酿酒酵母中β-胡萝卜素生物合成研究

发布时间：2018-05-13 08:29

本文选题：β-胡萝卜素 + 酿酒酵母　；参考：《北京化工大学》2016年硕士论文

【摘要】：酿酒酵母作为一种食用安全性的模式生物,已成为生物合成天然产物的首选宿主细胞。p-胡萝卜素因其高效抗氧化性和高附加值的特点备受人们关注。本文利用合成生物学的方法,引入不同来源p-胡萝卜素合成基因,旨在提高酿酒酵母中p-胡萝卜素的产量,具体工作如下：1、在菌株InvScl-GAB开始发酵后12 h、18 h、24 h添加麦角固醇抑制剂酮康唑,使其终浓度为30 mg·L-1。在发酵18 h后添加酮康唑,实验组的β-胡萝卜素的产量较对照组增长了20%,最高产量为20 mg·L-1。2、利用FMDV 2A序列连接途径中所有基因,实现其在同一读码框中的表达。构建菌株InvScl-2A-YBIE。InvScl-2A-BYIE、InvScl-2A-YBIG及InvScl-2A-BYIG,实现了欧文氏菌来源基因的表达及不同来源拢牛儿基r{牛儿基焦磷酸合成酶基因的替换。3、优化FMDV 2A序列,构建菌株InvScl-YBIE(2A), InvScl-YBIE(2A)/pR-t-2A-i, InvScl-IE-YB(2A), InvScl-ABG(2A), InvScl-AEB(2A)。通过优化连接顺序,导入上游限速酶基因,提高了β-胡萝卜素表达能力；通过比较欧文氏菌及三孢布拉氏霉菌的r{牛儿基r{牛儿基焦磷酸合成酶基因crtE和ggps,确定crtE的表达能力更强。4、在菌株InvScl-AEB(2A)的发酵过程中探究最适培养基、最适诱导时间。通过菌体生长情况及产量的比较,确定使用SD选择培养基,发酵至12 h诱导后产量最高；在探究的最优条件下发酵菌株InvScl-GAB与InvScl-AEB(2A),二者产量分别为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.
【学位授予单位】：北京化工大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：TS261.1;O629.4

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