Yarrowia lipolytica is a novel microbial chassis to upgrade renewable low-cost carbon feedstocks to high-value commodity chemicals and natural products. In this work, we systematically characterized and removed the rate-limiting steps of the shikimate pathway and achieved de novo synthesis of five aromatic chemicals in Y. lipolytica. We determined that eliminating amino acids formation and engineering feedback-insensitive DAHP synthases are critical steps to mitigate precursor competition and relieve the feedback regulation of shikimate pathway. Further overexpression of heterologous phosphoketolase and deletion of pyruvate kinase provided a sustained metabolic driving force that channels E4P (erythrose 4-phosphate) and PEP (phosphoenolpyruvate) precursors through the shikimate pathway. Precursor competing pathways and byproduct formation pathways were also blocked by inactivating chromosomal genes. To demonstrate the utility of our engineered chassis strain, three natural products, 2-phenylethanol (2-PE), p-coumaric acid and violacein, which were derived from phenylalanine, tyrosine and tryptophan, respectively, were chosen to test the chassis performance. We obtained 2426.22 ± 48.33 mg/L of 2-PE, 593.53 ± 28.75 mg/L of p-coumaric acid, 12.67 ± 2.23 mg/L of resveratrol, 366.30 ± 28.99 mg/L of violacein and 55.12 ± 2.81 mg/L of deoxyviolacein from glucose in shake flask. The 2-PE production represents a 286-fold increase over the initial strain (8.48 ± 0.50 mg/L). Specifically, we obtained the highest 2-PE, violacein and deoxyviolacein titer ever reported from the de novo shikimate pathway in yeast. These results set up a new stage of engineering Y. lipolytica as a sustainable biorefinery chassis strain for de novo synthesis of aromatic compounds with economic values.

中文翻译：

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工程解脂耶氏酵母作为从头合成五种芳烃衍生天然产物和化学品的底盘

解脂耶氏酵母是一种新型的微生物底盘，可将可再生的低成本碳原料升级为高价值的商品化学品和天然产品。在这项工作中，我们系统地表征并去除了iki草酸酯途径的限速步骤，并实现了解脂耶氏酵母中五种芳香族化学品的从头合成。我们确定消除氨基酸形成和工程反馈不敏感的DAHP合成酶是缓解前体竞争和缓解sh草酸途径的反馈调节的关键步骤。异源磷酸酮醇酶的进一步过表达和丙酮酸激酶的缺失提供了持续的代谢驱动力，该驱动力通过sh草酸酯途径引导E4P（4-磷酸赤藓糖）和PEP（磷酸烯醇丙酮酸）前体。灭活染色体基因也阻断了前体竞争途径和副产物形成途径。为了证明我们设计的底盘菌株的效用，分别选择了分别来自苯丙氨酸，酪氨酸和色氨酸的三种天然产物2-苯基乙醇（2-PE），对香豆酸和紫丁烯酸来测试底盘性能。我们获得了2426.22±48.33 mg / L的2-PE，593.53±28.75 mg / L的对香豆酸，12.67±2.23 mg / L的白藜芦醇，366.30±28.99 mg / L的紫杉醇和55.12±2.81 mg / L的摇瓶中葡萄糖中的脱氧卵磷脂。2-PE的产量比初始菌株（8.48±0.50 mg / L）高出286倍。具体来说，我们从酵母中的从头叶酸盐途径获得了最高的2-PE，紫精素和脱氧紫精素效价。