京都大学 / 新制・論文博士 / 博士(農学) / 乙第13458号 / 論農博第2899号 / 新制||農||1088(附属図書館) / 学位論文||R3||N5362(農学部図書室) / (主査)教授 小川 順, 教授 阪井 康能, 教授 栗原 達夫 / 学位規則第4条第2項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

Computational biology enabled us to manage vast amount of experimental data and make inferences on observations that we had not made. Among the many methods, predicting metabolic functions with genome-scale models had shown promising results in the recent years. Using sophisticated algorithms, such as flux balance analysis, OptKnock, and OptForce, we can predict flux distributions and design metabolic engineering strategies at a greater efficiency. The caveat of these current methods is the accuracy of the predictions. We proposed using flux balance analysis with flux ratios as a possible solution to improving the accuracy of the conventional methods. To examine the accuracy of our approach, we implemented flux balance analyses with flux ratios in five publicly available genome-scale models of five different organisms, including Arabidopsis thaliana, yeast, cyanobacteria, Escherichia coli, and Clostridium acetobutylicum, using published metabolic engineering strategies for improving product yields in these organisms. We examined the limitations of the published strategies, searched for possible improvements, and evaluated the impact of these strategies on growth and product yields. The flux balance analysis with flux ratio method requires a prior knowledge on the critical regions of the metabolic network where altering flux ratios can have significant impact on flux redistribution. Thus, we further developed the reverse flux balance analysis with flux ratio algorithm as a possible solution to automatically identify these critical regions and suggest metabolic engineering strategies. We examined the accuracy of this algorithm using an Arabidopsis genome-scale model and found consistency in the prediction with our experimental data. / Master of Science

Genetic variation was examined at 19 loci encoding enzymatic and general proteins Notropis lutrensis from the Brazos River in Texas. The thermal regime of the Brazos River below Possum Kingdom Reservoir is altered due to the release of water from the hypolimnion. Summer water temperatures fluctuate as much as 7^oC. Levels of heterozygosity at the malate dehydrogenase-2 locus were correlated with the degree of water temperature fluctuation at each locality. The isozymes from three homozygous patterns of supernatant malate dehydrogenase (Mdh-l, Mdh-2) exhibited different activities at different experimental temperatures.

Stomata are small pores that are located on the surface of epidermal leaves, and they can regulate the uptake of CO2 and prevent water lose by opening and closing the pores. Stomata of plants can be regulated by external condition such as CO2, biotic and abiotic stresses and internal factors. CAM (crassulacean acid metabolism) plants adapt to hot and dry environments by closing stomata during the day and opening stomata during the cool night. However, it is still unclear how CAM plants open their stomata during the night and close them during the day. In this study, a number of factors were evaluated for their potential roles in promoting stomatal opening in the model CAM plant Kalanchoe laxiflora. Citrate is an important organic acid and it accumulates during the night in CAM plants. It is shown in this study that citrate promoted stomatal opening in detached leaf epidermis of Kalanchoe laxiflora. Further, the cytokinin zeatin is also shown to stimulate stomatal opening in detached leave of Kalanchoe laxiflora. Melatonin is an important regulator of circadian rhythms in mammals and has been implicated in regulation of plant abiotic stress responses. Melatonin was detected in the leaves of Kalanchoe laxiflora. It promoted stomatal opening in detached epidermis of Kalanchoe laxiflora. Together, these results suggest that stomata of Kalanchoe laxiflora respond to citrate and malate which are the main organic acids accumulate during nighttime and also to some signaling molecules (zeatin, melatonin, and serotonin) by opening stomata during dark period.

