As an essential aromatic amino acid, L-phenylalanine (L-Phe) has a lot of applications as precursors for pharmaceuticals, artificial sweetner, etc. Previously, through metabolic engineering, we developed an Escherichia coli strain (YHP05) capable of overproducing L-Phe. In this study, we further engineered E. coli strain to develop an economic bioprocess for L-Phe production in large scale bioreactor. First, we developed E. coli YHP06 harboring pYHP which cultivation does not require supplementation of amino acids for cell growth. Next, the strong and constitutive gene expression systems for L-Phe biosynthesis were sequentially introduced into chromosome of E. coli, which allowed the antibiotic-free and inducer-free cultivation. Among the engineered strains, the H-02 strain showed the highest production of L-Phe (3.6 g/L) in the flask cultivation. The production of L-Phe in the engineered H-02 strain was also evaluated in lab-scale (5 L), and L-Phe as high as 30.7 g/L could be produced with 0.81 g/L/h of productivity. Finally, the pilot-scale (30 L) fed-batch cultivation was performed, and we could achieve the production of L-Phe as high as 38.6 g/L with 1.07 g/L/h of productivity.

作为一种必需的芳香族氨基酸，L-苯丙氨酸（L-Phe）作为药物、人造甜味剂等的前体有很多应用。以前，通过代谢工程，我们开发了一种能够过量生产 L-Phe 的大肠杆菌菌株 (YHP05)。在这项研究中，我们进一步改造了大肠杆菌菌株，以开发一种经济的生物工艺，用于在大型生物反应器中生产 L-Phe。首先，我们开发了含有 pYHP 的大肠杆菌YHP06，其培养不需要为细胞生长补充氨基酸。接下来，将用于 L-Phe 生物合成的强大的组成型基因表达系统依次引入大肠杆菌的染色体中，允许无抗生素和无诱导剂培养。在工程菌株中，H-02 菌株在烧瓶培养中表现出最高的 L-Phe (3.6 g/L) 产量。还在实验室规模 (5 L) 中评估了工程化 H-02 菌株中 L-Phe 的产量，并且可以以 0.81 g/L/h 的生产率生产高达 30.7 g/L 的 L-Phe。最后，进行了中试规模（30 L）补料分批培养，我们可以以 1.07 g/L/h 的生产率实现高达 38.6 g/L 的 L-Phe 产量。