Prof. Dr. Tanja Schneider

Leading Expert in the Discovery of Natural Products, Deputy Coordinator Novel Antibiotics in the German Center for Infection Research

Reason for contact

At the beginning of our project, we reached out to Prof. Schneider to seek guidance on selecting microbial clusters for our study. Nature harbors an immense treasure trove of bioactive compounds, much of which remains hidden in what is often called ‘microbial dark matter’—the countless microorganisms that cannot be grown under standard laboratory conditions. Prof. Schneider has successfully uncovered parts of this hidden world through her work on teixobactin, an antibiotic discovered from previously unculturable soil bacteria. Before our interview with her, we had already explored various strategies to access microbial dark matter, including advanced cultivation techniques, single-cell sequencing, metagenomics, and computational predictions of biosynthetic gene clusters (BGCs).

Key takeaways

Unlocking the Microbial Dark Matter: From Cultivation to Genome-Driven Discovery

When speaking with Prof. Schneider, we covered a broad range of strategies to access the ‘microbial dark matter’, but we focused the conversation on two methods: advanced cultivation and silent BGCs. She explained how the iChip allows bacteria to grow in their natural soil environment, providing nutrients and growth factors that cannot be replicated in the lab. This method has enabled the cultivation of previously inaccessible species and the discovery of novel antibiotics, including teixobactin.

We also discussed the challenges of cultivation. Despite its successes, growing microbes remains unpredictable and labor-intensive. While we could not implement iChip cultivation in our iGEM season, the discussion inspired us to center our project around soil as a rich and untapped microbial source.

Silent BGCs presented another exciting avenue. Many microbial genomes contain more biosynthetic pathways than the compounds they actually produce. Unlocking these hidden pathways could reveal entirely new classes of antibiotics. Yet, their activation is complex, often requiring environmental cues or precise regulatory signals. Traditional approaches like OSMAC are slow and empirical, limiting throughput.

Prof. Schneider emphasized that synthetic biology can overcome many of these limitations. By predicting BGCs computationally, extracting the DNA, and expressing the clusters in E. coli, we can explore microbial chemical diversity more systematically. This genome-driven approach allows us to rationalize traditional methods like OSMAC while increasing speed and efficiency.

Inclusion in our project

After our interview, we were amazed by the incredible diversity of this terrestrial treasure trove, yet we weren’t sure which specific organisms to focus on. That is why we looked for researchers in the field who could guide our cluster selection. For this purpose, she recommended reaching out to Prof. Kim Lewis who was also involved in the discovery of teixobactin.

Prof. Dr. Kim Lewis

Leading Scientist in Antimicrobial Discovery, Northeastern University

Reason for contact

We contacted Professor Kim Lewis as the director of the Center for Antimicrobial Discovery at Northeastern University because we recognized him as one of the leading experts in antibiotic discovery. His extensive body of work and numerous publications had sparked our interest and significantly contributed to shaping our understanding of the field. Through the interview, we aimed to explore the history of antibiotic discovery and to gain insights from past scientific challenges and mistakes. During our discussion, Professor Lewis provided a comprehensive overview of various discovery approaches, ranging from bioactivity-guided methods used in the Waksman platform to modern genome-guided strategies that are driving the current renaissance in natural product discovery.

The Waksman Platform: Bioactivity-guided Screening as a Revolutionary Idea in the 1940s

In the early 1940s, Selman Waksman developed a systematic method to search for new antibiotics. Unlike the discovery of penicillin, which happened by chance when a mold inhibited bacterial growth, Waksman’s approach was planned and reproducible. His team systematically screened soil-derived actinomycetes for antimicrobial activity. Extracts were tested on Petri dishes overlaid with target pathogens, and those producing zones of growth inhibition were selected for further study. Once activity was observed, the compounds responsible were isolated and chemically characterized, laying the foundation for a structured antibiotic discovery workflow.

Fig. 1: Workflow of the Waksman platform: Soil samples are collected as a source of diverse microorganisms, primarily actinomycetes. Individual strains are isolated, cultivated, and processed to generate crude extracts. These extracts undergo bioactivity screening to identify strains producing metabolites with potential therapeutic relevance.

