4-hydroxymandelate is a high-value aromatic compound used in the medicine, cosmetics, food, and chemical industry. However, existing natural extraction and chemical synthesis methods are costly and lead to environmental pollution. This study employed metabolic engineering and directed evolution strategies for de novo 4-hydroxymandelate biosynthesis. Two key challenges were addressed: insufficient precursor supply and limited activity of crucial enzymes. Through gene overexpression and multi-level gene interference using CRISPRi, An Escherichia coli chassis capable of producing the key precursor 4-hydroxyphenylpyruvate and the titer reached 5.05 mM (0.91 g/L). A mutant clone was obtained, HmaSV152G, which showed a 5.13-fold improvement in the catalytic rate. During fermentation, a high production of 194.87 mM (32.768 g/L) 4-hydroxymandelate was achieved in 76 h with a batch supply of glucose in a 5-L bioreactor. This study demonstrated the great potential of biosensors in protein engineering and provides a reference for large-scale production of other high-value aromatic compounds.

中文翻译：

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定向进化和代谢工程产生了大肠杆菌细胞工厂，用于从头生产 4-羟基扁桃酸


4-羟基扁桃酸酯是一种高价值芳香族化合物，用于医药、化妆品、食品和化学工业。然而，现有的天然提取和化学合成方法成本高昂且会导致环境污染。本研究采用代谢工程和定向进化策略进行 4-羟基扁桃酸从头生物合成。解决了两个关键挑战：前体供应不足和关键酶的活性有限。通过使用CRISPRi进行基因过表达和多水平基因干扰，大肠杆菌底盘能够生产关键前体4-羟基苯基丙酮酸，滴度达到5.05 mM（0.91 g/L）。获得了突变克隆HmaSV152G，其催化速率提高了5.13倍。发酵过程中，在 5 L 生物反应器中批量供应葡萄糖，76 小时内即可高产 194.87 mM (32.768 g/L) 4-羟基扁桃酸。该研究展示了生物传感器在蛋白质工程中的巨大潜力，为其他高价值芳香化合物的大规模生产提供了参考。