Journal
CHEMISTRY & BIOLOGY
Volume 18, Issue 10, Pages 1290-1299Publisher
CELL PRESS
DOI: 10.1016/j.chembiol.2011.06.014
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Funding
- MRC
- EU
- MRC [G0300193] Funding Source: UKRI
- Medical Research Council [G0300193] Funding Source: researchfish
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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.
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