Abstract

Concerned about the escalating threat of antimicrobial resistance (AMR), we investigated strategies to expand the antimicrobial pipeline. Conventional antibiotic discovery has slowed markedly, while resistant pathogens continue to spread globally, creating an urgent public‑health risk. We therefore pursued innovative approaches to accelerate the identification of novel antimicrobial compounds and alternatives to classical antibiotics.

We developed NRPieceS, a versatile synthetic biology platform that enables the generation of large peptide libraries with minimal cloning effort. Our approach outperforms existing methods in terms of efficiency and sustainability, allowing faster and easier production of potential bioactive peptides with antimicrobial properties.

At the core of our system lies a combinatorial biosynthesis library for nonribosomal peptides (NRPs), enabling biocatalytic peptide production through engineered nonribosomal peptide synthetases (NRPSs). Our approach integrates state-of-the-art methods in NRPS engineering, combining multiple established techniques to create a powerful and flexible platform for peptide design.

Our NRPieceS platform includes every step of an integrated Design-Build-Test-Learn engineering workflow, from heterologous expression, derivatization to activity testing and computational guidance via our software tool, mATChmaker. The NRPieceS plasmid collection comprises 160 plasmids, including our NRPieceS toolbox of 35 donor plasmids, providing an extensive resource for modular NRPS assembly and engineering.

mATChmaker, our software, provides insights to NRPS protein structures by generating and visualizing condensation complexes of native and engineered NRPS assembly lines. In addition, it provides guidance for choosing compatible NRPS Modules for the generation of hybrid NRPS assembly lines assessing phylogenetic relationships. Together, these features enable data-driven design, accelerating rational NRPS engineering.

Using this platform, we screened peptide libraries against ESKAPE pathogens, identifying several promising hit compounds with antimicrobial activity. To enhance therapeutic applicability, we further explored a Trojan horse drug delivery strategy, employing click chemistry to conjugate siderophores (1,2-dihydroxybenzene) to our peptides to improve bacterial uptake.

Our platform combines wet-lab innovation, computational tools, and novel delivery strategies into a unified workflow, paving the way for the discovery of next-generation antimicrobial agents. Our vision extends beyond developing a platform for antibiotics discovery, we strive to make our tools accessible to the iGEM community, empowering future teams to explore NRPS engineering projects. By lowering the barrier to combinatorial biosynthesis, NRPieceS opens the door to diverse applications, from antimicrobial discovery to sustainable biotechnological solutions.

The Challenge - Antimicrobial Resistance Crisis - Empty Pipeline

The golden age of natural product antibiotic discovery began with the discovery of penicillin in 1928 and spanned from the 1940s to the 1960s. During this time, most of the major antibiotic classes we still rely on today were identified. Penicillin and the antibiotics that followed fundamentally transformed modern medicine, contributing to an increase of the average human lifespan by more than two decades.

In the 1930s, Selman Waksman pioneered the study of antimicrobial compounds, defining them as natural products produced by microbes to defend themselves against other microbes. His groundbreaking work led to the discovery of several antibiotics from soil bacteria, many of which became cornerstones of clinical treatment^[1].

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After the 1960s, however, antibiotic discovery and development sharply declined. The widespread belief that all easily accessible antibiotics had already been found caused many pharmaceutical companies to shut down their natural product discovery programs. At the same time, the overuse and misuse of antibiotics in medicine and agriculture accelerated the emergence of resistant bacteria. This has culminated in today’s antimicrobial resistance (AMR) crisis^[1]. The projected global burden of AMR is severe: in 2050, an estimated 1.91 million deaths annually are directly attributable to AMR, with 8.22 million associated deaths worldwide^[2].

AMR is rapidly emerging as one of the greatest global health challenges of our time. Without effective action, infections once easily treated could again become deadly, with resistant pathogens spreading across healthcare and community settings. By 2050, AMR is projected to cause up to 10 million deaths per year, surpassing cancer as the leading cause of mortality worldwide.

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Modern antibiotic discovery is hampered by high research and development costs, lengthy timelines, and stringent regulatory hurdles for market approval. Even once approved, new antibiotics are typically reserved as last-resort treatments to delay resistance development, which reduces commercial incentives and further disincentivizes investment in the field^[3].