The Golden Era of Antibiotics

The success of this method was unprecedented and introduced the beginning of the Golden Era of Antibiotics. As Professor Kim Lewis described, there was a time when “people felt they had conquered the war against pathogens.”
Using this platform, Waksman’s group discovered streptomycin, the first effective treatment for tuberculosis, a breakthrough that earned him the Nobel Prize in 1952.

Encouraged by this success, the pharmaceutical industry adopted the Waksman platform on a large scale. Within two decades, it led to the discovery of nearly all major antibiotic classes still in use today, including aminoglycosides, tetracyclines, β-lactams, chloramphenicol, and macrolides. This period became known as the “golden age” of antibiotic discovery.

The Decline of the Platform

By the early 1960s, the Waksman platform began to lose its effectiveness. Despite intensive screening efforts, researchers were rediscovering known compounds rather than finding new ones. Many extracts also contained toxic “junk” molecules, making them unsuitable for therapeutic use. This diminishing return marked the end of the Waksman era.

Overmining: The Limits of a Once-Rich Resource

One major reason for this decline was overmining. The platform relied almost exclusively on Streptomyces and related actinomycetes, which proved highly productive but limited in diversity. As Prof. Lewis explained, these bacteria “exchange DNA among each other,” which means many species produce the same compounds, such as streptomycin. Over time, researchers mined all readily accessible, broad-spectrum antibiotics from this group, thereby effectively exhausting the resource.

The Rise of Synthetic Chemistry (1950s–1980s): From Whole-Cell Screening to the Fluoroquinolones

As the productivity of the Waksman platform declined in the late 1950s, attention began to shift from natural products to synthetic and semi-synthetic chemistry. Although the natural product well was beginning to run dry, synthetic efforts, which were guided largely by whole-cell phenotypic screening, delivered several notable successes. During this period, researchers identified a small but significant arsenal of synthetic antimicrobials effective against the most pressing infectious diseases of the time.

For tuberculosis, whole-cell screens yielded isoniazid, pyrazinamide, and ethambutol, compounds that remain cornerstones of therapy to this day. Around the same time, metronidazole, a synthetic nitroimidazole, was discovered and found to possess potent bactericidal activity against anaerobic and microaerophilic pathogens. These discoveries demonstrated that fully synthetic compounds could, under the right conditions, achieve clinically meaningful antimicrobial activity.

A decisive breakthrough occurred in the 1960s with the serendipitous discovery of nalidixic acid, a by-product of chloroquine synthesis that unexpectedly killed Escherichia coli. Although nalidixic acid itself was weak, its fluorinated derivatives - the fluoroquinolones - revolutionized antibacterial chemotherapy by targeting DNA gyrase and topoisomerase IV. These compounds combined broad-spectrum activity with favorable pharmacokinetics and rapidly became one of the most successful antibiotic classes, second only to β-lactams in clinical impact.

At the same time, synthetic and semi-synthetic chemistry proved invaluable in expanding the spectra and improving the properties of existing antibiotics. Structural modification of natural scaffolds transformed narrow-spectrum drugs into broad-spectrum agents; for example, penicillin into ampicillin, and erythromycin into azithromycin. Medicinal chemistry efforts also produced analogs that overcame emerging resistance mechanisms, giving the impression that synthetic optimization could indefinitely sustain the antibiotic arsenal.

By the 1970s, the momentum had clearly shifted. Natural product discovery appeared increasingly exhausted, while synthetic chemistry seemed ascendant. The prevailing belief was that with sufficient knowledge of chemical structure and biological targets, rational design could replace the empirical natural product screens of the past. This optimism set the stage for the next major transition: the Genomics Era, where high-throughput synthetic chemistry and molecular biology were expected to deliver a new generation of antibiotics. As later experience would show, however, this promise proved far more elusive than anticipated.

