天然資源由来炭素化合物を基質としたメタノール生成反応に関する合成生物学研究

京都大学新制・課程博士博士(農学)甲第23251号農博第2458号新制||農||1085(附属図書館)学位論文||R3||N5341京都大学大学院農学研究科応用生命科学専攻(主査)教授 阪井 康能, 教授 小川 順, 教授 井上 善晴学位規則第4条第1項該当Doctor of Agricultural ScienceKyoto UniversityDGA

Methanol production by reversed methylotrophy constructed in Escherichia coli

We constructed a reversed methylotrophic pathway that produces methanol, a promising feedstock for production of useful compounds, from fructose 6-phosphate (F6P), which can be supplied by catabolism of biomass-derived sugars including glucose, by a synthetic biology approach. Using Escherichia coli as an expression host, we heterologously expressed genes encoding methanol utilization enzymes from methylotrophic bacteria, i.e. the NAD⁺-dependent methanol dehydrogenase (MDH) from Bacillus methanolicus S1 and an artificial fusion enzyme of 3-hexulose-6-phosphate synthase and 6-phospho-3-hexuloisomerase from Mycobacterium gastri MB19 (HPS-PHI). We confirmed that these enzymes can catalyze reverse reactions of methanol oxidation and formaldehyde fixation. The engineered E. coli strain co-expressing MDH and HPS-PHI genes produced methanol in resting cell reactions not only from F6P but also from glucose. We successfully conferred reversed methylotrophy to E. coli and our results provide a proof-of-concept for biological methanol production from biomass-derived sugar compounds

Use of the illicium for age determination and verification of yellow goosefish Lophius litulon off Aomori Prefecture, northern Japan

青森県周辺海域におけるキアンコウの背鰭第一棘による年齢査定法を検証した。背鰭第一棘の付け根付近の横断面をエッチング処理した後，メチレンブルーで染色し，実体顕微鏡下で落射光と透過光の両者による比較観察した結果，不透明帯数の読み取り精度が向上した。同横断面には，1年に2本の不透明帯（主に6月と11–12月）が形成されていた。背鰭第一棘による年齢査定は脊椎骨によるものよりも読み取り誤差が小さく，標識放流魚の成長追跡結果と類似したことから，優れた年齢査定法と判定した。Yellow goosefish Lophius litulon is an economically valuable fish species around Japan. The aim of the present study was to evaluate a suitable technique for age determination of yellow goosefish using the illicium. Specimens of yellow goosefish were collected by commercial boats and research vessels from the Sea of Japan, Tsugaru Strait and the Pacific Ocean off Aomori Prefecture from November 2013 to March 2016 using bottom gill, set and trawl nets. Each specimen was measured and sexed, and the illicium and 8th vertebra were removed in the laboratory. The epidermis of each illicium was removed after boiling, and the illicia were dried, whereas each of the 8th vertebra was boiled and sectioned. We found that the best ageing method was a count of opaque zones in the illicia. This was facilitated using the cross-sectioned illicia that had been etched with 1 mol/L HCl for 30 s, stained with methylene blue for 4 h and viewed under transmitted and incident light. Opaque zones form twice per year (mainly during June and November-December) in the illicia. Age determination using opaque zones in the illicia was more accurate than that using opaque zones in the vertebral centra. Because the illicia of yellow goosefish are easy to collect and manipulate for age determination and the growth rate estimated using the illicia and tagging experiments is similar, this method of age determination may be useful for these fish in this region

Use of the illicium for age determination and verification of yellow goosefish <i>Lophius litulon</i> off Aomori Prefecture, northern Japan

