Directed evolution of a gatekeeper domain in nonribosomal peptide synthesis.
Modular natural products are biosynthesized by series of enzymes that activate, assemble, and process a nascent chain of building blocks. Adenylation domains are gatekeepers in nonribosomal peptide biosynthesis, providing the entry point for assembly of typical peptide-based natural products. We report the directed evolution of an adenylation domain based on a strategy of using a weak, promiscuous activity as a springboard for reprogramming the biosynthetic assembly line. Randomization of residues invoked in a "specificity-conferring code" and selection for a non-native substrate lead to mutant G2.1, favoring smaller amino acids with a specificity change of 10(5): a 170-fold improvement for L-alanine corresponds to a 10(3)-fold decrease for its original substrate (L-phenylalanine). These results establish directed evolution as a method to change gatekeeper domain specificity and suggest that adaptation of modules in combinatorial biosynthesis is achievable with few mutations during evolution.
Department of Biochemistry, University of Cambridge, Cambridge CB2 1GA, UK.
Benoit
B
Villiers
Florian
F
Hollfelder
D000409:Alanine / Q000378:metabolism; D000596:Amino Acids / Q000378:metabolism; D001665:Binding Sites; D019020:Directed Molecular Evolution; D057927:Hydrophobic and Hydrophilic Interactions; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D046908:Peptide Biosynthesis, Nucleic Acid-Independent; D010453:Peptide Synthases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010649:Phenylalanine / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011897:Random Allocation; D012997:Solvents / Q000737:chemistry; D013379:Substrate Specificity
D000596:Amino Acids; D012997:Solvents; D010649:Phenylalanine; D010453:Peptide Synthases; C035180:tyrocidine synthetase; D000409:Alanine