利用基因轉殖改良大腸桿菌酒精生產能力

Abstract

利用基因轉殖改良大腸桿菌酒精生產能力

學生：王琮凱 指導教授：曾慶平 博士

國立交通大學 生物科技 學系 (研究所) 碩士班

摘 要 生質酒精在經濟與環保方面是極具潛力之再生能源，有鑒於纖維素生質原料具有全球儲存量高、來源多樣化及不易與糧食生產發生衝突等優點，將纖維素轉化為酒精已被視為現階段生質酒精產製過程中最具潛力的發展方向，Escherichia coli本身具有可廣泛利用木質纖維素水解後的產物xylose及glucose，來進行酒精發酵的優點，為一具潛力的酒精生產菌株。 E. coili KO11為一能夠表現Zymomonas mobilis之pyruvate dehydrogenase(pdc) 及alchol dehydrogenaseⅡ(adhB)兩酵素基因的重組菌株，前人文獻中表示該菌株酒精生產力可達理論值的80~90％，但是其高酒精生產力及重組後基因的穩定性仍受質疑。 有鑒於此，本研究利用受厭氧訊號誘導的厭氧啟動子，配合發酵時以厭氧環境作為誘導因子，設計重組質體pNK與pNCDF-NC，希望能夠藉此維持並加強KO11的高酒精生產力。將此兩質體送入KO11後，發現此兩質體於厭氧狀態下，可以如預期地在細胞中表現，並可將KO11的酒精產量向上提升。另外將此二重組質體送入實驗室所篩選出的耐酒精突變株後發現，各菌株的酒精產量及菌株生長情形皆可獲得提升，而於培養液中額外添加2mM氧化劑，可以加強重組質體之表現效果。

Improving ethanol production in Escherichia coli by genetic engeering

Student：Tzong-Kai Wang Advisor：Dr. Ching-Ping Tseng

Institute of Biological Science and Technology National Chiao Tung University

ABSTRACT Bioethanol is a potential and renewable energy for economy and ecology. Lignocellulose comprising glucose and xylose represents the most abundant biomass in the world. It is important to study the bioconvertion of lignocellulose into ethanol. Escherichia coli can metabolize both glucose and xylose to ethanol under anaerobic condition. E. coli KO11 integrated pyruvate decaroxylase (pdc) and alcohol dehydrogenase II (adhB) genes from Zymomonas sp . can reach the ethanol yield with 80~90% of theoretical value. Although the productivity of ethanol of KO11 is high, the strain is unstable after long term fermentation. In this study, we constructed two plasmids, pNK containing pdc and adhB, and pCDF-NC containing citrate synthase gene (citZ) from Bacillus sp.. Both of them can be induced under anaerobic condition. The ethanol production of the strain including pNK and pCDF-NC in 5% glucose or xylose LB media was higher than that of wild type KO11. Moreover, the ethanol-tolerant mutants including pNK and pCDF-NC can produce high ethanol yield. The biomass of ethanol-tolerant mutants was also increased. Furthermore, the ethanol production and biomass were elevated by addition 2mM oxidizing agent in the medium.