Two-Step Production of Phenylpyruvic Acid from L-Phenylalanine by Growing and Resting Cells of Engineered Escherichia coli: Process Optimization and Kinetics Modeling

Abstract

Phenylpyruvic acid (PPA) is widely used in the pharmaceutical, food, and chemical industries. Here, a two-step bioconversion process, involving growing and resting cells, was established to produce PPA from l-phenylalanine using the engineered Escherichia coli constructed previously. First, the biotransformation conditions for growing cells were optimized (l-phenylalanine concentration 20.0 g·L−1, temperature 35°C) and a two-stage temperature control strategy (keep 20°C for 12 h and increase the temperature to 35°C until the end of biotransformation) was performed. The biotransformation conditions for resting cells were then optimized in 3-L bioreactor and the optimized conditions were as follows: agitation speed 500 rpm, aeration rate 1.5 vvm, and l-phenylalanine concentration 30 g·L−1. The total maximal production (mass conversion rate) reached 29.8 ± 2.1 g·L−1 (99.3%) and 75.1 ± 2.5 g·L−1 (93.9%) in the flask and 3-L bioreactor, respectively. Finally, a kinetic model was established, and it was revealed that the substrate and product inhibition were the main limiting factors for resting cell biotransformation.

A new approach for efficient synthesis of phenyllactic acid from L-phenylalanine: Pathway design and cofactor engineering

“…Compared to phenylalanine dehydrogenase (Brunhuber, Thoden, Blanchard, & Vanhooke, ; Ödman, Wellborn, & Bommarius, ) and aminotransferases (Hirotsu, Goto, Okamoto, & Miyahara, ), the oxidative deamination reaction l ‐AAD catalyzed requires only one amino acid, and can synthesize PPA without extra addition of coenzyme (FAD) or coenzyme regeneration system, because the FAD E.coli itself produced can support the entire reaction, so l ‐AAD was the optimal one. In addition, Hou et al has obtained the triple mutant D165K/F263M/L336M of l ‐AAD, which produced the highest PPA titer of 10.0 ± 0.4 g·L −1 of resting‐cell biotransformation, with a substrate conversion ratio of 100% (Hou et al, ; Hou, Hossain, Li, Shin, Du, et al, ; Hou, Hossain, Li, Shin, Liu, et al, ). Therefore, the triple mutant of l‐aad was cloned first to generate plasmid pRSF‐aad in this study.…”

“…In previous work, we found that l ‐amino acid deaminases ( l ‐AAD) from Proteus mirabilis KCTC 2566 can catalyze the efficient deamination of l ‐phenylalanine (Phe) to PPA and ammonia with a substrate conversion ratio of 100% (Hou et al, ; Hou, Hossain, Li, Shin, Du, et al, ; Hou, Hossain, Li, Shin, Liu, et al, ). The price of Phe is much lower than that of PPA (the price of PPA is about 500 times that of Phe), and thus the production cost of PLA can be significantly decreased if with Phe, not PPA, is used as the substrate.…”

Metabolism of Non-growing Cells and Their Application in Biotransformation

“…In this approach, cells are grown to a desired concentration, harvested and resuspended in a buffer supplemented with a substrate but not nitrogen source [1,5]. Besides increased productivities of product, possible reuse of the non-growing cells in multiple biotransformation and reduced consumption of NADPH in anabolic reactions also makes the approach appealing [1,[6][7][8]. Downstream product separation could also be made easier [6].…”

“…Besides increased productivities of product, possible reuse of the non-growing cells in multiple biotransformation and reduced consumption of NADPH in anabolic reactions also makes the approach appealing [1,[6][7][8]. Downstream product separation could also be made easier [6]. But biocatalyst stability is a limiting factor [1].…”

Efficient production of phenylpropionic acids by an amino‐group‐transformation biocatalytic cascade

“…Phenylpropionic acids are defined as aromatic carboxylic acids with the C6‐C3 skeleton, mainly including phenylalanines, phenylpyruvic acids, phenylacrylic acids, and phenyllactic acids. They are of great importance in the fields of fine chemicals synthesis, cosmetics, and pharmaceutical manufacturing (Busto et al, ; Busto, Simon, Richter, & Kroutil, ; Hou et al, ; Lütke‐Eversloh, Santos, & Stephanopoulos, ; Xue, Cao, & Zheng, ). For example, tyrosine derivatives play prominent roles in the synthesis of pharmaceuticals (Dennig, Busto, Kroutil, & Faber, ; Seisser et al, ).…”