The Genomics Era (1990s–20000s): The “Failure of Genomics”

The 1990s marked the beginning of what many believed would be a new golden age of antibiotic discovery - the Genomics Era. Advances in genomics, proteomics, and combinatorial chemistry promised a revolution. Industry assembled vast synthetic compound libraries, often containing millions of molecules, and combined them with rational, target-based screening approaches. Genomic data identified numerous “essential” bacterial genes, and high-throughput robotic systems enabled screening against both purified proteins and whole cells. The logic was compelling: by integrating structural biology, computational design, and rational optimization, researchers expected to create an efficient, high-tech discovery platform capable of outpacing resistance and replacing natural product discovery altogether (Lewis, 2013).

Initially, optimism ran high. This synthetic–genomic platform had proven successful in other therapeutic areas, such as oncology and cardiovascular disease, where target-based discovery reliably translated into clinical success. For antibiotics, however, this strategy failed almost completely. Despite enormous screening campaigns conducted by major pharmaceutical companies - notably GlaxoSmithKline and AstraZeneca - no new antibiotic classes emerged (Payne et al., 2007; Tommasi et al., 2015). The failure was not due to poor experimental design but to a fundamental biological barrier: the bacterial cell envelope.

Gram-negative bacteria, in particular, possess an exceptionally impermeable outer membrane composed of lipopolysaccharides (LPS), which form a dense, negatively charged, hydrophilic matrix stabilized by divalent cations. This structure effectively blocks large or hydrophobic compounds. Beneath it, the inner membrane - a hydrophobic phospholipid bilayer - restricts hydrophilic compounds, creating a dual barrier that excludes nearly all synthetic molecules. Even those that manage to cross are rapidly removed by trans-envelope multidrug resistance (MDR) efflux pumps, which recognize hydrophobic substrates and expel them before they reach their targets (Li et al., 2015; Lomovskaya & Lewis, 1992). Nutrient uptake through porins and transporters further constrains chemical entry, leaving little opportunity for drug accumulation.

Thus, despite immense technical sophistication, the genomics-based, target-driven paradigm proved incompatible with the complex permeability and resistance biology of bacteria. This widespread disappointment, often summarized as the “failure of genomics”, underscored a critical lesson: antibacterial discovery cannot be reduced to target inhibition. Unlike other therapeutic areas, antibiotic activity depends on bioavailability within the bacterial cell, evasion of efflux and degradation, and maintenance of intracellular potency.

Tab. 2: Overview of Antibiotic Discovery Attempts

Era / Platform	Source of Substances	Screening Strategy	Result
1940s–1960s (Waksman Era, “Golden Age”)	Natural products (NPs) from actinomycetes, fungi, soil samples	Cell-based screening: zone formation, growth inhibited/killed	Discovery of almost all classic antibiotic classes (β-lactams, aminoglycosides, tetracyclines, macrolides, etc.)
1960s–1980s	Semi-synthetic derivatives of known natural products	Still cell-based, but mainly clinically driven optimization	Improved pharmacokinetics, spectrum, resistance circumvention (e.g., penicillin → cephalosporins)
1980s–1990s	First fully synthetic molecules (e.g., quinolones, oxazolidinones)	Cell-based, but more rational approaches	Few, but important new classes
1990s–2000s (Genomics Era)	Large synthetic libraries, often not NP-based	Target-based screening (isolated enzymes, ribosomal proteins)	Many hits in vitro, hardly any clinical success → “Failure of genomics”

Key takeaways

A plea for platforms

Since the decline of the Waksman platform, antibiotic discovery has continued without a comparable systematic framework. Modern research often depends on ad hoc findings, synthetic chemistry, or genome-based approaches, but none have yet matched the reliability and productivity of Waksman’s original workflow. The story of the Waksman platform remains a powerful reminder of how a single structured method can revolutionize an entire field and of the challenges that arise once its limits are reached.

To effectively combat antimicrobial resistance (AMR), it is essential to maintain at least one reliable discovery platform capable of consistently generating new antibiotic leads. Since the decline of the Waksman-era natural product discovery pipeline, there has been no successful broad-spectrum antibiotic platform developed in over fifty years. This historical gap highlights the urgent need for a sustainable mechanism to continuously produce novel compounds. As discussed in the transcript, “we need at least one platform,” since without it, the discovery of even two or three promising candidates for combination therapies becomes nearly impossible. Combination approaches - such as those used successfully in the treatment of HIV, where three drugs are administered together to prevent resistance - demonstrate that multiple active agents can drastically reduce the likelihood of resistance development. Thus, a robust lead-generating platform would not only replenish the antibiotic pipeline but also provide the chemical diversity necessary to design synergistic combinations that actively mitigate the spread of resistance, rather than exacerbate it.

