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bioRxiv · 10.64898/2026.06.03.729908

Split-Indigoidine synthetase as optical reporter for benchmarking protein-protein interactions

Gonschorek, P.·Schelhas, C.·Flakowski, M.·Schenk, L.·Podolski, A.·Bode, H. B.
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Abstract

Indigoidine is a blue pigment biosynthesized by a single-module Non-Ribosomal Peptide Synthetase (NRPS) using L-glutamine as substrate. Despite its potential as a colorimetric reporter, no such system has been established from it to date. We used a recently characterized interdomain fusion site located between its adenylation (A) and thiolation (T) domains to develop the Indi2GO system, which provides a naked-eye detectable and quantitative optical readout of transient and covalent protein-protein-interaction (PPI) in living cells. Indi2GO enables high-throughput benchmarking and optimization of PPI tools in a standard 96-well plate reader format, without requiring exogenous substrates, specialized equipment or complex analytical workflows. We demonstrate its broad applicability with three widely used protein-protein interaction tools: SYNZIPS, inteins, and the SpyTag:SpyCatcher system. We used Indi2GO to validate novel SYNZIP pairs, which we used in NRPS engineering, highlighting its applicability for the development of novel PPI-mediating tools in the context of NRPS engineering and synthetic biology.

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Gonschorek, P., Schelhas, C., Flakowski, M., Schenk, L., Podolski, A., Bode, H. B.. 2026-06-04. Split-Indigoidine synthetase as optical reporter for benchmarking protein-protein interactions. https://doi.org/10.64898/2026.06.03.729908

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