【作者】 晏露；

【导师】 姜建国；

【作者基本信息】 华南理工大学 ， 生物化学与分子生物学， 2010， 硕士

【摘要】 β-胡萝卜素(β-carotene)是类胡萝卜素家族中的典型代表,不仅是体内维生素A的重要来源,而且其本身对人体也具有重要的生理功能,它可以预防、延缓和治疗某些疾病,尤其是癌症,同时也能提高机体的免疫功能。近年来,β-胡萝卜素的制备工艺,特别是发酵法生产,受到研究人员的重视。杜氏盐藻(Dunaliella salina,简称盐藻)是商业生产β-胡萝卜素的主要微生物之一,能在强光照,高盐度,低营养的条件下积累大量的β-胡萝卜素,是公认的产β-胡萝卜素最好的天然资源。因此从分子生物学角度研究盐藻合成和累积β-胡萝卜素的机理,将从盐藻中分离得到的功能基因用于类胡萝卜素的基因工程和代谢工程研究具有重大意义。盐藻番茄红素β-环化酶(LycB)的功能是催化番茄红素转化成β-胡萝卜素,盐藻高度累积β-胡萝卜素与该酶的基因调控及其酶催化机理直接相关。本研究根据盐藻LycB基因的开放阅读框(ORF)序列设计引物,采用RT-PCR的方法从盐藻中克隆得到LycB ORF,经测序确证后,将该段序列克隆入能合成LycB底物的原核表达载体pACCRT-EIB(携带欧文氏菌crtE,crtI和crtB基因,能合成番茄红素),构建得重组质粒pACCRT-EIB-LycB并转化大肠杆菌(Escherichia coli DH5α),经表达菌落的颜色筛选,获得了能稳定合成β-胡萝卜素的大肠杆菌基因工程菌株。通过对该菌株的培养条件进行优化研究,确定了影响β-胡萝卜素积累的主要因素,接着通过设计四因素三水平的响应面优化实验,确定了积累β-胡萝卜素的最佳培养基配方。实验结果表明,最优培养基成分为:蛋白胨20.43g/L,酵母膏10g/L,NaCl 5g/L,甘油1.97mL/L,NH4Cl 1.09g/L,Na2HPO4?7H2O 12.84g/L。在此条件下培养该菌株,β-胡萝卜素平均产量为2.3896mg/L,比相同培养条件下含有欧文氏菌(Erwinia uredovora)环化酶基因crtY的pACCRT16ΔX参照菌株累积的β-胡萝卜素提高了39.26%。更多还原

【Abstract】 As a typical representative of carotenoid family,β-carotene is not only an important source of vitamin A in human body, it also has some physiological functions to the health of a person. Recent research suggests thatβ-carotene is associated with reduced risk of certain disease, particularly in cacer. Simultaneouly, it can improve the immune ability of the body. In recent years, the producing technologies, especially fermentation, have been studied.Dunaliella salina is one of the major microorganisms commercially employed to produceβ-carotene, it can accumulate much moreβ-carotene than any other organisms under the conditions of high light, high salinity and nutrient deprivation. Therefor, it is of great economical significance to study the mechanisms ofβ-carotene synthesis and accumulation from the perspective of molecular biology, which may explain the molecular regulation of key genes and enzymes of D. salina in the carotenogenic pathway.Lycopeneβ-cyclase (LycB) is the key enzyme of catalyzing lycopene to formβ-carotene. The reason that D. salina highly accumulateβ-carotene is directly related to the catalytic mechanisms of LycB and the molecular regulation of its gene. In order to address the function of LycB, we cloned the open reading frame(ORF) of LycB by RT-PCR strategy. After confirmed by DNA sequencing, we constructed a recombinant plasmid pACCRT-EIB-LycB by cloning the ORF of LycB into the plasmid pACCRT-EIB which harbours the Erwinia uredovora genes necessary for lycopene formation. Than transformed the pACCRT-EIB-LycB into E.coli DH5α, so a genetically engineered bacteria strain which can accumulateβ-carotene stablely was gained.In order to obtain the maximum yield ofβ-carotene, response surface analysis methodology (RSM) was applied to optimize the composition of fermentation medium. Based on single-factor experiments, four-factors-three-levels Box-behnken central compositon experiments design were applied. The results showed that the best fermentation medium was tryptone 20.43g/L, yeast extract 10g/L, NaCl 5g/L, glycerol 1.97g/L, NH4Cl 1.09g/L, Na2HPO4?7H2O 12.84g/L. Validation experiments were carried out at these optimum conditions and the average yield ofβ-carotene is 2.3896mg/L. The experiment data shows the production ofβ-carotene has increased 39.26% compared with the E.coli strain containing pACCRT16ΔX which contains crtY gene of Erwinia uredovora under the same culture conditions.更多还原