2016年国際学会２件

ASM 2016 Microbe in Bostonで。いろんな微生物で寒天培地を芸術に？

国際代謝工学会(Metabolic Engineering 2016)

淡路島の夢舞台で開催。淡路島ですが、阿波踊りのエグジビション

アメリカ微生物学会、2016年はボストンで開催でした。

日本農芸化学会2016年度大会(Sapporo)

Halomonas sp. KM-1とその変異体を用いたピルビン酸生産の検討

Optimization of pyruvate production by Halomonas sp. KM-1 and its mutants

○岡本成平1、2、中薗沙也佳1、武井靖子1、2、倉本華歩1、辻彩花1、新川悠一1、西村拓3、松下功3、坪田潤3、河田悦和4､東慶直1

(1 近大・生物理工、2 JST・ALCA、3 大阪ガス、4 産総研・バイオメディカル)

○Naruhei OKAMOTO1,2, Sayaka NAKAZONO1, Yasuko TAKEI2, Kaho KURAMOTO1, Ayaka TSUJI1, Yuichi NIIGAWA1, Taku NISHIMURA3, Isao MATSUSHITA3, Jun TSUBOTA3, Yoshikazu KAWATA4, Yoshinao AZUMA1.

(1 BOST Kindai Univ., 2 JST ALCA, 3 Osaka Gas Co., Ltd., 4 Biomed. Res. Inst., AIST)

好塩好アルカリ性細菌の一種であるハロモナス菌Halomonas sp.KM-1は、スピルリナ培養時のコンタミ菌として分離された(1)。本菌は好気条件で菌体内に生分解性プラスチックPolyhydroxybutyrate(PHB)を高効率で生産し蓄積する。好気条件から微好気条件へと酸素供給量をシフトさせると、PHBのモノマーである3-Hydroxybutyrate(3HB)が培養液中に高濃度に蓄積する (2)。本菌の培養には炭素源となる糖以外にはアミノ酸やビタミンなどの有機物が不要であり、高アルカリ条件下において速やかに増殖し、OD600が100程度までの高密度となる。本菌の培養では、通常の発酵生産で必要とされる滅菌処理が不要であり、物質生産においては低コスト・低エネルギーでの産業化が期待される。また、通常では資化困難なC5糖キシロースやアラビノースでの生育、3HBの生産も可能であることが分かってきた(3)。

　一方、このHalomonas sp.KM-1菌を好気的条件で培養した場合、培養液中に高濃度のピルビン酸が蓄積することが確認された。ピルビン酸はファインケミカルの重要な中間体であり、現在は、酒石酸を硫酸水素ナトリウム存在下で加熱乾留する化学工業的方法などにより製造されている。本研究では、ピルビン酸を環境への負荷の少ない微生物発酵で、より安価に生産する方法の開発を目的として、ピルビン酸合成における最適な培養条件を検討するとともに、Halomonas sp.KM-1菌の変異株ライブラリーを作製し、ピルビン酸の高効率生産菌の分離を試みた。まず、簡便な培養条件では、10％グルコースを炭素源とすると約2日間の好気培養で2％程度のピルビン酸が培地中に蓄積し、続く2日間の培養でほぼ消失した。炭素源とする糖の選択や培養温度の検討により、ある炭素源を利用した場合にはグルコースでは増殖困難な45度でも増殖が可能で、同時に5％以上のピルビン酸を蓄積した。この条件下で、温度感受性変異体や糖代謝変異体など約100株の変異体を用いてピルビン酸合成の最適化を検討した。本研究大会では具体的な本発酵条件を紹介し、環境への負荷の少ないピルビン酸などの発酵に関してご議論頂きたい。

(1)Kawata Y and Aiba S. Biosci Biotechnol Biochem.2010;74(1):175-7.

(2)Kawata Y, Ando H, Matsushita I, Tsubota J. Bioresour Technol. 2014 ;156:400-403.

(3)Kawata Y, Nojiri M, Matsushita I, Tsubota J. Lett Appl Microbiol. 2015 ;61(4):397-402.

