Bahieldin Ahmed, Gadalla Nour O, Al-Garni Saleh M, Almehdar Hussein, Noor Samah, Hassan Sabah M, Shokry Ahmed M, Sabir Jamal S M, Murata Norio
Department of Biological Sciences, Faculty of Science, King Abdulaziz University (KAU), P.O. Box 80141, Jeddah 21589, Saudi Arabia; Department of Genetics, Faculty of Agriculture, Ain Shams University, Cairo, Egypt.
Department of Biological Sciences, Faculty of Science, King Abdulaziz University (KAU), P.O. Box 80141, Jeddah 21589, Saudi Arabia; Genetics and Cytology Department, Genetic Engineering and Biotechnology Division, National Research Center, Dokki, Egypt.
Plasmid. 2014 Mar;72:18-28. doi: 10.1016/j.plasmid.2014.03.001. Epub 2014 Mar 26.
Lycopene is an effective antioxidant proposed as a possible treatment for some cancers and other degenerative human conditions. This study aims at generation of a yeast strain (Saccharomyces cerevisiae) of efficient productivity of lycopene by overexpressing synthetic genes derived from crtE, crtB and crtI genes of Erwinia uredovora. These synthetic genes were constructed in accordance with the preferred codon usage in S. cerevisiae but with no changes in amino acid sequences of the gene products. S. cerevisiae cells were transformed with these synthetic crt genes, whose expression was regulated by the ADH2 promoter, which is de-repressed upon glucose depletion. The RT-PCR and Western blotting analyses indicated that the synthetic crt genes were efficiently transcribed and translated in crt-transformed S. cerevisiae cells. The highest level of lycopene in one of the transformed lines was 3.3mglycopene/g dry cell weight, which is higher than the previously reported levels of lycopene in other microorganisms transformed with the three genes. These results suggest the excellence of using the synthetic crt genes and the ADH2 promoter in generation of recombinant S. cerevisiae that produces a high level of lycopene. The level of ergosterol was reversely correlated to that of lycopene in crt-transformed S. cerevisiae cells, suggesting that two pathways for lycopene and ergosterol syntheses compete for the use of farnesyl diphosphate.
番茄红素是一种有效的抗氧化剂，被认为可能用于治疗某些癌症和其他人类退行性疾病。本研究旨在通过过表达源自番茄溃疡病菌crtE、crtB和crtI基因的合成基因，构建一株高效生产番茄红素的酵母菌株（酿酒酵母）。这些合成基因是根据酿酒酵母中偏好的密码子使用情况构建的，但基因产物的氨基酸序列没有变化。用这些合成的crt基因转化酿酒酵母细胞，其表达受ADH2启动子调控，该启动子在葡萄糖耗尽时去阻遏。RT-PCR和蛋白质免疫印迹分析表明，合成的crt基因在经crt转化的酿酒酵母细胞中能有效转录和翻译。其中一个转化株系中番茄红素的最高含量为3.3毫克番茄红素/克干细胞重量，高于此前报道的用这三个基因转化的其他微生物中番茄红素的含量。这些结果表明，在产生高水平番茄红素的重组酿酒酵母中，使用合成的crt基因和ADH2启动子具有优越性。在经crt转化的酿酒酵母细胞中，麦角固醇水平与番茄红素水平呈负相关，这表明番茄红素和麦角固醇合成的两条途径竞争法呢基二磷酸的使用。
