Introduction

Sharing resources and knowledge is a central principle of the iGEM competition. Contributions ensure that future teams can build upon past work instead of starting from scratch. In our project, we set out to tackle the complex problem of AMR.

Resistant pathogens are rapidly emerging, while the development pipeline for new antibiotics has nearly run dry, making the search for new antibiotics more urgent than ever. We aimed at advancing synthetic biology solutions against antimicrobial resistance (AMR), by providing tools and resources making nonribosomal Peptide Synthetase (NRPS) engineering more accessible.

Natural products remain a promising resource of bioactive compounds, and among them, nonribosomal peptide synthetases (NRPS) stand out in particular. NRPS are modular enzymes that produce unique peptides, some of which show diverse bioactivity. Most notably, valuable peptide antibiotics like cephalosporins, are assembled by NRPS, making them a promising source for the future discovery of novel antibiotics^[1].

The modular architecture of NRPS allows individual modules to be swapped, enabling the generation of new enzyme complexes that synthesize peptides with hybrid sequences^[2]. However, NRPS engineering has faced major obstacles like the difficulty in predicting the compatibility of NRPS units and therefore functionality. With our project, we aim to redefine the very concept of NRPS-based drug discovery: The NRPieceS Platform covers all steps, from heterologous biosynthetic gene cluster expression in E. coli, through NRPS engineering and peptide production, to peptide functionalization and bioactivity testing.

At the heart of our platform is the NRPieceS Plasmid Collection, comprising 160 modular plasmids that simplify NRPS engineering and library construction for future iGEM teams. Using our 105 tripartite NRPS expression plasmids, we generated thousands of novel cyclic peptides, opening up diverse opportunities for bioactivity screening and funcionalizing them by using our drug delivery appraoch.

To ensure our platform is fully accessible to other teams, we provided robust and reproducible protocols for NRPS cloning, expression, peptide purification, and bioactivity testing. In addition, we developed a computational tool that streamlines NRPS analysis and design, effectively bridging wet-lab and in silico approaches for future users.

The NRPieceS Parts Collection

We created the NRPieceS Parts Collection, a set of 160 plasmids designed to make NRPS engineering accessible to future iGEM teams.

To harness the potential of NRPS engineering for peptide derivatization for the community, we assembled a library of interchangeable, standardized parts that can be combined through Golden Gate cloning and further diversified using intein-mediated assembly (Fig. 1)^[3][4].

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The library is organized into two complementary vector sets: acceptors and donors. Acceptors are native NRPS clusters that have been split into three functional parts (initiator, elongator, and terminator) using inteins. Each acceptor has a defined site for the insertion of a donor which are single exchange units that can be introduced into these sites to diversify amino acid incorporation.

By providing a set of modular building blocks, we enable straightforward recombination of NRPS units into new hybrids. Thanks to this standardization, the collection can be easily extended by future teams, creating a growing resource for engineering new-to-nature peptides.

Explore the parts collection here, and find a detailed guide for using it here.

An Antibiotic Discovery Platform for the iGEM Community

Our project went beyond simply creating a collection of new plasmids for biotechnological peptide production. What initially drew us to NRPS was their unique bioactivity and we sought to explore this potential against ESKAPE pathogens by creating the necessary tools and sharing them^[5]. Addressing this challenge required considering the entire drug discovery process, including production, detection, characterization of compounds, their enhancement, functionalization and lastly bioactivity testing.

Therefore, we established the NRPieceS Platform, an easy and accessible workflow for antibiotic discovery (Fig. 2). It covers all steps, from heterologous biosynthetic gene cluster expression in E. coli, through NRPS engineering and peptide production, to peptide functionalization and bioactivity testing.

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This framework empowers future teams to generate new-to-nature peptides with novel bioactivities for drug discovery.

Sharing Protocols to Empower Future NRPS Engineering

NRPS engineering holds great promise due to its ability to generate diverse natural products with potential applications in medicine and biotechnology. However, NRPS enzymes are large, multi-domain complexes, making them challenging to manipulate and study. The production of natural products by these enzymes further adds complexity, as analyzing the resulting compounds requires specialized expertise in techniques such as LC/MS and peptide characterization.

To lower the barrier to NRPS engineering, we developed and openly shared detailed, robust protocols that guide teams through cloning, expression, and analysis, enabling broader participation in this field. Alongside these protocols, we provide an extensive reference dataset and clear instructions to ensure reproducible use of our platform, including guidance on data analysis and essential controls.

The protocol is openly shared on our protocol page and can be directly adapted by other labs. By lowering the technical threshold for NRPS expression, this contribution provides a practical foundation for teams interested in engineering natural product pathways.

Guiding hybrid NRPS design with the mATChmaker Software

A central challenge in NRPS engineering is that, although modules can be recombined, they are not inherently compatible and may fail to produce a functional enzyme. To address this, our dry lab developed mATChmaker, a software tool that improves the likelihood of functional hybrid NRPS by providing a guide for module comptability. mATChmaker is openly available for the iGEM community to support their own projects.

The mATChmaker software provides the iGEM community with two complementary pipelines: The T-TE pipeline extracts amino acid sequences from a GenBank file and compares them to a chosen reference. This comparison makes it possible to estimate the compatibility of NRPS clusters when recombining their units. The TCT pipeline focuses on analyzing the chemical interactions within the crucial condensation complex of recombined NRPS units.

We have integrated both pipelines into a user-friendly Docker environment, which is openly available on GitLab for the iGEM community and beyond. Within this environment, we have also included the functionality of PARAS, a prediction tool for A-domain specificity, as well as getcontacts, a tool for analyzing chemical interactions between molecules of interest. Both PARAS and getcontacts can either be used independently or as part of the pipelines, providing flexibility and accessibility for NRPS design and analysis^[6][7].

                        Choose what to run:
                            1) PARAS
                            2) TTE Extraction and Similarity Identification
                            3) TCT Extraction and Chemical Interaction Prediction
                            4) Run Get Contacts for predicted structures
                        Enter 1-4: []