Quantitative Analysis And Engineering Of L-Phenylalanine Biosynthesis By Identifying Key Enzymes In Vitro

L-Phenylalanine is an important amino acid used in both food and medicinal applications. With lower cost of raw materials and higher purity of product, fermentative production of L-phenylalanine is becoming more promising. The complicacy of L-phenylalanine biosynthesis pathway and its regulation mechanism results in low efficiency of L-phenylalanine production.We previously engineered an Escherichia coli cell line that produced 45 g/L total phenylalanine in a fed-batch fermentation. However, it was unclear which additional alterations to the strain would lead to further enhancement of phenylalanine biosynthesis. To determine a more direct strategy, we developed an in vitro system that allowed a direct, quantitative investigation of phenylalanine biosynthesis in Escherichia coli. Here, the absolute concentrations of six enzymes (AroK, AroL, AroA, AroC, PheA and TyrB) involved in the shikimate 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 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. Meanwhile, the phenylalanine yield on glucose increased from 0.186 g/g to 0.236 g/g.In summary, our quantitative findings provide a practical method and ideas 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, and accumulate basic datas for the targeted transformation of the strain.