【摘要】：D-乳酸作為一種重要的聚乳酸合成原料和手性中間前體物,在農(nóng)業(yè)、化工等各個(gè)領(lǐng)域均有廣泛應(yīng)用。目前新興的研究趨勢(shì)是開(kāi)發(fā)廉價(jià)木質(zhì)纖維素原料,然而存在的問(wèn)題是木質(zhì)纖維素水解液中的混合碳源在大腸桿菌利用過(guò)程中會(huì)對(duì)其他碳源產(chǎn)生分解代謝產(chǎn)物阻遏效應(yīng)。為了減輕該阻遏效應(yīng),本文將通過(guò)基因重組手段敲除Ecoli JH15(△pts G)的葡萄糖轉(zhuǎn)運(yùn)酶相關(guān)基因mgl B和galP,構(gòu)建兩株能利用混合碳源(五碳糖和六碳糖)大腸桿菌工程菌來(lái)發(fā)酵生產(chǎn)D-乳酸,研究?jī)?nèi)容及結(jié)果如下:(1)E.coli JH13是敲除了丙酮酸甲酸裂解酶(foc A-pfl B)、乙酸激酶(ack A)、乙醇脫氫酶(adh E)、延胡索酸還原酶(frd ABCD)、部分核酸酶(rng HSR2)基因的能高效利用木糖生產(chǎn)D-乳酸的基因工程菌,由JH13來(lái)構(gòu)建能同步利用混合糖發(fā)酵產(chǎn)D-乳酸的工程菌E.coli JH15(△pts G),再以JH15為出發(fā)菌株,通過(guò)同源重組技術(shù)敲除了甲基半乳糖苷結(jié)合蛋白基因(mgl B),構(gòu)建了E.coli JH18,在E.coli JH18基礎(chǔ)上又繼續(xù)敲除半乳糖轉(zhuǎn)運(yùn)蛋白基因(galP),構(gòu)建了E.coli JH20。(2)mgl B基因和galP基因的敲除確實(shí)能降低葡萄糖入胞速度。以10%葡萄糖作為糖源進(jìn)行發(fā)酵時(shí),JH18葡萄糖消耗速率為2.56 g/L,相比JH15和JH13分別降低了22.2%和26.9%,JH20葡萄糖消耗速度為2.37 g/L,相比JH15和JH13分別降低了28%和34.9%。(3)mgl B和galP基因的敲除對(duì)木糖消耗速率產(chǎn)生較大影響。以10%木糖作為糖源進(jìn)行發(fā)酵時(shí),JH18木糖消耗速度為1.58 g/L,相較于JH15和JH13快了一倍,JH20木糖消耗速度為1.2 g/L,比JH15、JH13提高了51.9%(4)mgl B和galP基因的敲除能在JH15基礎(chǔ)上進(jìn)一步降低葡萄糖效應(yīng),使混合碳源中木糖利用速度增加。以5%葡萄糖和5%木糖作為糖源進(jìn)行發(fā)酵時(shí),JH18葡萄糖消耗速度為1.1 g/L,比JH15和JH13分別降低了19.7%和53.4%,木糖消耗速度為0.83 g/L,比JH15和JH13分別提高了69.8%和160.7%;JH20葡萄糖消耗速度為0.85 g/L,比JH15和JH13分別降低了38%和63.7%,木糖消耗速度為0.48 g/L,比JH15和JH13分別增加了72.1%和164.3%。(5)在低糖環(huán)境下mgl B基因比pts G基因?qū)τ诮档推咸烟侨氚俾实某潭雀蟆Ｔ?%葡萄糖和1%木糖環(huán)境中,JH18耗費(fèi)12h發(fā)酵結(jié)束,葡萄糖糖耗速度為0.86 g/L,木糖消耗速度為0.79 g/L;JH15耗費(fèi)24h發(fā)酵結(jié)束,葡萄糖糖耗速度為1.62 g/L,木糖消耗速度為0.58 g/L。
[Abstract]:As an important raw material and chiral intermediate precursor of polylactic acid synthesis, D- lactic acid has been widely used in agriculture, chemical industry and other fields. At present, the emerging research trend is to develop cheap lignocellulose raw materials, however, the problem is that the mixed carbon sources in lignocellulose hydrolysate will produce catabolic metabolites repressor effect on other carbon sources during the use of Escherichia coli. In order to mitigate this repressor effect, we will knockout Ecoli JH15 (pts G) glucose transporter related genes mgl B and galP, by gene recombination. Two strains of Escherichia coli from mixed carbon sources (pentaccharide and hexaccharide) were constructed to ferment D- lactic acid. The main contents and results are as follows: (1) E.coli JH13 is a formic acid lyase from pyruvate (foc A-pfl B),). Acetate kinase (ack A), ethanol dehydrogenase (adh E), corydalis reductase (frd ABCD), partial nuclease (rng HSR2) gene can efficiently utilize xylose to produce Dlactic acid genetically engineered bacteria. JH13 was used to construct E.coli JH15 (pts G),) which could produce D-lactic acid by mixed sugar fermentation simultaneously. The homologous recombination technique was used to knockout the gene (mgl B), of methylgalactoside binding protein (MGBP) by homologous recombination technique. E.coli JH18, was constructed on the basis of E.coli JH18 and continued to knock out the galactose transporter gene (galP), to construct E.coli JH20. (2) mgl B gene and galP gene knockout) which can reduce glucose entry speed. When 10% glucose was used as sugar source, the glucose consumption rate of JH18 was 2.56 g / L, which was 22. 2% and 26. 9% lower than that of JH15 and JH13, respectively, and the glucose consumption rate of JH20 was 2. 37 g / L. Compared with JH15 and JH13, the rates of xylose consumption were decreased by 28% and 34.9% respectively. (3) the knockout of) mgl B and galP genes had a great effect on xylose consumption rate. When 10% xylose was used as sugar source, JH18 xylose consumption rate was 1.58 g / L, twice as fast as JH15 and JH13. JH20 xylose consumption rate was 1.2 g / L, which was higher than that of JH15,. JH13 increased 51.9% (4) mgl B and galP gene knockout) could further reduce glucose effect on the basis of JH15 and increase xylose utilization rate in mixed carbon source. When fermenting with 5% glucose and 5% xylose, the glucose consumption rate of JH18 was 1.1 g / L, which was 19.7% and 53.4% lower than that of JH15 and JH13, and the consumption rate of xylose was 0.83 g / L. Compared with JH15 and JH13, it increased by 69.8% and 160.7%, respectively. The glucose consumption rate of JH20 is 0.85 g / L, which is 38% and 63.7% lower than that of JH15 and JH13, respectively, and the xylose consumption rate is 0.48 g / L. Compared with JH15 and JH13, mgl B gene increased 72.1% and 164.3%, respectively. (5) in low glucose environment, mgl B gene decreased the rate of glucose entry more than pts G gene. In the environment of 1% glucose and 1% xylose, the glucose consumption rate was 0.86 g / L and the xylose consumption rate was 0.79 g / L after 12h fermentation. At the end of JH15 fermentation for 24 hours, the glucose consumption rate was 1.62 g / L, and xylose consumption rate was 0.58 g / L.
【學(xué)位授予單位】：湖北工業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：TQ921.3;Q93

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