A Rational Approach to Revive the Waksman Platform: Which Organisms to Mine?

Since synthetic chemistry has never established a reliable discovery platform, we discussed ways to revive natural product discovery with Professor Lewis. To frame this discussion on a scientifically grounded basis, we first examined another reason for the decline of the Waksman platform. The classical Waksman approach, mass screening of soil microorganisms for antibiotic production, has reached both a statistical and conceptual dead end. Most ecological niches have been exhaustively mined, and the likelihood of discovering new scaffolds with broad activity has become vanishingly small. What’s missing is not only a new library of strains but a rationale for which organisms to search. A rationalized antibiotic discovery strategy should deliberately focus on ecologically privileged bacterial groups - microbes whose natural life cycles impose the same selective pressures that define our pharmacological needs. For example, Photorhabdus and Xenorhabdus are symbiotic bacteria that live within entomopathogenic nematodes. When these nematodes infect insect larvae, the bacteria are released into the insect blood, where they kill the host and defend the carcass from competing microbes. This ecological context forces their metabolites to meet stringent functional criteria: they must diffuse systemically through the insect body (implying good tissue penetration and distribution), persist for days or weeks to maintain control of the cadaver (implying stability and long half-life), avoid harming the nematode host (indicating low host toxicity), and target mainly gram-negative competitors (matching the clinical need for new gram-negative agents). In other words, natural selection has already performed a kind of evolutionary pre-screening, enriching these symbiotic bacteria for compounds that inherently possess many of the properties we seek in ideal antibiotics—low toxicity, systemic efficacy, and favorable pharmacokinetics. By zeroing in on such ecologically informed “privileged groups,” antibiotic discovery can move beyond blind statistical screening toward a more rational, evolution-guided platform that exploits nature’s own optimization processes. Photorhabdus/Xenorhabdus bacteria, which kill and decompose the insect host, providing nutrients for nematode reproduction; nematode and bacteria have a mutualistic relationship, while the insect is parasitized.

Tab. 1: Translation of Ecological Features of Photorhabdus into pharmacological analogies based on their tripartite symbiosis

Requirement for meaningful antibiotics	Reason in ecological context	Pharmacological analogy
Diffuse throughout the insect body	The antibiotic must reach and suppress competing microbes across the insect cadaver, not just locally.	Good tissue penetration and distribution (a pharmacokinetic property).
Persist over time	The carcass must remain protected for days/weeks until the nematode completes its life cycle.	Good stability and half-life, avoiding rapid degradation (another PK-like feature).
Be non-toxic to the nematode host	The nematode depends on its bacterial symbiont and coexists with the compounds it produces.	Low host toxicity (good therapeutic index).
Be active against gram-negative bacteria	The main competitors in the nematode gut and in the insect cadaver are gram-negative microbes, similar to human pathogens.	Relevant antibacterial spectrum for clinical use.

The Benefit of Genomics-driven Discovery: Unlocking Hidden Natural Products

Part of our discussion with Prof. Lewis was also the differences between bioactivity-guided and genomics-driven discovery. We discussed that in bioactivity-guided discovery such as in the Waksman platform, researchers first prepare an extract from an organism and test it for biological activity, such as antibacterial effects. Only if activity is observed do they determine the chemical structure of the compound. In other words, the workflow is guided by activity first and structure second. In contrast, genomics-driven discovery works the other way around. Researchers use bioinformatics tools to predict biosynthetic gene clusters (BGCs) from DNA sequences and can even infer the potential structures of the compounds. These clusters are then expressed in heterologous hosts, and the resulting compounds are tested for biological activity. Here, the process is structured first and activity second.