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The development of new antibiotics has become increasingly unattractive for pharmaceutical companies. Research and clinical testing are extremely expensive, and the regulatory approval process is lengthy and resource-intensive. Moreover, newly approved antibiotics are usually reserved as last-resort treatments to prevent rapid resistance development, which means their use and therefore their sales remain limited. Unlike chronic medications that generate steady, long-term revenue, antibiotics are typically prescribed only for short treatment periods. As a result, companies often reach profitability only after around 23 years, while patent protection expires after 25 years, leaving merely a short window for return on investment before generic competition begins. This economic imbalance has led to a declining interest in antibiotic research, even as antimicrobial resistance continues to rise globally^[4].

Project Inspiration

Concerned about the escalating threat of antimicrobial resistance (AMR), we began to explore the urgent need for new antibiotics and alternative strategies to combat resistant pathogens. Our research revealed that traditional antibiotic discovery has slowed dramatically, while drug-resistant bacteria are rapidly spreading, posing a serious global health risk. Driven by a desire to address this pressing biomedical challenge, we sought innovative solutions to accelerate the discovery of novel antimicrobial compounds.

This journey led us to nonribosomal peptide synthetases (NRPSs), natural enzymatic systems capable of producing structurally diverse and biologically active peptides. We recognized the potential of combinatorial NRPS engineering to generate new bioactive peptides with antimicrobial properties and to expand the chemical diversity accessible for drug discovery. As a result, we set out to develop a platform combining a plasmid library, a part collection, predictive software tools, and delivery strategies, aiming to make NRPS-based peptide discovery more accessible, efficient, and impactful in the fight against AMR.

The Solution – Learning from Nature

Role of Natural Products in Drug Discovery

Nature has been a central source of therapeutics, particularly antibiotics. A prominent example is penicillin, produced by a nonribosomal peptide synthetase (NRPS)^[5]. NRPSs are large, multi-modular enzymes that catalyze the biosynthesis of nonribosomal peptides (NRPs) - a structurally diverse class of natural products with applications in medicine and agriculture, including antibiotics, antifungals, antitumor agents, and immunosuppressants^[6]. More than 20 nonribosomal peptides are already marketed as approved drugs, highlighting their clinical relevance.

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Peptides represent a unique class of therapeutics derived from natural products. They combine characteristics of both small molecules and biologics, such as high specificity for difficult targets like protein–protein interactions (PPIs), tunable half-lives, reduced toxicity, and potent biological activity^[7][8].

NRPS as Native Antibiotic Producers

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NRPS-derived metabolites have been optimized by evolution in their native microbial hosts to fulfill specialized ecological functions, such as defense against competing organisms. These natural scaffolds therefore serve as valuable reference points for drug discovery and development^[9]. Unlike ribosomal synthesis, which is limited to 20 canonical amino acids, NRPSs can incorporate over 400 different building blocks into their products^[10]. This capacity results in an immense chemical and structural diversity, making NRPSs one of the richest sources of bioactive compounds.

Potential and Challenges of NRPS Engineering- MATChmaker

Nonribosomal peptide synthetases (NRPSs) are encoded in biosynthetic gene clusters (BGCs) and act as modular enzymatic assembly lines. Each module contains distinct catalytic domains that work in a coordinated, stepwise fashion, enabling the synthesis of structurally diverse nonribosomal peptides (NRPs). Typically, an elongation module comprises three domains essential to peptide elongation. Each module of the NRPS is responsible for a specific amino acid in the final NRP. The modular architecture of NRPSs offers exceptional potential for bioengineering, as each module corresponds to a defined step in peptide assembly. This modularity can be exploited to recombine modules, enabling the design of new-to-nature peptides with therapeutic potential (NRPS Engineering).

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However, despite their promise, NRPS engineering remains challenging. Efficient recombination of modules and the reliable production of designed peptides often face low yields, loss of enzyme activity, module incompatibility or impaired assembly-line functionality. Overcoming these challenges is key to unlocking the full potential of NRPS systems for next-generation drug discovery.

To tackle module incompatibility and assembly-line limitations, we developed Matchmaker, a software tool designed to guide NRPS analysis and engineering. Matchmaker integrates two complementary pipelines: One to analyze the chemical interactions at the crucial condensation site of an NRPS, and another to assess the phylogenetic relation between clusters. Together, they enable predictions of compatibility when recombining units from different NRPS clusters (Software).

This approach provides a computational framework to support experimental NRPS engineering, reducing trial-and-error and accelerating the discovery of novel antimicrobial compounds.

