This study aimed to establish a high-level phenol bioproduction system from glycerol through metabolic engineering of the yeast Pichia pastoris (Komagataella phaffii). Introducing tyrosine phenol-lyase to P. pastoris led to a production of 59 mg/L of phenol in flask culture. By employing a strain of P. pastoris that overproduces tyrosine—a precursor to phenol—we achieved a phenol production of 1052 mg/L in glycerol fed-batch fermentation. However, phenol concentrations exceeding 1000 mg/L inhibited P. pastoris growth. A phenol pertraction system utilizing a hollow fiber membrane contactor and tributyrin as the organic solvent was developed to reduce phenol concentration in the culture medium. Integrating this system with glycerol fed-batch fermentation resulted in a 214 % increase in phenol titer (3304 mg/L) compared to glycerol fed-batch fermentation alone. These approaches offer a significant framework for the microbial production of chemicals and materials that are highly toxic to microorganisms.

本研究旨在通过酵母毕赤酵母（ Komagataella phaffii ）的代谢工程，建立以甘油为原料的高水平苯酚生物生产系统。将酪氨酸苯酚裂解酶引入巴斯德毕赤酵母，在烧瓶培养物中产生了 59 mg/L 的苯酚。通过使用过量生产酪氨酸（苯酚的前体）的巴斯德毕赤酵母菌株，我们在甘油补料分批发酵中实现了 1052 mg/L 的苯酚产量。然而，苯酚浓度超过1000 mg/L会抑制毕赤酵母的生长。开发了一种利用中空纤维膜接触器和三丁酸甘油酯作为有机溶剂的苯酚萃取系统，以降低培养基中的苯酚浓度。与单独的甘油分批补料发酵相比，将该系统与甘油分批补料发酵相结合，苯酚滴度 (3304 mg/L) 增加了 214%。这些方法为微生物生产对微生物具有剧毒的化学品和材料提供了重要的框架。