成果報告書詳細
管理番号20110000000591
タイトル*平成22年度中間年報 新エネルギー技術研究開発 バイオマスエネルギー等高効率転換技術開発(先導技術開発) 大腸菌によるイソプロパノール生産の研究開発
公開日2011/6/7
報告書年度2010 - 2010
委託先名国立大学法人九州大学 国立大学法人神戸大学
プロジェクト番号P07015
部署名新エネルギー部
和文要約和文要約等以下本編抜粋:1.研究開発の内容及び成果等 本研究では、セルロースおよびキシランからイソプロパノールを直接発酵により生産する大腸菌の分子育種およびバイオプロセスの開発を実施する。そのため、大腸菌を用いた糖からのイソプロパノール生産向上を目指した「代謝工学によるイソプロパノール生産向上の研究開発」を九州大学が、セルロースおよびキシランからの直接発酵を目指した「細胞表層工学によるセルラーゼの表層提示大腸菌の研究開発」を神戸大学が担当する。
英文要約Title: Development of Technology for High-efficiency Conversion of Biomass and Other Energy/Development of Preparatory Basic Bioenergy Technologies/Research and Development to Improve Production through the Use of Engineered E. coli (FY 2008-2011) FY 2010 annual report.
1) To improve isopropanol production from conifer model medium mixed glucose (85%) and xylose (15%), strain which cannot use glucose or xylose was made. A strain deleted xylA used only glucose from model medium and a strain deleted manZ, ptsG, and gak did only xylose.
To improve isopropanol production rate from glucose, modification of glucose uptake system was carried out. We made a strain deleted pts glucose uptake system and over expressed galP and glk. Growth inhibition by IPTG for this strain was reported, so promoter for isopropanol production gene repressed by lacI was changed to that by tetR. This strain grew and used glucose almost same as a wild type but did not produce any isopropanol.
To produce isopropanol from cellobiose, a strain using beta-glucosidase-displaying E. coli with isopropanol production plasmid was created. This strain produced small amount of isopropanol successfully. To our knowledge, this is the first report to produce something from cellobiose by E. coli.
2) We demonstrated direct assimilation of cellobiose using beta-glucosidase (BGL)-displaying E. coli. After screening active BGLs, Tfu0937, which is a BGL from Thermobifida fusca YX, was successfully displayed on the E. coli cell surface and directly assimilated cellobiose as a carbon source. Then, we screened for a suitable anchor protein for BGL cell-surface display and found a novel anchor protein, Blc, which allowed for both N- and C-terminal fusion of Tfu0937. The pNPG activity of Tfu-0937 using Blc as an anchor protein was significantly improved, up to 7535 U/O.D. 600/ml, which is approximately 65-fold higher than using PgsA as the anchor protein. Finally, Tfu0937-displaying E. coli JCM20137 was successfully grown on 0.2% cellobiose, and the O.D. 600 was 1.05 after 20 h, which is almost the same rate of growth on 0.2% glucose after 16 h. Tfu0937-displaying E. coli JCM20137 was also successfully grown on 0.2 % cellotriose, cellotetraose, cellopentaose and cellohexaose.
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