Kyoto University (京都大学) / 0048 / 新制・課程博士 / 博士(人間・環境学) / 甲第19069号 / 人博第722号 / 新制||人||173 / 32020 / 京都大学大学院人間・環境学研究科相関環境学専攻 / (主査)准教授 土屋 徹, 教授 宮下 英明, 教授 川本 卓男 / 学位規則第4条第1項該当

The family of Deg/HtrA proteases is present in a wide range of organisms from bacteria, archaea to eukaryota. These ATP-independent serine endopeptidases play key roles in the cellular protein quality control. The cyanobacterium Synechocystis sp. PCC 6803, a model organism for studies on photosynthesis, metabolism and renewable energy, contains three Deg proteases known as HhoA, HhoB and HtrA. The three proteases are important for survival in stress conditions, such as high light or temperature. In my work the biochemical characteristics of each protease were revealed in vitro and in vivo. In vitro studies performed using recombinant Synechocystis Deg proteases allowed conclusions about their oligomerization states, proteolytic activities and tertiary structure. The in vivo studies addressed their sub-cellular localization, expression and physiological importance by comparing wild-type Synechocystis cells with the three single mutants lacking one of the Deg proteases. HhoA seems to be involved in the cytoplasmic protein quality control. This protease is regulated post-transcriptional and post-translational: oligomerization, pH and/or cation-binding are some of the important factors to stimulate its proteolytic activity. Instead HhoB acts on periplasmic proteins and seems to be important for the transportation/secretion of proteins. While it has low proteolytic capacity, it may act as a chaperone. The stress-induced HtrA functions in the cellular tolerance against photosynthetic stress; additionally it might act as a protease partner of HhoB, generating a protease/chaperone complex. The results presented in this thesis lay the foundation for a better understanding of the dynamic protein quality control in cyanobacteria, which is undoubtedly important for various cellular metabolic pathways.

The diminishing of fossil fuels and growing concerns towards climate change have intensified biofuel production from renewable resources. Recently, progresses are made in microbial production of biofuels. Among various biofuels, isobutanol is gaining an increasing attention due to its high energy content and suitable chemical and physical properties, enabling it to be a suitable substitution of fossil fuel. In this study, instead of using heterotrophic microorganisms, we performed metabolic engineering of Synechocystis PCC 6803 (Synechocystis) for isobutanol production under autotrophic condition. After introduced 2-keto acid pathway, Synechocystis is able to produce isobutanol when provided with water, carbon dioxide and solar energy. When cultivated in an optimal condition (50 μmol photons m-1s-2 and adjusted pH to 7-8 with HCl), the engineered strain pEEK2-ST was able to produce 425 mg L-1 in-flask isobutanol titer and 911 mg L-1 cumulative isobutanol titer, respectively, in 46 days. There should be bottlenecks existing in 2-keto acid pathway based on the similar isobutanol production of strain pEEK2-ST with and without pyruvate addition. However, the attempt to identify potential bottlenecks of upstream genes by overexpressing ST and one of the three upstream genes failed, instead what we conclude is that the isobutanol production is tightly correlated to Kivd (ST) expression level. Thus, more strategies will be employed for identifying bottlenecks successfully and further improvement of isobutanol production in the future. In conclusion, this study demonstrates the importance of cultivation condition on isobutanol production in Synechocystis.

abstract: Lipids and free fatty acids (FFA) from cyanobacterium Synechocystis can be used for biofuel (e.g. biodiesel or renewable diesel) production. In order to utilize and scale up this technique, downstream processes including culturing and harvest, cell disruption, and extraction were studied. Several solvents/solvent systems were screened for lipid extraction from Synechocystis. Chloroform + methanol-based Folch and Bligh & Dyer methods were proved to be "gold standard" for small-scale analysis due to their highest lipid recoveries that were confirmed by their penetration of the cell membranes, higher polarity, and stronger interaction with hydrogen bonds. Less toxic solvents, such as methanol and MTBE, or direct transesterification of biomass (without pre-extraction step) gave only slightly lower lipid-extraction yields and can be considered for large-scale application. Sustained exposure to high and low temperature extremes severely lowered the biomass and lipid productivity. Temperature stress also triggered changes of lipid quality such as the degree of unsaturation; thus, it affected the productivities and quality of Synechocystis-derived biofuel. Pulsed electric field (PEF) was evaluated for cell disruption prior to lipid extraction. A treatment intensity > 35 kWh/m3 caused significant damage to the plasma membrane, cell wall, and thylakoid membrane, and it even led to complete disruption of some cells into fragments. Treatment by PEF enhanced the potential for the low-toxicity solvent isopropanol to access lipid molecules during subsequent solvent extraction, leading to lower usage of isopropanol for the same extraction efficiency. Other cell-disruption methods also were tested. Distinct disruption effects to the cell envelope, plasma membrane, and thylakoid membranes were observed that were related to extraction efficiency. Microwave and ultrasound had significant enhancement of lipid extraction. Autoclaving, ultrasound, and French press caused significant release of lipid into the medium, which may increase solvent usage and make medium recycling difficult. Production of excreted FFA by mutant Synechocystis has the potential of reducing the complexity of downstream processing. Major problems, such as FFA precipitation and biodegradation by scavengers, account for FFA loss in operation. Even a low concentration of FFA scavengers could consume FFA at a high rate that outpaced FFA production rate. Potential strategies to overcome FFA loss include high pH, adsorptive resin, and sterilization techniques. / Dissertation/Thesis / Ph.D. Civil and Environmental Engineering 2011

