番茄红素(lycopene)是一种抗氧化性较强的天然色素,已广泛应用于食品、保健品及化妆品等领域。该研究首先在酿酒酵母YPH499△gal80中分别整合3个拷贝数的3种不同的外源牻牛儿基牻牛儿基焦磷酸合酶(geranylgeranyl diphosphate synthase, GGPPS)编码基因CrtE。然后分别选取不同来源的4种八氢番茄红素合酶(phytoene synthase, CrtB)编码基因CrtB和4种八氢番茄红素脱氢酶(phytoene dehydrogenase, CrtI)编码基因CrtI进行组合,得到16个不同的番茄红素游离表达质粒,转化上述突变株。此外对番茄红素测定方法也进行了探索和优化:分析在5种不同溶剂中番茄红素和β-胡萝卜素的吸收情况,确定合适的溶剂、波长、检测限和线性范围。通过肉眼观察菌落色泽深浅和酶标仪测定,最终筛选得到1株最优组合,即表达来源于曼地亚红豆杉(Taxus x media,Tm)的TmCrtE、成团泛菌(Pantoea agglomerans,Pa)的PaCrtB和三孢布拉霉氏菌(Blakeslea trispora,Bt)的BtCrtI的突变菌株。最终重组菌株摇瓶发酵,产量用酶标仪检测472 nm处的吸光度,达到1.33 mg/g DCW。该研究筛选得到的最优组合菌株,可强化番茄红素生产能力,为进一步获得高产番茄红素细胞工厂提供基础。
Lycopene is a natural pigment with strong oxidation resistance. It has been widely used in food, health products and cosmetic industries. In this study, three copies of three different heterologous CrtE genes coding geranylgeranyl diphosphate synthase (GGPPS) were integrated into the genome DNA of Saccharomyces cerevisiae YPH499△gal80. Four CrtB genes coding phytoene synthase (CrtB) and four CrtI genes coding phytoene dehydrogenase (CrtI) from diverse species were then combined into 16 different lycopene-producing episomal plasmids and then transformed into the above-mentioned mutant strains. Finally, the lycopene detection method was also explored and optimized. The absorbance in five different solvents was analyzed. The optimal solvent, wavelength, detection limit and linear range were defined. One optimal combination mutant was screened (CrtE from Taxus x media, CrtB from Pantoea agglomerans and CrtI from Blakeslea trispora) through visual observation of the color of colonies and micro plate reader detection. According to the absorbance at 472 nm by micro plate reader, the highest titer of lycopene reached 1.33 mg/g DCW (dry cell weight). The screened recombinant strain in this work enhanced the productivity of lycopene-producing strains and provided the basis for construction of cell factories for lycopene production.
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