利用大腸桿菌在溫度調控系統下提升生質異丁醇發酵產量之研究

Abstract

能源的匱乏與環保的議題使得生質能源的開發成為近年來重要的研究。生質異丁醇比起生質乙醇有相對多的優勢，例如較高的熱質、較低的侵蝕性以及揮發性等，這使得異丁醇可能取代乙醇成為下一代的生質醇類。而以大腸桿菌作為生產生質丁醇的菌株則是因為其對碳源廣泛的使用能力。本研究在大腸桿菌DH5α中以alsS, ilvC, ilvD, kivd四個基因將胺基酸valine合成途徑中的中間產物KIV導出合成異丁醇。為了提高生質異丁醇的產量，本研究使用一個溫度敏感的核糖體結合位（ribosome biding site）搭配tetR抑制系統去建立一個溫度誘導基因表現系統，並以此系統去調控異丁醇合成末期的kivd基因表現的時機，降低產物異丁醇對表現菌生長初期的生長抑制狀況，藉以提高異丁醇的產量。實驗結果顯示單純表現四個基因再培養24小時後可以得到約1.6 g/L的產量，然而加入溫度誘導系統後卻可以得到8 g/L，約五倍的產量增加。再加上培養條件的改良，可以在培養48小時得到近20 g/L的產量。此研究證明此溫度誘導基因表現系統可在合適培養條件下成功的大幅提升生質異丁醇在大腸桿菌中的產量。

Biofuel synthesis is of increasing interest because of global energy and environmental problems. Isobutanol is a renewable and clean biofuel. Compared with ethanol, isobutanol has high energy density to burn, low corrosiveness to be stored and long carbon chain, which is similar to gasoline. Furthermore, isobutanol can completely replace gasoline without engine modification. Since Escherichia coli can broadly utilize 5-C sugar (xylose, arabinose, mannose) and 6-C sugar (glucose, galactose); therefore, it is a promising host to produce isobutanol. In this study, four genes (alsS, ilvC, ilvD, kivd) were cloned and transformed into E. coli DH5α to direct glycolysis pathway for isobutanol production. In order to improve the yield of isobutanol, a thermo- sensitive ribosome biding site and tetR repressor were used to repress kivd gene expression and overcome the barrier of isobutanol tolerance of E. coli. The result revealed that E. coli harboring four genes overexpression could produce 0.4 g/L (v/v) isobutanol after 48 h cultivation. Moreover, E. coli harboring the modified plasmid with temperature control could achieve 20 g/L isobutanol yield. This result indicates that thermo-sensitive RBS for expressing kivd gene in E. coli may enhance isobutanol production by temperature.