This thesis focuses on the functional and structural characterization of FtsH proteases in Synechocystis PCC 6803. One of the aims was to determine localization and subunit organization of FtsH homologues in Synechocystis cells using GST and GFP tagged FtsH derivatives. The main result of the thesis is identification of two FtsH hetero-oligomeric complexes and one homo-oligomeric complex in Synechocystis cells. The large part of the thesis is aimed at establishing the role of the first hetero-oligomeric complex, FtsH2/FtsH3, in quality control of Photosystem II and at identification of a mechanism, how its substrate proteins D1 and D2 are recognized. Another part is dedicated to characterization of the second hetero-oligomeric complex, FtsH1/FtsH3, which consists of two essential FtsH homologues and which is here identified as an important regulatory element in maintaining iron homeostasis.

abstract: Photosynthesis converts sunlight to biomass at a global scale. Among the photosynthetic organisms, cyanobacteria provide an excellent model to study how photosynthesis can become a practical platform of large-scale biotechnology. One novel approach involves metabolically engineering the cyanobacterium Synechocystis sp. PCC 6803 to excrete laurate, which is harvested directly. This work begins by defining a working window of light intensity (LI). Wild-type and laurate-excreting Synechocystis required an LI of at least 5 µE/m2-s to sustain themselves, but are photo-inhibited by LI of 346 to 598 µE/m2-s. Fixing electrons into valuable organic products, e.g., biomass and excreted laurate, is critical to success. Wild-type Synechocystis channeled 75% to 84% of its fixed electrons to biomass; laurate-excreting Synechocystis fixed 64 to 69% as biomass and 6.6% to 10% as laurate. This means that 16 to 30% of the electrons were diverted to non-valuable soluble products, and the trend was accentuated with higher LI. How the Ci concentration depended on the pH and the nitrogen source was quantified by the proton condition and experimentally validated. Nitrate increased, ammonium decreased, but ammonium nitrate stabilized alkalinity and Ci. This finding provides a mechanistically sound tool to manage Ci and pH independently. Independent evaluation pH and Ci on the growth kinetics of Synechocystis showed that pH 8.5 supported the fastest maximum specific growth rate (µmax): 2.4/day and 1.7/day, respectively, for the wild type and modified strains with LI of 202 µE/m2-s. Half-maximum-rate concentrations (KCi) were less than 0.1 mM, meaning that Synechocystis should attain its µmax with a modest Ci concentration (≥1.0 mM). Biomass grown with day-night cycles had a night endogenous decay rate of 0.05-1.0/day, with decay being faster with higher LI and the beginning of dark periods. Supplying light at a fraction of daylight reduced dark decay rate and improved overall biomass productivity. This dissertation systematically evaluates and synthesizes fundamental growth factors of cyanobacteria: light, inorganic carbon (Ci), and pH. LI remains the most critical growth condition to promote biomass productivity and desired forms of biomass, while Ci and pH now can be managed to support optimal productivity. / Dissertation/Thesis / Doctoral Dissertation Civil and Environmental Engineering 2015

京都大学 / 0048 / 新制・課程博士 / 博士(人間・環境学) / 甲第19069号 / 人博第722号 / 新制||人||173(附属図書館) / 26||人博||722(吉田南総合図書館) / 32020 / 京都大学大学院人間・環境学研究科相関環境学専攻 / (主査)准教授 土屋 徹, 教授 宮下 英明, 教授 川本 卓男 / 学位規則第4条第1項該当 / Doctor of Human and Environmental Studies / Kyoto University / DFAM

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L-valine is a branched chain amino acid often used in food, pharmaceutical, cosmetic, and animal feed industries. The most used production method for L-valine and other branched chain amino acids is bacterial fermentation through Escherichia coli or Corynebacterium glutamicum, both of which are heterotrophic bacteria in need of added sugars and energy demanding bioreactors. Synechocystis sp. PCC 6803 is a model cyanobacteria that only uses carbon dioxide and sunlight as energy source and naturally can biosynthesize L-valine, which makes it a suitable platform for sustainable production. The regulation of the L-valine biosynthesis pathway is not fully understood why more research is needed to be able to optimize the production of L-valine. In other organisms, there is feedback inhibition by L-valine that limit the biosynthesis which might be the case for Synechocystis as well. During this project, adaptive laboratory evolution was used to increase the valine tolerance of Synechocystis sp. PCC 6803, by evolving the cells to grow in increasing concentrations of L-valine over multiple generations. This resulted in a final strain that had a tenfold increase in tolerance compared to non-evolved wild type Synechocystis. Whole genome sequencing was used to determine if and what mutations had led to the increased tolerance. Another aim of the project was to evolve a strain that overproduced L-valine. This was done by adaptive laboratory evolution with norvaline as a selection pressure. Norvaline is an amino acid analogue that has a very similar structure to valine, why it can be mis incorporated during aminoacyl-tRNA synthetase. We hypothesized that the Synechocystis cells would overproduce L-valine to outcompete the increased norvaline, thereby increasing the norvaline tolerance. Through adaptive laboratory evolution the norvaline tolerance was increased, but the mechanism behind the tolerance could not be determined during this project. The production of all branched chain amino acids by the evolved strains first needs to be measured to determine if they are in fact producing more L-valine. Then, transcriptomics and/or whole genome sequencing can be used to investigate what genes are regulated or mutated to obtain the increased L-valine production.