Efficient synthesis of Ala-Tyr by L-amino acid ligase coupled with ATP regeneration system.

The multi-enzyme coupling reaction system has become a promising biomanufacturing platform for biochemical production. Tyr is an essential amino acid, but the limited solubility restricts its use. Tyrosyl dipeptide has been paid more attention due to its higher solubility. In this study, an efficient enzymatic cascade of Ala-Tyr synthesis was developed by a L-amino acid ligase together with polyphosphate kinase (PPK). Two L-amino acid ligases from Bacillus subtilis and Bacillus pumilus were selected and applied for Ala-Tyr synthesis. The L-amino acid ligase from B. subtilis (Bs) was selected and coupled with the PPK from Sulfurovum lithotrophicum (PPKSL) for regenerating ATP to produce Ala-Tyr in one pot. In the optimization system, 40.1 mM Ala-Tyr was produced within 3 h due to efficient ATP regeneration with hexametaphosphate (PolyP(6)) as the phosphate donor. The molar yield was 0.89 mol/mol based on the substrates added, while the productivity of Ala-Tyr achieved 13.4 mM/h, which were the highest yield and productivity ever reported about Ala-Tyr synthesis with L-amino acid ligase.