A major limitation of classical bioactivity-guided antibiotic discovery, including the Waksman platform, is that it overlooks a large portion of microbial chemical diversity. Many bacteria cannot be cultured in the lab, and many biosynthetic gene clusters (BGCs) remain silent under standard laboratory conditions. Genomics-driven discovery addresses both of these challenges. By sequencing all the DNA in an environmental sample, such as soil, and assembling it into contigs, researchers can identify BGCs from uncultivable bacteria as well as silent clusters from cultivable species. These sequences can then be synthetically reconstructed and expressed in heterologous hosts, making compounds that were previously inaccessible available for study.

This approach thus provides access to natural products that would be missed by classical bioactivity-guided methods, significantly expanding the pool of potential new antibiotics and other bioactive compounds.

Beyond Genomic-driven Antibiotic Discovery: Engineering is the way to go

When we asked Kim Lewis about engineering in the context of recombining BGCs to create novel compounds, he confidently stated that it is the way to go because nature does it anyway. Nature has spent billions of years generating chemical diversity, constantly recombining biosynthetic gene clusters (BGCs) to produce an astonishing variety of natural products with unique biological activities. This natural combinatorial process underlies the discovery of many antibiotics. However, the pace of natural evolution is slow, and in combinatorial NRPS biosynthesis, our current ability to generate novel compounds is still far below what would be needed to explore the full chemical space. Through engineering, we can imitate or emulate what nature has already perfected, accelerating the generation of chemical diversity.

Inclusion in our project

The interview proved transformative for our understanding and significantly shaped the scientific rationale behind our project. Before speaking with Professor Lewis, our choice to work with Photorhabdus BGCs was primarily guided by the remarkable terrestrial diversity of soil organisms. After the discussion, however, we gained a much deeper understanding of why Photorhabdus represents an ecologically and pharmacologically privileged group. The interview challenged us to critically examine the rationale behind our selections, ultimately establishing a solid scientific foundation for our focus.

Professor Lewis also emphasized that genomics-driven discovery is a powerful approach and that engineering offers substantial potential. However, he noted that both strategies face limitations in generating sufficient molecular diversity. This insight directly confirmed our planned approach: by combining two complementary diversification strategies, at the DNA level and at the protein level, we can expand library breadth far beyond what is naturally accessible in the lab. This nature-inspired engineering approach enables genome-driven discovery of new antibiotics in a systematic and rational way, effectively compressing billions of years of natural evolution into practical experimental timelines.

Dr. Joachim Hug

Expert in Natural Product Research, European Molecular Biology Laboratories

Reason for contact

Our earlier discussion with Prof. Dr. Kim Lewis helped us identify Photorhabdus as an ecologically and pharmacologically privileged bacterial genus, making it an attractive candidate for genome-driven drug discovery. Inspired by his insights, we aimed to explore the biosynthetic gene clusters (BGCs) of Photorhabdus as potential sources of novel bioactive compounds.

A major challenge in natural product discovery that we identified in the interview with Prof. Kim Lewis is the generation of molecular diversity. To address this, our strategy integrates methods that induce diversity both at the DNA level and at the protein level. This combined approach represents a modern implementation of combinatorial biosynthesis. Our conversation with Dr. Joachim Hug focused on how such strategies could overcome the limitations of earlier approaches, such as precursor-directed biosynthesis and mutasynthesis. Therefore, we wanted to conduct a comparative analysis between our approach and already existing ones with him.

In addition to tackling the diversity challenge, we also needed to carefully consider the choice of host organism for heterologous expression of Photorhabdus BGCs. Initially, we thought of using E. coli due to its status as a well-established heterologous expression host and its accessibility to iGEM teams worldwide. Nonetheless, Prof. Lewis emphasized that heterologous expression remains a major bottleneck in natural product discovery, and we were unsure whether E. coli could effectively express complex BGCs from Photorhabdus. During our research on alternative strategies, we came across the publication “Bacteria as genetically programmable producers of bioactive natural products”, which prompted us to contact Dr. Hug.