A resposta metabólica do bagre Genidens genidens de três estuários (rios Grande, Indaiá e Escuro) do município de Ubatuba SP foi estudada comparativamente em relação ao impacto da atividade humana na região. Encéfalo, fígado e músculo epaxial foram utilizados para determinar a atividade específica das enzimas lactato desidrogenase (LDH), malato desidrogenase (MDH) e arginase, como prováveis marcadoras desse impacto. As diferenças entre os níveis teciduais de LDH dos bagres coletados nos três estuários não foram significativamente diferentes, provavelmente devido a sua capacidade adaptativa, associada à sazonalidade das concentrações de oxigênio nas águas estuarinas. Os níveis de arginase hepática e muscular dos bagres coletados nos estuários dos rios Escuro e Indaiá, respectivamente, foram diferentes em relação aos dois outros estuários. Comportamento semelhante a esse foi constatado em relação aos níveis da enzima MDH, marcadora do potencial gerador de ATP desses tecidos. Nesse sentido, os níveis de arginase e MDH do fígado e músculo epaxial podem estar refletindo o esforço adaptativo metabólico desse bagre às condições ambientais dessas regiões estuarinas. / The metabolic response of catfish Genidens genidens of the three estuaries (Grande, Indaia and Escuro rivers) in the municipality of Ubatuba - SP was studied in comparison to the impact of human activity in the region. Brain, liver and epaxial muscle were used to determine the specific activity of the enzymes lactate dehydrogenase (LDH), malate dehydrogenase (MDH) and arginase, as probable markers of that impact. The differences between the tissues levels of LDH of the catfishes collected in the three estuaries were not significantly different, probably due to its adaptive capacity, associated with the seasonality of the oxygen concentrations in estuarine waters. Arginase levels of liver and muscle of the catfishes collected in estuaries of rivers Escuro and Indaia, respectively, were different in the relation to the other two estuaries. Behavior was similar to that observed in the levels of the enzyme MDH, marker of the potential generator of ATP of those tissues. Accordingly, the levels of arginase and MDH of the liver and epaxial muscle might reflect the effort of that adaptive metabolic catfish to environmental conditions such estuarine areas.

Les triacylglycérols d'algues (TAG) représentent une source prometteuse de biocarburants. Les principales étapes de la synthèse des acides gras et du métabolisme du TAG des algues ont été déduites de celles des plantes terrestres, mais on en sait peu sur les sources de carbones et d’énergie intervenant dans la synthèse de lipides de réserve. Nous avons donc étudié la synthèse des acides gras chez l’algue modèle Chlamydomonas reinhardtii en utilisant une combinaison d'approches génétiques, biochimiques et microscopiques. Plus précisément, j'ai d'abord examiné la localisation subcellulaire de gouttelettes de lipides dans des cellules d'algues exposées à une forte lumière, conditions où une plus grande quantité de pouvoir réducteur est produite. J'ai ensuite contribué à mettre en évidence que la bêta-oxydation des acides gras est un processus peroxysomal, et que pendant une carence en azote réalisée en conditions photoautotrophe, des mutants dépourvus de la malate déshydrogénase 2 peroxysomale (mdh2) accumulent 50% plus TAG que les souches parentales. Ces résultats nous ont permis de mettre en évidence l'importance du contexte redox cellulaire sur la synthèse lipidique. Cette étude a également permis de révéler l’existence d'un échange d’énergie entre le peroxysome et le chloroplaste. Enfin, en caractérisant des mutants déficients dans la dégradation des acides aminés à chaîne ramifiée (BCAA), j'ai montré que le catabolisme des BCAAs joue un double rôle dans la synthèse de TAG en fournissant des précurseurs carbonés et de l'ATP. L'ensemble de ces travaux ouvert de nouvelles pistes pour l'amélioration génétique future de souches d'algues pour la production de biocarburants. / Algal triacylglycerols (TAG) represent a promising source for biofuel. The major steps for fatty acid synthesis and TAG metabolism have been deduced based on that of land plants, but little is known about carbon and energy sources. To address this question, we investigated fatty acid synthesis in algal cells using a combination of genetic, biochemical and microscopic approaches in the model microalga Chlamydomonas reinhardtii. Specifically, I first examined subcellular localization of lipid droplets in algal cells exposed to high light, a condition favoring production of reducing power. Secondly, I contributed to put on evidence that the beta-oxidation of fatty acids is a peroxisomal process, and that during photoautotrophic nitrogen starvation, knock-out mutants of the peroxisomal malate dehydrogenase 2 (mdh2) made 50% more TAG than parental strains, highlighting the importance of cellular redox context on lipid synthesis. This study also revealed for the first time the occurrence of an energy trafficking pathway from peroxisome to chloroplast. And finally, by characterizing mutants defected in degradation of branched-chain amino acids (BCAAs), I showed that BCAA catabolism plays a dual role in TAG synthesis via providing carbon precursors and ATP. Taken together, this work highlighted the complex interplay between carbon and energy metabolism in green photosynthetic cells, and pointed future directions for genetic improvement of algal strains for biofuel productions.

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