Engineering the pH optimum of tyrosine ammonia lyase from Rhodobacter sphaeroides via computationally guided rational strategy

Tyrosine Ammonia Lyase (TAL) is a key enzyme used for the commercial production of p-coumaric acid. The currently known TAL enzymes encoded by diverse microbial species showed optimal activity at alkaline pH 9.0–10.5. However, efficient TAL variants that function at neutral pH are required to meet biorefinery demands. In this study, a computationally guided rational strategy was used to perform site-directed mutagenesis by decreasing negative charges on the surface residues and enhancing the positive charges near the active site of the TAL enzyme from the bacterium Rhodobacter sphaeroides. In total, 21 residues, including six near the active site and 15 on the enzyme's surface, were selected and subjected to site-directed mutagenesis. The mutants P68H, P9D, and P484E, demonstrated a pH optimal shift from 9.0 to 8.0 and increased activity by 0.8-, 4.8-, and 4.0-fold, respectively. These single optimal mutants were combined in different combinations (P9D/P68H, P9D/P484E, and P68H/P484E), and double mutants were designed. The double mutant P9D/P484E showed a shift in the pH from 9.0 to 7.0, with a 6-fold increase in the enzyme's activity at neutral pH. The double mutant (P9D/P484E) of the TAL enzyme from R. sphaeroides demonstrates potential for application in the industrial-scale production of p-coumaric acid.