Protocatechuic acid (3, 4-dihydroxybenzoic acid, PCA) is widely used in the pharmaceuticals, health food, and cosmetics industries owing to its diverse biological activities. However, the inhibition of 3-dehydroshikimate dehydratase (AroZ) by PCA and its toxicity to cells limit the efficient production of PCA in . In this study, a high-level strain of 3-dehydroshikimate, DHS01, was developed by blocking the carbon flow from the shikimate-overproducing strain SA09. Additionally, the PCA biosynthetic pathway was established in DHS01 by introducing the high-activity AroZ. Subsequently, the protein structure and catalytic mechanism of 3-dehydroshikimate dehydratase from PHEA-2 (AroZ) were clarified. The variant AroZ, achieved by modulating the conformational dynamics of AroZ, effectively relieved product inhibition. Additionally, the tolerance of the strain PCA04 to PCA was enhanced by adaptive laboratory evolution, and a biosensor-assisted high-throughput screening method was designed and implemented to expedite the identification of high-performance PCA-producing strains. Finally, in a 5 L bioreactor, the final strain PCA05 achieved the highest PCA titer of 46.65 g/L, a yield of 0.23 g/g, and a productivity of 1.46 g/L/h for PCA synthesis from glucose using normal fed-batch fermentation. The strategies described herein serve as valuable guidelines for the production of other high-value and toxic products.

中文翻译：

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利用大肠杆菌系统工程高效生产原儿茶酸

原儿茶酸（3, 4-二羟基苯甲酸，PCA）因其多种生物活性而广泛应用于医药、保健食品和化妆品行业。然而，PCA对3-脱氢莽草酸脱水酶（AroZ）的抑制及其对细胞的毒性限制了PCA的有效生产。在本研究中，通过阻断过度生产莽草酸的菌株SA09的碳流，开发了高水平的3-脱氢莽草酸菌株DHS01。此外，通过引入高活性AroZ，在DHS01中建立了PCA生物合成途径。随后，阐明了来自PHEA-2（AroZ）的3-脱氢莽草酸脱水酶的蛋白质结构和催化机制。通过调节 AroZ 的构象动力学实现的变体 AroZ 有效缓解了产物抑制。此外，通过适应性实验室进化增强了菌株PCA04对PCA的耐受性，并设计并实施了生物传感器辅助的高通量筛选方法，以加快高性能PCA生产菌株的鉴定。最后，在 5 L 生物反应器中，最终菌株 PCA05 使用正常补料从葡萄糖合成 PCA 时达到了 46.65 g/L 的最高 PCA 滴度、0.23 g/g 的产量和 1.46 g/L/h 的生产率。分批发酵。本文描述的策略可以为生产其他高价值和有毒产品提供有价值的指导。