Integrated Human Practices

To achieve our goal of combating antimicrobial resistance (AMR), we knew from the very beginning that our work could not remain confined to the laboratory. The challenge of developing new antibiotics goes far beyond scientific discovery — it is deeply connected to economic realities, translational barriers, and global health priorities.

That’s why we built our project around continuous engagement with key stakeholders from science, policy, and industry. Through interviews and discussions, we sought to understand three central questions guiding our Human Practices work:

• Why did the antibiotic pipeline run dry?

• How can we refill it?

• And what else must be done to effectively combat AMR?

We learned that refilling the pipeline, while essential, is only part of the solution. The decline in antibiotic innovation stems not only from scientific difficulties but also from economic disincentives and structural barriers between academic discovery and industrial translation. Insights from Prof. Dr. Christoph Gradmann helped us understand these systemic roots, while experts from academia and industry guided us in translating discoveries into applicable therapies.

During this process, we identified outer membrane penetration as one of the key scientific bottlenecks in antibiotic development — especially in gram-negative bacteria. To better understand this challenge, we conducted further expert interviews, which highlighted the complexity of crossing these biological barriers. Based on these insights, we decided to establish drug delivery as an additional pillar of our project, aiming to explore strategies that improve compound uptake and intracellular access.

We were also determined not to let our project end in the lab. To ensure that our platform has real-world impact, we analyzed its industrial relevance and explored how it compares to established methods such as solid-phase peptide synthesis (SPPS). This helped us assess how our approach can expand chemical diversity, improve efficiency, and make antibiotic discovery more attractive for future applications in both academia and industry. These reflections directly inspired our Future Implementation, where we outlined concrete pathways for translating our work from concept to application.

By integrating these insights, we shaped a project that goes beyond the discovery void, addressing both scientific and translational challenges to build a foundation for sustainable and impactful antibiotic innovation.

Outlook & Vision

Our journey with NRPieceS began with a simple goal: to create a modular and accessible platform for the biosynthetic production of antimicrobial peptides. Along the way, we realized that nonribosomal peptide synthetases (NRPSs) represent one of the most powerful and versatile enzyme systems in nature, capable of assembling countless bioactive molecules with therapeutic and biotechnological potential. Nevertheless, NRPSs remain difficult to engineer, limiting their application. With NRPieceS, we set out to change that, making NRPS engineering accessible, more efficient, and drug discovery sustainable as stated in SDG3.

Looking ahead, we envision several key directions to further advance our work. The next logical step would be to expand the NRPieceS plasmid library. This can be achieved by increasing the NRPieceS donor toolbox including modules from the 400 building blocks incorporated by NRPS. Furthermore the technology of NRPieceS can be applied to any other NRP derived compound, accelerating biosynthetic drug discovery. Integrating mATChmaker, our prediction tool, could refine the design process even further, providing data-driven guidance for future peptide discovery. Moreover, developing a biosynthetic siderophore-peptide production system directly within microbial hosts could make large-scale biological manufacturing possible, minimizing the need for chemical synthesis.

Beyond the technical aspects, we also considered the broader context of antibiotic development. Developing new antibiotics is a slow and costly process, research and approval can take decades, while market incentives are low because antibiotics are short-term, last-resort medications. By providing an open-source, modular system for drug discovery, NRPieceS aims to democratize antibiotic innovation and empower academic research groups and small biotech initiatives to contribute to this critical global challenge. Our approach integrates several state-of-the-art methods in NRPS engineering, combining multiple established techniques to create a powerful and flexible platform for peptide design.

Our vision reaches beyond antimicrobial discovery. NRPieceS has the potential to become a universal platform for combinatorial peptide biosynthesis, enabling sustainable exploration of bioactive compounds for applications in medicine, agriculture, and environmental science. We imagine a future where iGEM teams, researchers, and students worldwide can use NRPieceS to design and produce their own custom peptides, not only to fight infections, but also to create dyes, biosurfactants and other drugs.

While our work laid the foundation, we see NRPieceS as an evolving system that grows through collaboration. By sharing our plasmid library, conjugation toolkit, and software openly with the iGEM community, we hope to spark a network of teams building upon each other’s work. Together, we can accelerate drug discovery, rethink how antibiotics are developed, and contribute to a more sustainable, accessible, and innovative future for synthetic biology.