ASM Microbe 2016 (Boston)

Molecular Engagements for Hyper Glucose-Tolerance of Acetic Acid Bacteria, Tanticharoenia sakaeratensis and Asaia bogorensis

Hiromi Hadano1,2, Naruhei Okamoto1,2, Sou Takebe1, Kazunobu Matsushita3 and Yoshinao Azuma1*

1Faculty of Biology-oriented Science and Technology, Kindai University, Kinokawa, Wakayama, Japan, 649 6493

2Advanced Low Carbon Technology Research and Development Program (ALCA), Japan Science and Technology Agency (JST)

3Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University, Yamaguchi, Yamaguchi, Japan

Acetic acid bacteria (AAB) form a divergent phylogenetic group, and two phylogenetically closed AAB, Tanticharoenia sakaeratensis and Asaia bogorensis, show a distinctive ability to grow in media containing 30% glucose. To clarify mechanisms for the hyper glucose-tolerance, comparative omics analyses, including genome DNA sequencing, transcriptome and proteome analyses, were performed. The results illustrated that the two AAB altered expressions of similar genes and systems under different conditions with low and high glucose concentrations. In both AAB gene expressions for glycolysis and pentose phosphate pathways decreased, and ones for antioxidant enzymes (such as superoxide dismutase (SOD) and peroxidase) and cytochrome o ubiquinol oxidase increased. On the contrary, the omics analyses indicated a variety uniqueness of each bacterium. For instance, under high glucose conditions, T. sakaeratensis repressed a main energy metabolism including NADH dehydrogenase (complex I) and induced gene expression of pyruvate decarboxylase, whereas A. bogorensis promoted expressions of more stress responsible genes involved in anti-oxidation, such as DNA starvation/stationary phase protection protein (Dps) and osmotically inducible peroxiredoxin OsmC. Because the complex I is a main superoxide producer, T. sakaeratensis needs less antioxidant enzymes than A. bogorensis. We herein propose that enzymes managing oxidative stresses are engaged in overcoming the high glucose stress, and the acquisitions of the glucose tolerance systems in the two AAB were evolutionally independent.

This work was financially supported by the Advanced Low Carbon Technology Research and Development Program (ALCA), and a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (KAKEN-HI: 22510222).

Metabolic Engineering 11

Omics analyses for Hyper Glucose-Tolerance using Acetic Acid Bacteria

Hiromi Hadano1,2, So Takebe1, Kazunobu Matsushita3 and Yoshinao Azuma1*

1Faculty of Biology-oriented Science and Technology, Kindai University, Kinokawa, Wakayama, Japan, 649 6493

2Advanced Low Carbon Technology Research and Development Program (ALCA), Japan Science and Technology Agency (JST)

3Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University, Yamaguchi, Yamaguchi, Japan

Acetic acid bacteria (AAB), forming a divergent phylogenetic group within alpha-proteobacteria, are characterized by their abilities to transform alcohols and sugars into the corresponding organic acids. The capabilities have been historically used for vinegar fermentation from ethanol. Whereas, natural habitats of AAB are widespread niches accumulating sugars, e.g. fruits, flowers and soils near fruit plants. Among AAB, two phylogenetically closed species, Tanticharoenia sakaeratensis and Asaia bogorensis, show a distinctive ability to grow in media containing 30% glucose. Interestingly, both of AAB could not multiply in media containing even 2% of salt, which gives lower osmotic pressure than glucose 30%. It indicates that osmotic tolerance of the AAB is not for general substances but specific to sugars, and that the AAB must include mechanisms for the hyper glucose tolerance. To clarify mechanisms for the hyper glucose-tolerance, comparative omics analyses, including genome DNA sequencing, transcriptome and proteome analyses, were performed. The results illustrated that the two AAB altered expressions of similar genes and systems under different conditions with low and high glucose concentrations. In both AAB gene expressions for glycolysis and pentose phosphate pathways decreased, and ones for antioxidant enzymes (such as superoxide dismutase (SOD) and peroxidase) and cytochrome o ubiquinol oxidase increased. On the contrary, the omics analyses indicated a variety uniqueness of each bacterium. For instance, under high glucose conditions, T. sakaeratensis repressed a main energy metabolism including NADH dehydrogenase (complex I) and induced gene expression of pyruvate decarboxylase, whereas A. bogorensis promoted expressions of more stress responsible genes involved in anti-oxidation, such as DNA starvation/stationary phase protection protein (Dps) and osmotically inducible peroxiredoxin OsmC. Because the complex I is a main superoxide producer, T. sakaeratensis needs less antioxidant enzymes than A. bogorensis. We herein propose that enzymes managing oxidative stresses are engaged in overcoming the high glucose stress, and the acquisitions of the glucose tolerance systems in the two AAB were evolutionally independent.

