Construction of hybrid peptide synthetases for the production of alpha-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 alpha-l-aspartyl-l-phenylalanine (Asp-Phe), the precursor of the high-intensity sweetener alpha-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 alpha-Asp-Phe and the by-product beta-Asp-Phe. Dependent on the turnover rates ranging from 0.01-0.7 min-1, the amount of alpha-Asp-Phe was between 75 and 100% of overall product, indicating a direct correlation between the turnover numbers and the ratios of alpha-Asp-Phe to beta-Asp-Phe. Taken together these results provide useful guidelines for the rational construction of hybrid peptide synthetases.
Duerfahrt
Thomas
T
Philipps-Universität Marburg, Fachbereich Chemie/Biochemie, Marburg, Germany.
Doekel
Sascha
S
Sonke
Theo
T
Quaedflieg
Peter J L M
PJ
Marahiel
Mohamed A
MA
D001218:Aspartame / Q000378:metabolism*; D001224:Aspartic Acid / Q000378:metabolism; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D004151:Dipeptides / Q000096:biosynthesis*; D004926:Escherichia coli; D013058:Mass Spectrometry; D010453:Peptide Synthases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010649:Phenylalanine / Q000378:metabolism; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity
D004151:Dipeptides; D011994:Recombinant Proteins; D001224:Aspartic Acid; D010649:Phenylalanine; D010453:Peptide Synthases; C104385:non-ribosomal peptide synthase; D001218:Aspartame