Key takeaways

Early Strategies: Precursor-Directed Biosynthesis (PDB) and Mutasynthesis Limitations

When we asked Dr. Hug about precursor-directed biosynthesis (PDB), he explained that it is one of the simplest ways to generate modified natural products. Instead of altering the genome, the producing organism is fed with synthetic or chemically modified building blocks. The native NRPS enzymes are often flexible enough to accept these precursors, so the organism produces structural analogues of its native products.

Despite its simplicity and independence from genetic engineering, PDB has clear limitations. Only certain positions in a molecule tolerate precursor incorporation. Additionally, uptake and availability of precursors can limit efficiency, and the exact site of substitution is often unpredictable, leading to mixtures that are hard to purify and characterize. As a result, PDB mainly produces derivatives of existing compounds rather than truly investigating unexplored chemical space.

Mutasynthesis was developed as a more controlled alternative to address some of these shortcomings. In this approach, the organism is first genetically modified so that natural biosynthesis is blocked at a key intermediate. The resulting dependency on externally supplied unnatural precursors allows incorporation of new building blocks and production of structurally novel compounds. Compared to PDB, mutasynthesis provides greater selectivity and control, although it still faces challenges such as precursor uptake and the need for detailed pathway knowledge.

Together, PDB and mutasynthesis illustrate both the promise and constraints of early combinatorial biosynthesis attempts: they can expand chemical repertoires, but fall short of reliably accessing truly novel chemical space.

Site-Specific Mutagenesis: A-Domain Engineering

Next, Dr. Hug described site-specific mutagenesis as a way to alter the substrate specificity of biosynthetic enzymes, particularly NRPS adenylation (A) domains. Unlike precursor-based methods, this strategy modifies the enzymes themselves, for example by engineering the binding pocket or swapping subdomains, to produce new products.

While powerful for point-specific changes, A-domain engineering has proven difficult to scale. Initially thought to depend mainly on the Stachelhaus code, a set of key amino acid residues within the adenylation domain that determine which substrate is recognized, substrate specificity is now understood to involve many additional residues. This complexity makes high-throughput applications challenging, as predictions are unreliable and outcomes hard to generalize.

As Dr. Hug reflected on these strategies, he noted the central challenge that drives this field:

"One major challenge is to access truly new chemical space that cannot be reached by other methods."

To implement an effective combinatorial biosynthesis strategy, a suitable microbial host is essential as a ‘living factory’ that can accommodate engineered pathways. We wanted to use E.coli as a model organism and were looking for suitable soil-dwelling source organisms. Based on sequence similarity, Dr. Hug advised us to look into Xenorhabdus and Photorhabdus as source organisms as these offered the closest match to E.coli.

Choosing the Right Host: Practical Considerations for NRPS Expression

To implement an effective combinatorial biosynthesis strategy, selecting a suitable microbial host is essential: a “living factory” capable of accommodating engineered pathways. Although we already wanted to choose E. coli as our heterologous expression host, our discussion with Dr. Joachim Hug, strongly reinforced this decision. Dr. Hug emphasized the value of using a host with high sequence similarity to Photorhabdus and Xenorhabdus, which aligns perfectly with our approach. His insights and the engineering techniques he discussed further supported that our intended strategies could be effectively implemented in E. coli, confirming it as an ideal platform for our combinatorial biosynthesis experiments.

Inclusion in Our Project

The insights from Dr. Hug provided valuable support for our project. His discussion of PDB, mutasynthesis, and site-specific mutagenesis highlighted both the potential and limitations of various combinatorial biosynthesis strategies. This perspective helped us understand where previous methods have succeeded, where challenges remain.

Additionally, the conversation confirmed the plausibility of our choice of E. coli as a host due to its sequence similarity with Photorhabdus, thereby supporting its suitability for our approach.

Overall, these insights allowed us to position our project more strategically: by carefully selecting hosts, we are not only addressing known limitations but also establishing a platform that exceeds existing approaches in generating natural product diversity.

Ultimately, these discussions clarified our project’s novelty and refined our vision of enabling E. coli to serve as a reliable platform for producing diverse and bioactive natural products.

