Chen Yan, Xiao Wenhai, Wang Ying, Liu Hong, Li Xia, Yuan Yingjin
Key Laboratory of Systems Bioengineering (Ministry of Education), Tianjin University, Tianjin, 300072, People's Republic of China.
SynBio Research Platform, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), School of Chemical Engineering and Technology, Tianjin University, Tianjin, 300072, People's Republic of China.
Microb Cell Fact. 2016 Jun 21;15(1):113. doi: 10.1186/s12934-016-0509-4.
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.
近年来，番茄红素的微生物生产受到了越来越多的关注，番茄红素是一种具有商业和医学重要性的化合物。与大肠杆菌相比，酿酒酵母被认为是生产番茄红素更安全的宿主。然而，迄今为止，酿酒酵母中的番茄红素产量（mg/g干细胞重）低于大肠杆菌，且不利于下游提取过程，这可能归因于宿主细胞与异源途径之间的不相容性。因此，为了在酿酒酵母中实现番茄红素的过量生产，宿主细胞和异源途径都需要进行精细改造。
在本研究中，通过将CrtE、CrtB和CrtI整合到酿酒酵母CEN.PK2中构建了番茄红素生物合成途径。当删除一个远缘基因座YPL062W时，几乎没有乙酸积累，相对于亲本菌株，胞质乙酰辅酶A池增加了约100%。通过从不同物种中筛选CrtE、CrtB和CrtI，获得了一种最佳的类胡萝卜素生成酶组合，发现来自三孢布拉氏霉菌（BtCrtI）的CrtI在番茄红素生产以及类胡萝卜素中番茄红素比例方面具有优异性能。然后，对BtCrtI的表达水平进行了微调，并评估了细胞交配类型的影响。最后，删除了潜在的远缘基因靶点（YJL064W、ROX1和DOS2），并上调了一个应激反应转录因子INO2。通过上述宿主细胞与类胡萝卜素生成途径之间的改造，番茄红素产量提高了约22倍（从2.43提高到54.63mg/g干细胞重）。最终，在补料分批发酵中，番茄红素产量达到55.56mg/g干细胞重，这是酵母中报道的最高产量。
本研究对酿酒酵母进行了工程改造以生产番茄红素。通过将宿主工程（远缘基因座和细胞交配类型）与途径工程（酶筛选和基因微调）相结合，与起始菌株相比，番茄红素产量逐步提高了22倍。在5升生物反应器中实现了酵母中最高的番茄红素产量（55.56mg/g干细胞重）。本研究为微生物过量生产药物和化学产品的宿主细胞和异源途径组合工程提供了良好参考。
