【作者】 宋熙；

【导师】 赵学明；

【作者基本信息】 天津大学 ， 制药工程， 2010， 硕士

【摘要】 虾青素是一种类胡萝卜素,具有极强的淬灭自由基的能力并且还具有极强的着色能力,因此在医药,化妆品,食品及饲料行业都有广泛的应用前景。红发夫酵母的发酵生产虾青素,以培养时间短,不需光照而且可以进行高密度培养等优点得到广泛关注。代谢工程方法改造菌种常用手段为敲除某些旁路的关键酶基因以减少碳的代谢分流,或者过表达某些关键酶基因。本文以野生型红法夫酵母As2.1557作为出发菌株,确定应该在上游基因编码的关键酶上入手,通过crtE基因的扩增,增加虾青素的前体物,进而考查其对红发夫酵母的生长及虾青素的产量带来的影响。crtE基因编码牻牛儿基牻牛儿基焦磷酸合成酶(GGPP),GGPP是多种初生和次生代谢物的共同前体,为许多二萜、类胡萝卜素、叶绿素、醌和维生素E侧链等提供构建骨架。所以GGPP合酶能够起到调节碳流的作用,从而成为初生和次生代谢途径中的关键酶之一。本实验通过同源重组的方法,构建了两个转化载体pSX-pc,pSX-gc。这两个载体均以rDNA为同源臂,分别将自带启动子的crtE基因,以及替换了强启动子gpd的crtE基因转化进红发夫酵母,并整合至其基因组上,以G418作为抗性筛选标记,最终筛选出成功扩增crtE基因的菌株。通过测定其生长曲线,类胡萝卜素和虾青素的含量,发现,与野生菌株相比,对GGPP合成酶进行过表达后的转化株生长速度稍有下降;以替换gpd为启动子的crtE基因扩增载体改造的菌种GC,由于基因表达水平提高,从而使虾青素含量提升,最高达到了464.37μg/gDCW;以未替换启动子的crtE基因扩增载体改造的菌种PC,虾青素含量提升最高达到了503.22μg/gDCW。更多还原

【Abstract】 Astaxanthin is a carotenoid, has strong ability to quench free radicals and also has a strong coloring ability, so in medicine, cosmetics, food and feed industry has a wide range of applications. Phaffia yeast fermentation of astaxanthin, in order to cultivate a short time, without light and can get the advantages of high-density culture attention.Metabolic engineering methods for the transformation of a common means of knockout strains of certain key genes bypass to reduce the diversion of carbon metabolism or overexpression of certain key genes. This wild-type Phaffia yeast As2.1557 as the original strain, should determine the key enzyme in the upstream gene encoded on the start, through crtE gene amplification, increased astaxanthin precursors, and then examine its Phaffia yeast growth and astaxanthin production impact.crtE gene coding based Mang Mang ox ox base pyrophosphate synthase (GGPP), GGPP is a variety of primary and secondary metabolites of a common precursor for many diterpenes, carotenoids, chlorophyll, and vitamin E quinone side chain, etc. to provide building skeleton. Therefore, GGPP synthase could play a role in regulating carbon flow in order to become primary and secondary metabolic pathways in one of the key enzymes.In this study, the method by homologous recombination was constructed of two transformation vectors, pSX-pc and pSX-gc,. The two carriers are rDNA as homologous arms, respectively crtE own promoter gene, and the replacement of a strong promoter of crtE gpd gene was transformed into the yeast Phaffia, and integrated into its genome to G418 as anti- sexual selection marker, the ultimate success of selected gene amplification crtE strains.By measuring its growth curve, carotenoids and astaxanthin content, found that compared with the wild strain, the overexpression of GGPP synthase for the conversion of strain after the growth rate declined slightly; to replace the gpd gene as the promoter crtE amplified strain vector transformation, due to increased gene expression, thereby enhancing astaxanthin content, up to a 464.37μg/gDCW; to not replace the promoter crtE gene amplification vector transformation strain, as integrated copies few more, GGPP synthase and a corresponding increase in the expression, enhance the astaxanthin content of the most significant to the 503.22μg/gDCW.更多还原