One of the challenges that nonribosomal peptide synthetase (NRPS) engineering faces is high throughput screening. This year we are leveraging the chromophore properties of IndC, a NRPS module, and improving its robustness to easily tag any NRPS product. Shown on the right is a depiction of IndC, which can be incorporated in a NRPS system to tag peptide products (image from Heidelberg iGEM 2013).
Figure 1. The mechanism of indigoidine synthesis from L-glutamine. Glutamine is activated by the adenylation domain of IndC, while the thioesterase (TE) domain of IndC catalyzes intramolecular cyclization of the glutamine thioester. The cyclized compound is then oxidized by the oxidation domain of IndC, forming an intramolecular double bond. The oxidized compound then dimerizes via condensation to form the complete indigoidine blue pigment molecule.
The product of the IndC module is the molecule indigoidine, a dimer of cyclized L-glutamine that is oxidized to form a conjugated system, creating a blue pigment. If incorporated into a complex NRPS system, we hope to successfully tag peptide products by attaching indigoidine using the IndC module. However, previous work on the IndC module have revealed restrictions with its tagging abilities. Heidelberg's 2013 iGEM team observed steric hinderance affecting indigoidine incorporation when the precursor polypeptide possessed a bulky structure.
Figure 2. a) The IndC module, an NRPS module expressed as a gene product of bpsA, isolated from Streptomyces lavendulae. L-glutamine is taken as the substrate and converted into indigoidine. b) Indigoidine functions as a blue pigment when expressed in bacteria cultures.
The IndC module itself contains a unique tailoring domain, the oxidation domain, inserted within a hypervariable region of the adenylation domain. This oxidation domain is responsible for oxidizing the L-glutamine substrate taken in by IndC, a crucial step in the biosynthesis of indigoidine. We plan on expressing the isolated oxidation domain from IndC to test its oxidation capability on cyclized L-glutamine as well as complex NRPS organic molecules containing cyclized glutamine-like residues, most notably padanamide B.
1. Reverchon, S., C. Rouanet, D. Expert, and W. Nasser. 2002. Characterization of indigoidine biosynthetic genes in Erwinia chrysanthemmi and role of this blue pigment in pathogenicity. Journal of Bacteriology 184(3):654-665.
2. Team Heidelberg. Modular organisation of NRPS. 2013. Non-Ribosomal Peptide Synthesis - Get to know the theory. iGEM. Web. 17 Jun. 2016.