Pyruvic acid production using thermotolerant Halomonas sp. KM-1

○ Ayaka TSUJI1, Sayaka NAKAZONO1, Kaho KURAMOTO1, Naruhei OKAMOTO1,2, Yasuko TAKEI2, Yuichi NIIGAWA1, Taku NISHIMURA3, Isao MATSUSHITA3, Jun TSUBOTA3, Yoshikazu KAWATA4, Takaaki Kiryu5, Taro Kiso5, Hiromi Murakami5, Kazunobu Matsushita6, Yoshinao AZUMA1.

(1 BOST Kindai Univ., 2 JST ALCA, 3 Osaka Gas Co., Ltd., 4 Biomed. Res. Inst., AIST, 5 Osaka MTRI, 6 Yamaguchi Univ.)

One of most important processes in industrial fermentation is temperature controlling of fermenters, actually huge electric energy is used to cool the fermenters to appropriate temperatures. While massive consumption of energy can become a factor causing climate change, it must be considered to decrease energy consumption in any fields, including even in the fields known as white-biotechnology, which requires less energy and creates less waste and environmental impact during the fermentative production. Thereupon we have attempted to develop a fermentation system without or with less temperature controlling using thermotolerant microbes. Halomonas sp. KM-1, isolated as a contaminant of Spirulina platensis culture, is an alkaliphilic, halophilic, aerobic, thermophilic, and heterotrophic bacterium requesting no organic nutrients including amino acids and vitamins except a carbon source for its cultivation. The KM-1 strain, which characteristics provide useful features, i.e. no requirement of sterilization and less cost for fermentation, accumulates poly-3-hydroxybutyrate (PHB) under aerobic fermentation and secret its monomer, 3-hydroxybutyrate (3-HB) under successive microaerobic one using varied carbon sources (1,2). Besides PHB and 3-HB, 2-oxopropanoic acid (pyruvic acid) was found to be accumulated in media of KM-1 culture using glucose as a carbon source. Pyruvic acid is known for having numerous benefits as a fine chemical compound to produce medicines and cosmetic products. It is also a relatively new supplement for human to decrease in body weight and fat mass and for athletes as an ergogenic compound to improve endurance during aerobic activity and increase ATP/energy production, but the roles are still controversial. To produce pyruvic acid at a commercial level using an environmentally benign process, we attempted to optimize the production of pyruvic acid by Halomonas sp. KM-1 and selected better mutants derived from the strain mutagenized using UV irradiation. Under a small-scale culturing of the KM-1 using 10% glucose at 33˚C, pyruvic acid was accumulated up to 2% in the culture medium after 2-day incubation, and vanished within another 2-day culturing. First we tested a variety of carbon sources, their concentrations, and culturing temperatures. As a result, we could clarify a condition under which pyruvic acid was accumulated up to 5% in the culture at 45˚C and no diminution of the product was observed even after longer incubation. We have selected mutants, which could not grow in media including glucose as a sole carbon source. Based on tests of growth on different carbon sources and productivities of pyruvic acid, it was shown that the mutants carried mutations on sugar transporters and enzymes for the glycolytic pathway. We also isolated several dozens of thermotolerant mutants, which could grow at 47˚C. Those mutants might be useful to produce pyruvic acid or other chemicals in the glycolytic pathway at high temperatures, and capabilities of those mutants will be presented in this meeting.