成果報告書詳細
管理番号20090000001134
タイトル*平成20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2010/2/10
報告書年度2008 - 2008
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技術開発部 バイオマスグループ
和文要約以下本編抜粋: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 2008-2009) FY 2008 annual report.
1) To improve isopropanol production from glucose using E. coli, observation of growth inhibition by isopropanol, optimization of fermentation condition, and optimization of metabolic pathway were carried out.
A. Growth inhibition by isopropanol
To investigate how much concentration of isopropanol is critical for growth of E. coli, growth experiment of E. coli under 0, 2.5, 5, 7, 8, 9, 10 vol/vol% isopropanol concentrations in LB medium using 96 well plate was carried out. Comparing with control (0% isopropanol) experiment, even lower concentration (2.5-7 %) experiment shows growth inhibition but E. coli growth well until 7%. Over 8%, E. coli can not grow at all. Now, our highest concentration of isopropanol production is about 0.5%. Therefore, it is high possibility that the limiting factor for isopropanol production is not production inhibition.
B. Optimization of fermentation condition (temperature and initial glucose concentration)
To optimize fermentation temperature and initial glucose concentration, isopropanol production experiment changing fermentation temperature (30, 30°C) and initial glucose concentration (0, 0.5, 1, 2, 5 weight/vol%) were carried out. As the result, 30°C fermentation temperature and 2% initial glucose concentration is the best for isopropanol production.
C. Optimization of metabolic pathway (secondary alcohol dehydrogenase)
To optimize expression level of alcohol dehydrogenase, we developed a new plasmid which has all genes for isopropanol production with high copy origin. Our previous plasmids for isopropanol production are all genes except for last step (secondary alcohol dehydrogenase) with high copy origin and gene for last step with medium copy origin. Comparing isopropanol production from a new plasmid with that from a previous one, productions from both plasmids were almost same.
2) We developed novel cell surface displaying system on E.coli. Previously reported anchor proteins are too large size and it is not suitable for cellulase display. In this research, we focused on pgsA as an anchor protein. Alpha-amylase fused pgsA was successfully expressed on the E.coli surface and its enzymatic activity was also retained. We also found Porin was suitable for another anchor protein, because its small size. Porin was also suitable for displaying alpha-amylase on the E.coli cell surface.
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