Non-ribosomal peptide synthetases

Non-ribosomal peptide synthetases

Non-ribosomal peptides (NRPs) are small, secondary metabolites not produced by the ribosome but by specialized enzymes called non-ribosomal peptide synthetases (NRPSs). They are found mostly in bacteria and fungi and often serve as bioactive reagents against microbes, fungi, mycoplasmas and bacteria. This makes NRPs greatly interesting for biomedical and biotechnological applications. The chemical diversity of NRPs is due to the presence of non-proteinogenic as well as D- amino acids, α-hydroxy-, and carboxylic acids among others (for simplicity all building blocks will be refered as AA in the following). NRPSs are intrinsically modular enzymes: each NRPS is comprised of several modules, each in turn comprised of several domains that perform various catalytic reactions. Each module specifically incorporates one AA, therefore – at least for linear NRPSs – the sequence and number of modules determines the sequence and length of the peptide chain. Modules are generally composed of at least three domains: the adenylation (A) domain, which determines and activates the right AA; the thiolation (T, also called peptidyl carrier protein, PCP) domain, which carries the intermediate NRP and presents it to the catalytic centers; and the condensation (C) domain, which catalyzes the elongation of the upstream intermediate NRP by the downstream activated AA through formation of a peptide bond. Additional domains such as epimerization or N-methylation can be present as well, and they facilitate further modifications of the peptide product. Finally, a terminal thioesterase (TE) domain releases the final product from the NRPS. Typically, an NRP is produced by several interacting NRPSs, which communicate by so-called communication (COM) domains.

Despite the fact that a great variety of bioactive NRPs are endogenously produced by various organisms, an effective procedure to extract and use them, for instance as therapeutics, is still missing. On the one hand, the small, endogenous NRPs are hard to purify. On the other hand, the target of endogenous NRPs cannot be changed. However, the increasing threat to health caused by multiple resistant bacteria or by cancer calls for novel therapeutics targeting different molecular mechanisms. Being able to engineer NRPSs to specify which AAs are incorporated, in which sequence and with which modifications, would mean being able to produce custom peptides that could be later on tested for specific biological activities.

A very promising approach for the full exploitation of their therapeutic potential is the use of bacterial cells to produce engineered NRPs. Previous work has shown that it is possible to modify NRPSs in various ways, for instance by making A domains accept non-cognate AAs, by removing or adding modules at specific locations, or by re-wiring the communication between COM domains. Yet, these attempts so far were tailor-made to specific cases and general rules, which can be applied all the times to create peptides of any desired composition and length, are missing.

We are currently trying to understand the design principles that will allow us one day to create peptides à la carte using engineered NRPSs.

As model system we are using the unimodular NRPS that produces a blue pigment called indigoidine.

Indigoidine can be produced by E. coli cells. Liquid cultures of bacterial cells non expressing (left) or expressing (right) the indigoidine synthetase. The presence of the NRP indigoidine is clearly visible by the naked eye.
A more artistic view of indigoidine. Pictures of blue ink dissolving in water. Photos taken by Aidan and Toby Gibson.


Context-dependent activity of A domains in the tyrocidine synthetase
A. Degen, F. Mayerthaler, H. D. Mootz and B. Di Ventura
Scientific Reports 2019

Creating functional engineered variants of the single-module non-ribosomal peptide synthetase IndC by T domain exchange
R. Beer, K. Herbst, N. Ignatiadis, I. Kats, L. Adlung, H. Meyer, D. Niopek, T. Christiansen, F. Georgi, N. Kurzawa, J. Meichsner, S. Rabe, A. Riedel, J. Sachs, J. Schessner, F. Schmidt, P. Walch, K. Niopek, T. Heinemann, R. Eils, B. Di Ventura
Mol Biosyst, 2014