizu, Rama Shanker Dubey, Takahisa Miyamoto, Shoji Hatano, Masayoshi Iio, Purification and characterization of two isoforms of glucose 6-phosphate dehydrogenase (G6PDH) from Chlorella vulgaris C-27, Bioscience, Biotechnology and Biochemistry, 10.1271/bbb.67.1888, 67, 9, 1888-1896, 67(9): 1888-1896, 2003.09, Two kinds of isoforms of glucose 6-phosphate dehydrogenase (G6PDH) were purified from cells of a freezing-tolerant strain, Chlorella vulgaris C-27, by sequential steps of chromatography on five kinds of columns, including a HiTrap Blue column which showed excellent separation of the isoforms from each other. The two isoforms (G6PDH1 and G6PDH2) were purified up to 109-fold and 197-fold with specific activity of 14.4 and 26.0 U/mg-protein, respectively. G6PDH1 showed an apparent Mr of 200,000 with a subunit Mr of about 58,000, whereas G6PDH2 showed an apparent Mr of 450,000 with a subunit Mr of about 52,000. The kinetic parameters were measured and several enzymatic features of the isoforms, such as effects of metal ions on the enzyme activity, were clarified, which showed that the two isoforms were different from each other in many respects. Among the effective ions, Cd2+ showed marked stimulating effects on both isoforms. G6PDH1 and G6PDH2 seem to be a cytosolic and a chloroplastic type, respectively, as judged by their sensitivity to DTT, and also from the results of sequence similarity searches using their N-terminal and internal amino acid sequences.. |
| 17. | Ken Ichi Honjoh, Koushirou Suga, Fuminori Shinohara, Isao Maruyama, Takahisa Miyamoto, Shoji Hatano, Masayoshi Iio, Preparation of protoplasts from Chlorella vulgaris K-73122 and cell wall regeneration of protoplasts from C. vulgaris K-73122 and C-27, Journal of the Faculty of Agriculture, Kyushu University, 47, 2, 257-266, 47(2): 257-266, 2003.02, Protoplasts from Chlorella vulgaris K-73122 were obtained by enzymatic digestion with a mixture of Acromopeptidase, Cellulase ONOZUKA R-10, Chitosanase KI, Gluczyme, and Uskizyme. The formation of naked protoplasts was confirmed by fluorescence microscopy using fluorescent brightner 28, which stains cell walls. About 88% of C. vulgaris K-73122 cells were converted into osmotically-labile cells. Furthermore, a method for regeneration of intact cells from the protoplasts was developed. Utilization of 0.5 M sucrose as an osmoticum, Fe-EDTA as an iron source, and bacto-agar as a supporting was shown to help regeneration of the cell walls of two strains, C. vulgaris K-73122 and C-27.. |
| 18. | Suga, K., Honjoh, K., Furuya, N., Shimizu, H., Nishi, K., Shinohara, F., Hirabaru, Y., Maruyama, I., Miyamoto, T., Hatano, S., and Iio, M.,, Two low-temperature-inducible Chlorella genes for D12 and w-3 fatty acid desaturase (FAD): Isolation of D12 and w-3 fad cDNA clones, expression of D12 fad in Saccharomyces cerevisiae , and expression of w-3 fad in Nicotiana tabacum, Biosci. Biotechnol. Biochem., 66, 6, 1314-1327, 66(6): 1314-1327, 2002.06. |
| 19. | Koushirou Suga, Ken-ichi Honjoh, Naoki Furuya, Hideyuki Shimizu, Koutarou Nishi, Fuminori Shinohara, Yoshie Hirabaru, Isao Maruyama, Takahisa Miyamoto, Two low-temperature-inducible chlorella genes for delta 12 and omega-3 fatty acid desaturase (FAD): isolation of delta 12 and omega-3 fad cDNA clones, expression of delta12 fad in Saccharomyces cerevisiae, and expression of omega-3 fad in Nicotiana tab・・・, Biosci Biotechnal Biochem, 10.1271/bbb.66.1314, 66, 6, 1314-1327, 2002.01, Two low-temperature-inducible chlorella genes for delta 12 and omega-3 fatty acid desaturase (FAD): isolation of delta 12 and omega-3 fad cDNA clones, expression of delta12 fad in Saccharomyces cerevisiae, and expression of omega-3 fad in Nicotiana tabacum. |
