Directed Evolution of an Adenylation Domain Specificity Code

Non‐ribosomal peptides (NRPs) are a medically and agriculturally important class of natural products found in bacteria and fungi that are biosynthesized by non‐ribosomal peptide synthetases (NRPSs). Adenylation (A) domains of NRPSs have a high degree of specificity for the substrate they recognize and are therefore referred to as the gatekeepers of NRPS enzymology. The specificity of A domains is determined by ten residues in the substrate‐binding pocket; thus, these residues are known as the A domain specificity code. To produce new NRPs, efforts have been made to rationally alter A domain specificity by changing the specificity code, but these experiments have typically resulted in proteins with broadened specificity or poor function. We hypothesize that an NRPS‐based genetic selection system is needed to better understand the specificity code by allowing us to identify functional A domains with altered specificity codes in a high‐throughput manner. We used directed evolution to examine the flexibility of the specificity code of EntF from the enterobactin (ENT) siderophore biosynthetic system in E. coli. Proper assembly of ENT requires L‐Ser incorporation and thereby provides a genetic selection for functional L‐Ser recognition. We created several entF A domain libraries, each randomizing different but overlapping subsets of five specificity code residues using NNK‐containing primers. These libraries were used to complement an E. coli ΔentF mutant in trans, selecting for functional A domain variants on iron‐limiting media. We determined that certain positions in the specificity code have strict requirements, while others are tolerant of substitutions and often contain residues not observed in known L‐Ser codes. All the isolated EntF variants retain specificity solely for L‐Ser and have biological and kinetic activity comparable to that of wildtype EntF. This work expands our understanding of A domain specificity and will aid in engineering of NRPSs to produce new natural products.Support or Funding InformationThis work is supported by the National Science Foundation grant 1716594.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.