本文選題：β-胡蘿卜素 + 釀酒酵母��； 參考：《北京化工大學(xué)》2016年碩士論文

【摘要】：釀酒酵母作為一種食用安全性的模式生物,已成為生物合成天然產(chǎn)物的首選宿主細(xì)胞。p-胡蘿卜素因其高效抗氧化性和高附加值的特點備受人們關(guān)注。本文利用合成生物學(xué)的方法,引入不同來源p-胡蘿卜素合成基因,旨在提高釀酒酵母中p-胡蘿卜素的產(chǎn)量,具體工作如下：1、在菌株InvScl-GAB開始發(fā)酵后12 h、18 h、24 h添加麥角固醇抑制劑酮康唑,使其終濃度為30 mg·L-1。在發(fā)酵18 h后添加酮康唑,實驗組的β-胡蘿卜素的產(chǎn)量較對照組增長了20%,最高產(chǎn)量為20 mg·L-1。2、利用FMDV 2A序列連接途徑中所有基因,實現(xiàn)其在同一讀碼框中的表達(dá)。構(gòu)建菌株InvScl-2A-YBIE。InvScl-2A-BYIE、InvScl-2A-YBIG及InvScl-2A-BYIG,實現(xiàn)了歐文氏菌來源基因的表達(dá)及不同來源攏牛兒基r{牛兒基焦磷酸合成酶基因的替換。3、優(yōu)化FMDV 2A序列,構(gòu)建菌株InvScl-YBIE(2A), InvScl-YBIE(2A)/pR-t-2A-i, InvScl-IE-YB(2A), InvScl-ABG(2A), InvScl-AEB(2A)。通過優(yōu)化連接順序,導(dǎo)入上游限速酶基因,提高了β-胡蘿卜素表達(dá)能力；通過比較歐文氏菌及三孢布拉氏霉菌的r{牛兒基r{牛兒基焦磷酸合成酶基因crtE和ggps,確定crtE的表達(dá)能力更強(qiáng)。4、在菌株InvScl-AEB(2A)的發(fā)酵過程中探究最適培養(yǎng)基、最適誘導(dǎo)時間。通過菌體生長情況及產(chǎn)量的比較,確定使用SD選擇培養(yǎng)基,發(fā)酵至12 h誘導(dǎo)后產(chǎn)量最高；在探究的最優(yōu)條件下發(fā)酵菌株InvScl-GAB與InvScl-AEB(2A),二者產(chǎn)量分別為2.9 mg·g-1和2.8 mg·g-1。
[Abstract]:Saccharomyces cerevisiae (Saccharomyces cerevisiae) as a model organism for food safety has become the preferred host cell for the biosynthesis of natural products. In order to increase the yield of pcarotene in Saccharomyces cerevisiae, we introduced pcarotene synthesis genes from different sources by means of synthetic biology. The specific work was as follows: 1. The final concentration of ketoconazole was 30 mg L ~ (-1) at 12 h ~ (18) h ~ (-1) and 24 h after fermentation of the strain InvScl-GAB, and the final concentration of ketoconazole was 30 mg 路L ~ (-1). After fermentation for 18 h, the 尾 -carotene production of the experimental group increased by 20% compared with that of the control group, and the highest yield was 20 mg / L ~ (1.2). The expression of 尾 -carotene in the same reading frame was realized by using the FMDV 2A sequence to connect all the genes in the pathway. The strains InvScl-2A-YBIE.InvScl-2A-BYIEE InvScl-2A-YBIG and InvScl-2A-BYIGs were constructed. The expression of Erwinia genes and the substitution of the genes of different sources of calf-base pyrophosphatase were achieved. The FMDV 2A sequence was optimized to construct the strains InvScl-YBIE2An, InvScl-YBIE2AP-rpt-2A-ip, Invcl-IE-YB2A, Invcl-IE-YB2A4, Invcl-ABGG 2AU, InvScl-YBIE2AN, InvScl-YBIE2Ae / pt-2A-i. The expression of 尾 -carotene was improved by the introduction of upstream rate-limiting enzyme gene by optimizing the ligation sequence. By comparing the r {bovine pyrophosphate synthase genes crtE and GPs of Irvine's strain and A. trispora, it was determined that the expression of crtE was stronger. 4. The optimum medium and the optimal induction time were explored in the fermentation process of the strain InvScl-AEB2A (InvScl-AEB2A). According to the comparison of the growth and yield of bacteria, it was determined that the optimum fermentation medium was SD, and the yield of fermentation strain InvScl-GAB and InvScl-AEB2AN was 2.9 mg / g ~ (-1) and 2.8 mg / g ~ (-1), respectively, under the optimum conditions of 12 h fermentation, and the yield of them was 2.9 mg / g ~ (-1) and 2.8 mg / g ~ (-1), respectively.
【學(xué)位授予單位】：北京化工大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：TS261.1;O629.4

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