Lycopene overproduction in Saccharomyces cerevisiae through combining pathway engineering with host engineering.
Lycopene overproduction in Saccharomyces cerevisiae through combining pathway engineering with host engineering.
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DOI：
10.1186/s12934-016-0509-4
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发表时间：
2016-06-21
影响因子：
6.4
通讯作者：
Yuan Y
中科院分区：
文献类型：
--
作者：
Chen Y;Xiao W;Wang Y;Liu H;Li X;Yuan Y
近年来，番茄红素的微生物生产受到越来越多的关注，番茄红素是一种重要的商业和医学化合物。酿酒酵母被认为是比大肠杆菌更安全的番茄红素生产宿主。然而，迄今为止，酿酒酵母中番茄红素的产量(mg/gDCW)低于在大肠杆菌中的产量，并且不利于下游提取过程，这可能是由于宿主细胞与外源途径的不亲和性所致。因此，要在酿酒酵母中实现番茄红素的过量生产，必须对宿主细胞和外源途径进行精细的工程改造。本研究通过整合CrtE、CrtB和CrtI在酿酒酵母CEN.PK2中构建了番茄红素生物合成途径。远缘遗传位点YPL062W缺失后，几乎没有乙酸盐积累，胞质乙酰-辅酶A池比亲本增加约100%。通过对不同菌种中CrtE、CrtB和CrtI的筛选，得到了一种最佳的促胡萝卜素酶组合，其中三孢链霉菌CrtI(BtCrtI)对番茄红素的产量和番茄红素在类胡萝卜素中的比例都有较好的效果。然后，对BtCrtI的表达水平进行了微调，并对细胞交配类型的影响进行了评估。最后，潜在的远距离遗传目标(YJL064W、ROX1和DOS2)被删除，逆境反应转录因子INO2也被上调。通过对宿主细胞和胡萝卜素合成途径的上述修饰，番茄红素产量提高了约22倍(从2.43 mg/g DCW提高到54.63 mg/g DCW)。最终，在补料分批发酵中，番茄红素产量达到55.56 mg/g DCW，这是酵母中报道的最高产量。本研究以酿酒酵母生产番茄红素为研究对象。通过将寄主工程(远端遗传位点和细胞交配类型)与途径工程(酶筛选和基因微调)相结合，番茄红素产量比出发菌株逐步提高了22倍。在5-L生物反应器中，酵母中番茄红素的产量最高(55.56 mg/gDCW)。本研究为医药化工产品微生物生产过剩的宿主细胞和异源途径的组合工程提供了良好的参考。本文的在线版本(doi：10.1186/s12934-0160509-4)包含补充材料，授权用户可以使用。
Microbial production of lycopene, a commercially and medically important compound, has received increasing concern in recent years. Saccharomyces cerevisiae is regarded as a safer host for lycopene production than Escherichia coli. However, to date, the lycopene yield (mg/g DCW) in S. cerevisiae was lower than that in E. coli and did not facilitate downstream extraction process, which might be attributed to the incompatibility between host cell and heterologous pathway. Therefore, to achieve lycopene overproduction in S. cerevisiae, both host cell and heterologous pathway should be delicately engineered. In this study, lycopene biosynthesis pathway was constructed by integration of CrtE, CrtB and CrtI in S. cerevisiae CEN.PK2. When YPL062W, a distant genetic locus, was deleted, little acetate was accumulated and approximately 100 % increase in cytosolic acetyl-CoA pool was achieved relative to that in parental strain. Through screening CrtE, CrtB and CrtI from diverse species, an optimal carotenogenic enzyme combination was obtained, and CrtI from Blakeslea trispora (BtCrtI) was found to have excellent performance on lycopene production as well as lycopene proportion in carotenoid. Then, the expression level of BtCrtI was fine-tuned and the effect of cell mating types was also evaluated. Finally, potential distant genetic targets (YJL064W, ROX1, and DOS2) were deleted and a stress-responsive transcription factor INO2 was also up-regulated. Through the above modifications between host cell and carotenogenic pathway, lycopene yield was increased by approximately 22-fold (from 2.43 to 54.63 mg/g DCW). Eventually, in fed-batch fermentation, lycopene production reached 55.56 mg/g DCW, which is the highest reported yield in yeasts. Saccharomyces cerevisiae was engineered to produce lycopene in this study. Through combining host engineering (distant genetic loci and cell mating types) with pathway engineering (enzyme screening and gene fine-tuning), lycopene yield was stepwise improved by 22-fold as compared to the starting strain. The highest lycopene yield (55.56 mg/g DCW) in yeasts was achieved in 5-L bioreactors. This study provides a good reference of combinatorial engineering of host cell and heterologous pathway for microbial overproduction of pharmaceutical and chemical products. The online version of this article (doi:10.1186/s12934-016-0509-4) contains supplementary material, which is available to authorized users.