TAILIEUCHUNG - Báo cáo khoa học: Construction of hybrid peptide synthetases for the production of a-L-aspartyl-L-phenylalanine, a precursor for the high-intensity sweetener aspartame

Microorganisms produce a large number of pharmaco-logically 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 recom-binant 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 a-L-aspartyl-L-phenylalanine (Asp-Phe), the precursor of the high-intensity sweetener a-L-aspartyl-L-phenylalanine methyl ester (aspartame). . | Eur. J. Biochem. 270 4555-4563 2003 FEBS 2003 doi Construction of hybrid peptide synthetases for the production of a-L-aspartyl-L-phenylalanine a precursor for the high-intensity sweetener aspartame Thomas Duerfahrt1 Sascha Doekel1 Theo Sonke2 Peter J. L. M. Quaedflieg2 and Mohamed A. Marahiel1 1 Philipps-Universitat Marburg Fachbereich ChemieỊBiochemie Marburg Germany 2DSM Research Life Sciences - Advanced Synthesis Catalysis and Development Geleen the Netherlands 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 a-L-aspartyl-L-phenylalanine Asp-Phe the precursor of the high-intensity sweetener a-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 a-Asp-Phe and the by-product b-Asp-Phe. Dependent on the turnover rates ranging from min-1 the amount of a-Asp-Phe was between 75 and 100 of overall product indicating a direct correlation between the turnover numbers and the ratios of a-Asp-Phe to b-Asp-Phe. Taken together these results provide useful guidelines for the rational construction of hybrid

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