产番茄红素Yarrowia lipolytica基因工程菌的构建

发布时间：2018-01-17 15:08

本文关键词：产番茄红素Yarrowia lipolytica基因工程菌的构建　出处：《中国医药工业研究总院》2016年硕士论文　论文类型：学位论文

【摘要】：番茄红素是一种天然类胡萝卜素,具有多种生理功能,在食品、药品等领域有着重要的应用价值,利用合成生物学技术构建基因工程菌发酵生产番茄红素是目前的一个研究热点。耶氏解脂酵母由于其卓越的脂质合成和积累能力,在脂类物质生产方面具有很好的应用潜力。本文的研究目的是构建生产番茄红素的耶氏解脂酵母工程菌。首先,构建含有番茄红素合成途径关键基因crtB(编码八氢番茄红素合成酶,催化两分子r{牛儿基r{牛儿基焦磷酸缩合生成八氢番茄红素)、crtI(编码八氢番茄红素去饱和酶,催化八氢番茄红素脱氢生成番茄红素)、GGS1(编码r{牛儿基r{牛儿基焦磷酸合成酶,催化法尼基焦磷酸和异戊二烯焦磷酸缩合生成r{牛儿基r{牛儿基焦磷酸)的质粒pBIGgut2、pBIGInt F,这两个质粒能够把外源基因整合于耶氏解脂酵母染色体GUT2(位于B染色体,编码甘油三磷酸脱氢酶,催化甘油醛-3-磷酸转化为二羟基丙酮磷酸)和IntF(位于F染色体,目前尚未发现有任何基因功能)位点。然后,通过LiAc转化方法将线性化质粒pBIGgut2导入耶氏解脂酵母20362(Hmg)感受态细胞,利用MM(基本培养基)平板筛选重组子,得到9个阳性转化子,进行摇瓶发酵培养,其中LY3001-1在摇瓶中番茄红素发酵产量最高,为49mg/L。最后,初步研究了LY3001-1菌种在5L发酵罐上发酵的代谢规律。通过补料培养,番茄红素发酵48h的产量达到492.5mg/L。
[Abstract]:Lycopene is a kind of natural carotenoid, has many physiological functions, and has important application value in food, medicine and other fields. The production of lycopene by using synthetic biological technology of genetically engineered bacteria is a research hotspot at present. Yeasts have excellent ability of lipid synthesis and accumulation due to their excellent lipid synthesis and accumulation ability. It has good application potential in the production of lipids. The purpose of this study is to construct yeast-lycopene lycopene yeast engineering bacteria. First of all. The key gene of lycopene synthesis pathway crtB (encoding octahydrolycopene synthase, which catalyzes the condensation of two molecules r {bovine base r {bovine base pyrophosphoric acid) to produce octahydrolycopene was constructed. CrtI (encoding lycopene desaturase) catalyzes the dehydrogenation of lycopene to lycopene GGS1. The plasmid pBIGgut2pBIGInt F was synthesized by catalytic condensation of pyrophosphoric acid with isoprene pyrophosphoric acid. These two plasmids can integrate foreign genes into GUT2 (located on chromosome B, encoding glycerol triphosphate dehydrogenase). Catalytic conversion of glyceraldehyde-3-phosphoric acid to dihydroxy acetone phosphoric acid) and IntF (located on the F chromosome, no gene function has been found so far). Then. The linearized plasmid pBIGgut2 was introduced into the receptive cells of Lipiomyces yeast-20362Hmg by LiAc transformation, and the recombinant was screened by using MM2 (basic medium) plate. Nine positive transformants were obtained and cultured in shake flask. The yield of lycopene was the highest in shaking flask, which was 49mg / L. The metabolic law of LY3001-1 strain fermenting on 5L fermenter was studied preliminarily. The yield of lycopene reached 492.5 mg / L after 48h fermentation.
【学位授予单位】：中国医药工业研究总院
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：Q78;Q939.9

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