High-Level Production of Hydroxytyrosol in Engineered Saccharomyces cerevisiae

Hydroxytyrosol (HT) is a valuable aromatic compound with numerous applications. Herein, we enabled the efficient and scalable de novo HT production in engineered Saccharomyces cerevisiae (S. cerevisiae) from glucose. Starting from a tyrosol-overproducing strain, six HpaB/HpaC combinations were investigated, and the best catalytic performance was acquired with HpaB from Pseudomonas aeruginosa (PaHpaB) and HpaC from Escherichia coli (EcHpaC), resulting in 425.7 mg/L HT in shake flasks. Next, weakening the tryptophan biosynthetic pathway through downregulating the expression of TRP2 (encoding anthranilate synthase) further improved the HT titer by 27.2% compared to the base strain. Moreover, the cytosolic NADH supply was improved through introducing the feedback-resistant mutant of the TyrA (the NAD+-dependent chorismate mutase/prephenate dehydrogenase, TyrA*) from E. coli, which further increased the HT titer by 36.9% compared to the base strain. The best performing strain was obtained by optimizing the biosynthesis of HT in S. cerevisiae through a screening for an effective HpaB/HpaC combination, biosynthetic flux rewiring, and cofactor engineering, which enabled the titer of HT reaching 1120.0 mg/L in the shake flask. Finally, the engineered strain produced 6.97 g/L of HT by fed-batch fermentation, which represents the highest titer for de novo HT biosynthesis in microorganisms reported to date.羟基酪醇（Hydroxytyrosol，HT）是一种极具应用价值的芳香族化合物。本研究实现了以葡萄糖为碳源，在工程化酿酒酵母（Saccharomyces cerevisiae，S. cerevisiae）中高效、可规模化的HT从头合成。以酪醇高产菌株为初始底盘，本研究共测试了6组HpaB/HpaC酶组合，其中来自铜绿假单胞菌（Pseudomonas aeruginosa）的PaHpaB与来自大肠杆菌（Escherichia coli）的EcHpaC组合展现出最优催化性能，摇瓶发酵中HT效价可达425.7 mg/L。随后，通过下调编码邻氨基苯甲酸合酶的TRP2基因表达以弱化色氨酸生物合成途径，相较于基础菌株，HT效价进一步提升27.2%。此外，通过引入来自大肠杆菌的反馈抑制抗性型TyrA突变体（即NAD+依赖型分支酸变位酶/预苯酸脱氢酶，TyrA*）以强化胞质NADH供给，相较于基础菌株，HT效价再次提升36.9%。最终，通过筛选高效HpaB/HpaC组合、重构生物合成通量以及辅因子工程，本研究完成了酿酒酵母中HT生物合成途径的系统优化，获得的最优工程菌株在摇瓶发酵中HT效价可达1120.0 mg/L。最后，该工程菌株经补料分批发酵可实现6.97 g/L的HT产量，为目前已报道的微生物从头合成HT的最高效价。