青森県周辺海域におけるキアンコウの背鰭第一棘による年齢査定法を検証した。背鰭第一棘の付け根付近の横断面をエッチング処理した後，メチレンブルーで染色し，実体顕微鏡下で落射光と透過光の両者による比較観察した結果，不透明帯数の読み取り精度が向上した。同横断面には，1年に2本の不透明帯（主に6月と11–12月）が形成されていた。背鰭第一棘による年齢査定は脊椎骨によるものよりも読み取り誤差が小さく，標識放流魚の成長追跡結果と類似したことから，優れた年齢査定法と判定した。Yellow goosefish Lophius litulon is an economically valuable fish species around Japan. The aim of the present study was to evaluate a suitable technique for age determination of yellow goosefish using the illicium. Specimens of yellow goosefish were collected by commercial boats and research vessels from the Sea of Japan, Tsugaru Strait and the Pacific Ocean off Aomori Prefecture from November 2013 to March 2016 using bottom gill, set and trawl nets. Each specimen was measured and sexed, and the illicium and 8th vertebra were removed in the laboratory. The epidermis of each illicium was removed after boiling, and the illicia were dried, whereas each of the 8th vertebra was boiled and sectioned. We found that the best ageing method was a count of opaque zones in the illicia. This was facilitated using the cross-sectioned illicia that had been etched with 1 mol/L HCl for 30 s, stained with methylene blue for 4 h and viewed under transmitted and incident light. Opaque zones form twice per year (mainly during June and November-December) in the illicia. Age determination using opaque zones in the illicia was more accurate than that using opaque zones in the vertebral centra. Because the illicia of yellow goosefish are easy to collect and manipulate for age determination and the growth rate estimated using the illicia and tagging experiments is similar, this method of age determination may be useful for these fish in this region

Identification of B cell adaptor for PI3-kinase (BCAP) as an Abl interactor 1-regulated substrate of Abl kinases

AbstractIn previous work we showed that Abl interactor 1 (Abi-1), by linking enzyme and substrate, promotes the phosphorylation of Mammalian Enabled (Mena) by c-Abl. To determine whether this mechanism extends to other c-Abl substrates, we used the yeast two-hybrid system to search for proteins that interact with Abi-1. By screening a human leukocyte cDNA library, we identified BCAP (B-cell adaptor for phosphoinositide 3-kinase) as another Abi-1-interacting protein. Binding experiments revealed that the SH3 domain of Abi-1 and the C-terminal polyproline structure of BCAP are involved in interactions between the two. In cultured cells, Abi-1 promoted phosphorylation of BCAP not only by c-Abl but also by v-Abl. The phosphorylation sites of BCAP by c-Abl were mapped to five tyrosine residues in the C-terminal region that are well conserved in mammals. These results show that Abi-1 promotes Abl-mediated BCAP phosphorylation and suggest that Abi-1 in general coordinates kinase-substrate interactions

Systematic Expression Profiling of the Mouse Transcriptome Using RIKEN cDNA Microarrays

The number of known mRNA transcripts in the mouse has been greatly expanded by the RIKEN Mouse Gene Encyclopedia project. Validation of their reproducible expression in a tissue is an important contribution to the study of functional genomics. In this report, we determine the expression profile of 57,931 clones on 20 mouse tissues using cDNA microarrays. Of these 57,931 clones, 22,928 clones correspond to the FANTOM2 clone set. The set represents 20,234 transcriptional units (TUs) out of 33,409 TUs in the FANTOM2 set. We identified 7206 separate clones that satisfied stringent criteria for tissue-specific expression. Gene Ontology terms were assigned for these 7206 clones, and the proportion of `molecular function' ontology for each tissue-specific clone was examined. These data will provide insights into the function of each tissue. Tissue-specific gene expression profiles obtained using our cDNA microarrays were also compared with the data extracted from the GNF Expression Atlas based on Affymetrix microarrays. One major outcome of the RIKEN transcriptome analysis is the identification of numerous nonprotein-coding mRNAs. The expression profile was also used to obtain evidence of expression for putative noncoding RNAs. In addition, 1926 clones (70%) of 2768 clones that were categorized as “unknown EST,” and 1969 (58%) clones of 3388 clones that were categorized as “unclassifiable” were also shown to be reproducibly expressed