Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, People's Republic of China.
Department of Food Science and Engineering, School of Food, Nanchang University, Nanchang 330029, People's Republic of China.
Sci Rep. 2016 Aug 25;6:32208. doi: 10.1038/srep32208.
L-Phenylalanine (L-Phe) is an important amino acid used in both food and medicinal applications. We developed an in vitro system that allowed a direct, quantitative investigation of phenylalanine biosynthesis in E. coli. Here, the absolute concentrations of six enzymes (AroK, AroL, AroA, AroC, PheA and TyrB) involved in the shikimate (SHIK) pathway were determined by a quantitative proteomics approach and in vitro enzyme titration experiments. The reconstitution of an in vitro reaction system for these six enzymes was established and their effects on the phenylalanine production were tested. The results showed that the yield of phenylalanine increased 3.0 and 2.1 times when the concentrations of shikimate kinase (AroL) and 5-enolpyruvoyl shikimate 3-phosphate (EPSP) synthase (AroA) were increased 2.5 times. Consistent results were obtained from in vivo via the overexpression of AroA in a phenylalanine-producing strain, and the titer of phenylalanine reached 62.47 g/l after 48 h cultivation in a 5-liter jar fermentor. Our quantitative findings provide a practical method to detect the potential bottleneck in a specific metabolic pathway to determine which gene products should be targeted to improve the yield of the desired product.
L-苯丙氨酸（L-Phe）是一种在食品和医药应用中都很重要的氨基酸。我们开发了一种体外系统，可以直接定量研究大肠杆菌中的苯丙氨酸生物合成。在这里，通过定量蛋白质组学方法和体外酶滴定实验确定了参与莽草酸（SHIK）途径的六种酶（AroK、AroL、AroA、AroC、PheA 和 TyrB）的绝对浓度。建立了这些六种酶的体外反应系统的重组，并测试了它们对苯丙氨酸生产的影响。结果表明，当莽草酸激酶（AroL）和 5-烯醇丙酮酰莽草酸 3-磷酸合酶（AroA）的浓度增加 2.5 倍时，苯丙氨酸的产量分别增加了 3.0 倍和 2.1 倍。通过在苯丙氨酸生产菌株中过表达 AroA，在 5 升罐式发酵罐中培养 48 小时后，苯丙氨酸的效价达到 62.47g/l，体内结果也得到了一致的验证。我们的定量发现为检测特定代谢途径中的潜在瓶颈提供了一种实用方法，以确定应针对哪些基因产物来提高所需产物的产量。
