Construction of hybrid peptide synthetases for the production of α‐l‐aspartyl‐l‐phenylalanine, a precursor for the high‐intensity sweetener aspartame

Microorganisms produce a large number of pharmacologically and biotechnologically important peptides by using nonribosomal peptide synthetases (NRPSs). Due to their modular arrangement and their domain organization NRPSs are particularly suitable for engineering recombinant proteins for the production of novel peptides with interesting properties. In order to compare different strategies of domain assembling and module fusions we focused on the selective construction of a set of peptide synthetases that catalyze the formation of the dipeptide α‐l‐aspartyl‐l‐phenylalanine (Asp‐Phe), the precursor of the high‐intensity sweetener α‐l‐aspartyl‐l‐phenylalanine methyl ester (aspartame). The de novo design of six different Asp‐Phe synthetases was achieved by fusion of Asp and Phe activating modules comprising adenylation, peptidyl carrier protein and condensation domains. Product release was ensured by a C‐terminally fused thioesterase domains and quantified by HPLC/MS analysis. Significant differences of enzyme activity caused by the fusion strategies were observed. Two forms of the Asp‐Phe dipeptide were detected, the expected α‐Asp‐Phe and the by‐product β‐Asp‐Phe. Dependent on the turnover rates ranging from 0.01–0.7 min−1, the amount of α‐Asp‐Phe was between 75 and 100% of overall product, indicating a direct correlation between the turnover numbers and the ratios of α‐Asp‐Phe to β‐Asp‐Phe. Taken together these results provide useful guidelines for the rational construction of hybrid peptide synthetases.