Biosynthesis of 4-hydroxyphenylpyruvic acid from l-tyrosine using recombinant Escherichia coli cells expressing membrane bound l-amino acid deaminase.

4-Hydroxyphenylpyruvic acid (4-HPPA), a kind of α-keto acid, is an intermediate in the metabolism of tyrosine and has a wide range of application in food, pharmaceutical and chemical industry. Using amino acids as raw material to produce the corresponding α-keto acid is thought to be both economic and efficient. Among the enzymes that convert amino acid to α-keto acid, membrane bound l-amino acid deaminase (ml-AAD), which is anchored to the outer side of the cytomembrane, become an ideal enzyme to prepare α-keto acid since there is no cofactors needed and H2O2 production during the reaction. In this study, the ml-AAD from Proteus vulgaris was used to prepare whole-cell catalysts to produce 4-HPPA from l-tyrosine. The secretory efficiency of ml-AAD conducted by its own twin-arginine signal peptide (twin-arginine translocation pathway, Tat) and integrated pelB (the general secretory pathway, Sec)-Tat signal peptide were determined and compared firstly, using two pET systems (pET28a and pET20b). It was found that the Tat pathway (pET28a–mlaad) resulted in higher cell-associated ml-AAD activity and cell biomass, and was more beneficial to prepare biocatalyst. In addition, expression host Bl21(DE3) and 0.05 mmol·L− 1 IPTG were found to be suitable for ml-AAD expression. The reaction conditions for ml-AAD were optimized and 72.72 mmol·L− 1 4-HPPA was obtained from 100 mmol·L− 1 tyrosine in 10 h under the optimized conditions. This bioprocess, which is more eco-friendly and economical than the traditional chemical synthesis ways, has great potential for industrial application.