A bi-enzymatic cascade to yield pyruvate as co-substrate for l-tyrosine production

Abstract l -Tyrosine is a versatile compound used in the fine chemical, pharmaceutical, and functional food industries. Here, we report a bi-enzymatic cascade involving alanine racemase (ALR) and d -amino acid oxidase (DAAO) to produce pyruvate, as co-substrate for l -tyrosine production, from the cheap substrate l -alanine. The BpALR (ALR from Bacillus pseudofirmus ) was used as a whole-cell biocatalyst, converting l -alanine to d , l -alanine. The FsDAAO (DAAO from Fusarium solani ) was immobilized to oxidize the d -alanine generated in the first step to pyruvate. Both systems were combined as a continuous-flow reactor for maximized l -alanine-to-pyruvate conversion rates. The optimal parameters and appropriate conditions for FsDAAO immobilization were investigated. The pyruvate concentration of 86.6 g/L was achieved within 17 h. Subsequently, a whole-cell biocatalyst system for l -tyrosine production, catalyzed by the tyrosine phenol-lyase (TPL) from Erwinia herbicola (EhTPL), was developed, and a fed-batch approach was applied with phenol and the pyruvate produced with the ALR/DAAO system mentioned above. The concentration of phenol and pyruvate in the reactor should not exceed 7.5 g/L and 10 g/L, respectively. Significantly, the l -tyrosine concentration of 152.5 g/L was achieved within 10 h, demonstrating the great potential for high-efficiency production of l -tyrosine through the approach we established in this paper. Graphical abstract Key points • A specific bioreactor system for pyruvate produced from l -alanine was developed • The appropriate condition for immobilization of FsDAAO was investigated • A fed-batch process was established to produce l -tyrosine with recombinant E. coli • The bi-enzymatic cascade was successfully used for l -tyrosine production at low cost