NRPS systems contain a variety of tailoring domains that modify the amino acid substrate before it is incorporated into the peptide chain. QGEM is exploiting the power of CRISPR-Cas9 to insert these tailoring domains into NRPS genetic clusters. Specifically, these tailoring domains will be inserted into hypervariable regions within the adenylation domains that nature has already used to insert cis tailoring domains.
Figure 4. Some examples of enzymatic tailoring domains in NRPS with their functions. A) The cyclization domain (Cy) in the VibF module from Vibrio cholerae catalyzes the cyclization of threonine into an oxazoline ring. B) The oxidation domain (Ox) in the EpoB module oxidizes the thiazoline ring to thiazole (adding a doube bond) in the presence of cofactor FMN. C) The reduction domain (R) of PchG (a trans domain) reduces the thiazoline ring to thiazolidine on PchF in the presence of NADPH.
Some examples of tailoring domains that can be inserted within the adenylation domain of NRPS modules include oxidation domains (as shown above in B), which can help form double bonds in the molecule to potentially create a conjugated system (for stability). Other tailoring domains reduce, halogenate, epimerize, methylate, or cyclize peptides to create finished compounds with varying properties. Our goal is to manipulate the hypervariable regions of the adenylation domains in NRPS systems at the genetic level to incorporate new, "auxiliary" domains that could potentially increase the variety and decrease toxicity of synthetic compounds produced by NRPS. As a result, insertion of tailoring domains will be a pipeline towards establishing and expanding NRPS drug libraries while optimizing their therapeutic function.
1. Hur, Gene H., C. R. Vickery, and M. D. Burkart. Enzymatic reactions of the tailoring domains. 2012. Explorations of catalytic domains in non-ribosomal peptide synthetase enzymology. Royal Society of Chemistry. Web. 17 Jun. 2016.