Optimizing Oleaginous Yeast Cell Factories for Flavonoids and Hydroxylated Flavonoids Biosynthesis
Optimizing Oleaginous Yeast Cell Factories for Flavonoids and Hydroxylated Flavonoids Biosynthesis
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DOI：
10.1021/acssynbio.9b00193
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发表时间：
2019-11-01
影响因子：
4.7
通讯作者：
Xu, Peng
中科院分区：
文献类型：
--
作者：
Lv, Yongkun;Marsafari, Monireh;Xu, Peng
植物拥有无数的次级代谢产物，具有广泛的健康促进益处。迄今为止，植物提取仍然是生产高价值天然产品的主要途径，这固有地受到经济和可扩展性问题的影响。植物生物合成基因簇在微生物宿主中的异源表达被认为是克服这些限制的可行途径。产油酵母产生大量的脂质体，丰富的膜结构和亲脂环境为许多植物源P450酶的区域选择性和立体选择性提供了理想的环境。在这项工作中，我们使用模块化的方法来构建，表征和优化Yarrowia lipolytica中的黄酮途径。我们还评估了各种前体生物合成途径，并释放了Y的代谢潜力。以产生类黄酮和羟基化类黄酮。具体来说，我们已经确定，查尔酮合成酶（CHS）和细胞色素P450还原酶（CPR）的羟基化类黄酮生产的瓶颈。我们确定CHS和CPR的最佳基因拷贝数分别为5和2。我们通过表达与分支酸和丙二酰辅酶A供应相关的基因进一步消除了前体途径的限制。在摇瓶发酵条件下，工程菌以葡萄糖为底物，经pH值和碳氮比（C/N）优化后，可分别产生252.4mg/L的柚皮素、134.2mg/L的圣草酚和110.5mg/L的紫杉叶素。黄酮及其羟基化衍生物最显著地被称为抗氧化剂和抗衰老剂。这些发现证明了我们有能力利用产油酵母作为微生物主力来扩大自然界的生物合成潜力，使我们能够弥合药物发现和天然产品制造之间的差距。
Plants possess myriads of secondary metabolites with a broad spectrum of health-promoting benefits. To date, plant extraction is still the primary route to produce high-value natural products which inherently suffers from economics and scalability issues. Heterologous expression of plant biosynthetic gene clusters in microbial host is considered as a feasible approach to overcoming these limitations. Oleaginous yeast produces a large amount of lipid bodies, the abundant membrane structure and the lipophilic environment provide the ideal environment for the regioselectivity and stereoselectivity of many plant-derived P450 enzymes. In this work, we used modular method to construct, characterize, and optimize the flavonoid pathways in Yarrowia lipolytica. We also evaluated various precursor biosynthetic routes and unleashed the metabolic potential of Y. lipolytica to produce flavonoids and hydroxylated flavonoids. Specifically, we have identified that chalcone synthase (CHS) and cytochrome P450 reductases (CPR) were the bottlenecks of hydroxylated flavonoid production. We determined the optimal gene copy number of CHS and CPR to be 5 and 2, respectively. We further removed precursor pathway limitations by expressing genes associated with chorismate and malonyl-CoA supply. With pH and carbon-nitrogen ratio (C/N) optimization, our engineered strain produced 252.4 mg/L naringenin, 134.2 mg/L eriodictyol, and 110.5 mg/L taxifolin from glucose in shake flasks. Flavonoid and its hydroxylated derivatives are most prominently known as antioxidant and antiaging agents. These findings demonstrate our ability to harness the oleaginous yeast as the microbial workhorse to expand nature's biosynthetic potential, enabling us to bridge the gap between drug discovery and natural product manufacturing.