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.

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L -Tyrosine derivatives are widely applied in the pharmaceutical, food, and chemical industries. Their production is mainly confined to chemical synthesis and plant extract. Microorganisms, as cell factories, exhibit promising advantages for valuable chemical production to fulfill the increase in the demand of global markets. Yeast has been used to produce natural products owing to its robustness and genetic maneuverability. Focusing on the progress of yeast cell factories for the production of L -tyrosine derivatives, we summarized the emerging metabolic engineering approaches in building L -tyrosoineoverproducing yeast and constructing cell factories of three typical chemicals and their derivatives: tyrosol, p-coumaric acid, and L -DOPA. Finally, the challenges and opportunities of L -tyrosine derivatives production in yeast cell factories were also discussed.aromatic amino acids, cell factory, L -tyrosine derivatives, synthetic biology, yeastis an essential amino acid nutrient with diverse applications in food, medicine, and chemical industries. Its derivatives, including Hydroxytyrosol S. cerevisiae Glucose 5L fed-batch 6970 H. Liu, Wu, et al. (2022) Salidroside S. cerevisiae Glucose 5L fed-batch 26,550 H. Liu, Tian, et al. (2021) p-Coumaric acid derivatives p-coumaric acid S. cerevisiae Glucose 1L fed-batch 12,500 Q. Liu, Yu, et al. (2019) p-coumaric acid S. cerevisiae Xylose 1L fed-batch 242 Borja et al. (2019) Caffeic acid S. cerevisiae Carboxymethylcellulose Shake flask 16.91 M. Cai et al. (2022) Caffeic acid S. cerevisiae Glucose 1.2L fed-batch 5500 R. Chen et al. (2022) Caffeic acid Candida glycerinogenes Glucose and Xylose 5L fed-batch 431.45 X.-H. Wang et al. (2022) Rosmarinic acid S. cerevisiae Glucose Shake flask 208 P. Zhou et al. (2022) Ferulic Acid S. cerevisiae Glucose 1.2L fed-batch 3800 R. Chen et al. (2022) Resveratrol S. cerevisiae Glucose and Ethanol shake flask 187 Costa et al. (2021) Resveratrol S. cerevisiae Lactose Shake flask 210 Costa et al. (2022) Resveratrol Scheffersomyces stipitis Cellobiose Shake flask 529.8 Kobayashi et al. (2021) Resveratrol S. stipitis Sucrose Shake flask 668.6 Kobayashi et al. (2021) Resveratrol Pichia pastoris Glycerol 250 mL fed-batch 1825 Kumokita et al. (2022) Pterostilbene S. cerevisiae Glucose Shake flask 34.93 M. Li et al. (2016) Pinostilbene S. cerevisiae Glucose Shake flask 5.52 M. Li et al. (2016) Naringenin S. cerevisiae Glucose Shake flask 203.49 Tong et al. (2022) Naringenin S. cerevisiae Glucose 5L fed-batch 1184.1 H. Li et al. (2022) Naringenin Yarrowia lipolytica Glucose 3L fed-batch 898 Palmer et al. (2020) Naringenin P. pastoris Glycerol 250 mL fed-batch 1067 Kumokita et al. (2022) Naringenin Y. lipolytica Glucose Shake flask 252.4 Lv et al. (2019) Eriodictyol Y. lipolytica Glucose Shake flask 134.2 Lv et al. (2019) Phloretin S. cerevisiae Glucose 5L fed-batch 619.5 C. Jiang et al. (2020) Apigenin Y. lipolytica Glucose Shake flask 80.74 Vanegas et al. (2018) Luteolin Y. lipolytica Glucose Shake flask 47.9 Vanegas et al. (2018) Kaempferol S. ...

Abstract3-deoxy-D-arabino-heptulosonate-7-phosphate synthase (DAHPS) is a key enzyme in the shikimate pathway for the biosynthesis of aromatic compounds. L-Phe and L-Tyr bind to the two main DAHPS isoforms and inhibit their enzyme activities, respectively. Synthetic biologists aim to relieve such inhibitions in order to improve the productivity of aromatic compounds. In this work, we reported a point mutant of yeast DHAPS, Aro3D154N, which retains the wild type enzyme activity but converts it highly inert to the inhibition by L-Phe. The Aro3 crystal structure along with the molecular dynamics simulations analysis suggests that the D154N mutation distant from the inhibitor binding cavity may reduce the binding affinity of L-Phe. Growth assays demonstrated that substitution of the conserved D154 with asparagine suffices to relieve the inhibition of L-Phe on Aro3, L-Tyr on Aro4, and the inhibitions on their corresponding homologues from diverse yeasts. The importance of our discovery is highlighted by the observation of 29.1% and 43.6% increase of yield for the production of tyrosol and salidroside respectively upon substituting ARO3 with ARO3D154N. We anticipate that this allele would be used broadly to increase the yield of various aromatic products in metabolically diverse microorganisms.