| 20. | Ken Ichi Honjoh, Hideyuki Shimizu, Noriko Nagaishi, Hiroko Matsumoto, Koushirou Suga, Takahisa Miyamoto, Masayoshi Iio, Shoji Hatano, Improvement of freezing tolerance in transgenic tobacco leaves by expressing the hiC6 gene, Bioscience, Biotechnology and Biochemistry, 10.1271/bbb.65.1796, 65, 8, 1796-1804, 65(8): 1796-1804, 2001.08, A cryoprotective protein, HIC6, was expressed transgenically in tobacco, a cold-sensitive plant, and the localization of the protein within the cell as well as freezing tolerance of the transgenic tobacco was investigated. For constitutive expression of HIC6 in tobacco, its corresponding gene was subcloned into pBI121. Through the transformation with pBI121/hiC6, fifteen transgenic tobacco lines were acquired, out of which twelve lines expressed the HIC6 protein. None of the transgenic tobacco lines, however, showed significant differences in freezing tolerance from the control plants (wild-type and transformed with pBI121) at -1, -3, and -4°C, with the exception that their freezing temperature was -2°C. In order to increase the accumulation level of HIC6, pBE2113 with a stronger promoter was used. Eight lines expressed the protein out of thirteen lines transformed with pBE2113/hiC6. The accumulation levels of the protein were clearly higher in the tobacco plants transformed with pBE2113/hiC6 than in those with pBI121/hiC6. The HIC6 protein seemed to be localized in mitochondria of the transgenic tobacco plants. Freezing-tolerance tests at -1-4°C showed that the degree of electrolyte leakage was significantly lower in the plants with pBE2113/hiC6 than in the control plants. A leaf browning observation also showed that high accumulation of HIC6 significantly suppressed injury caused by freezing to the transgenic tobacco at -3°C.. |
| 21. | Honjoh, K., Matsumoto, H., Shimizu, H., Ooyama, K., Tanaka, K., Oda, Y., Takata, R., Joh, T., Suga, K., Miyamoto, T., Iio, M. and Hatano, S., Cryoprotective activities of group3 late embryogenesis abundant proteins from Chlorella vulgaris C-27, Biosci. Biotechnol. Biochem., 10.1271/bbb.64.1656, 64, 8, 1656-1663, 64(8): 1656-1663, 2000.08. |
| 22. | Ken Ichi Honjoh, Yuichi Oda, Ryoji Takata, Takahisa Miyamoto, Shoji Hatano, Introduction of the hiC6 gene, which encodes a homologue of a late embryogenesis abundant (LEA) protein, enhances freezing tolerance of yeast, Journal of Plant Physiology, 10.1016/S0176-1617(99)80046-7, 155, 4-5, 509-512, Vol.155, No.4,5, 509-512, 1999.10, The hiC6 gene of Chlorella vulgaris, sharing sequence similarity with a late embryogenesis abundant (lea) gene, was introduced into Saccharomyces cerevisiae. It was inserted on a multicopy plasmid under the transcriptional control of the yeast GAL1 promoter. Expression of HIC6 protein was confirmed by immunochemical methods. Expression level of the protein was increased gradually with the induction-time by galactose. With maximum induction time, the freeze-tolerance of yeast transformed with hiC6 gene was approximately 3.3 times (from 20% to 66% survival rate) higher than that of the control yeast.. |
| 23. | Joh, T., Yoshimoto, M., Honjoh, K., Miyamoto, T., Hatano, S., Changes in translatable RNA population during hardening of Chlorella ellipsoidea C-27., J. Fac. Agr., Kyushu Univ., 37, 3-4, 257-263, 37(3,4), 257-263, 1993.02. |
Presentations
- Japanese Society for Safety Education
- Japanese Society for Plant Cell and Molecular Biology
- Japan Society for Bioscience, Biotechnology, and Agrochemistry
- The Japanese Society for Food Science and Technology
- The Japanese Society of Plant Physiologists
- Japanese Society of Food Microbiology
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