Prof. Dr. Felix Hausch

Expert for Rational Drug Design at the Technical University Darmstadt and Coordinator of the Macrocycles for Drug Discovery (MC4DD) consortium

Reason for Contact

Before our interview with Prof. Felix Hausch, we faced the task of selecting biosynthetic gene clusters (BGCs) for our derivatization platform. The collection of BGCs included NRPS clusters producing both linear and cyclic peptides. To make a well-considered decision about which peptide scaffold of our collection would be the most promising for our platform, we recognized the need to better understand the different types of peptides and their properties. To establish a solid scientific rationale for determining which peptide would form the core of our library, we sought the guidance of a leading expert at the intersection of peptide science and drug discovery.

Key takeaways

Macrocycles as a Tool to Expand the ‘Druggable Space’

As coordinator of the Macrocycles for Drug Discovery consortium (MC4DD), Prof. Felix Hausch explained that his research originated from a specific challenge: finding a drug-like molecule capable of selectively targeting FKBP51, a protein that conventional small molecules could not adequately address. This led him to the concept of the ‘druggable space’, which is a term referring to the set of molecular targets that are accessible to specific classes of compounds, such as small molecules, peptides, or antibodies. Within this framework, macrocycles stand out because they can engage targets that remain inaccessible to classical small molecules, thereby occupying a crucial area between small molecules and larger biologics (Fig. 1)

🔍

“Macrocycles are one of the best tricks that medicinal chemists can play to target difficult-to-tackle proteins.”

Understanding the Scientific Background

To explore the differences between small molecules and macrocycles in terms of target accessibility, we conducted further research. We found that classical small molecules typically rely on concave binding pockets in proteins, which are characterized as deep, well-defined grooves or cavities that can envelop the ligand (Fig 2a). In such pockets, a small molecule can make multiple, tight contacts that yield both high affinity and good specificity. However, many ‘difficult’ targets such as protein– protein interactions (PPIs) occur across large, relatively flat surfaces and lack such well-defined pockets. Consequently, classical small molecules often struggle to bind these flat interfaces: they cannot make enough favorable contacts, so the affinity and specificity are typically low (Fig 2b).

By contrast, macrocyclic peptides (in the 500–2000 Da range) are large enough to reach across broad, flat interfaces and make multiple contact points across the surface, yet still small and flexible enough to adapt to the target region. Thus, they can span flat binding surfaces in a way small molecules cannot and thereby achieve antibody-like affinity and specificity (Fig 2c). In effect, macrocycles can convert a flat interface into a pseudo‐“concave” interaction by molding themselves over the surface and engaging multiple side chains across that interface.

🔍

Beyond the Usual Targets: New Paths in Antibiotic Design

These insights are relevant for expanding the limited set of bacterial targets currently exploited by antibiotics. While the bacterial proteome includes about 200 conserved essential proteins, most antibiotics target only a few pathways, such as the ribosome, cell wall synthesis, and DNA gyrase/topoisomerase. By expanding the accessible "druggable space", macrocycles could enable entirely new modes of antibacterial intervention.

The Better Drugs: Macrocycles’ Superior Pharmacokinetic Properties

Prof. Hausch also highlighted the challenges peptides face as drugs, such as short metabolic half-lives exemplified by endogenous peptide hormones.

To improve stability and pharmacokinetics, medicinal chemists employ modifications like D-amino acids, unnatural amino acids, and N-terminal capping. These strategies align with our own NRPS-based approach, which can incorporate D- and other non-proteinogenic building blocks and N-terminal acyl chains. A striking example for a NRP that contains these features is cyclosporin (Fig. 3), where these structural characteristics translate into outstanding stability and bioavailability.

🔍

“A bottleneck in applying peptides as therapeutic drugs is their proteolytic lability, and macrocyclization is a superb way to address that because they very often immediately get substantially more stable.”

This statement motivated us to explore cases where macrocyclization has been particularly successful. Phage display, although primarily used for monoclonal antibody development, has also yielded peptide drugs like romiplostim and ecallantide. Many of these peptides initially suffered from poor stability and limited bioavailability. Through macrocyclization, these limitations were effectively overcome, demonstrating its critical role in transforming phage display hits into viable therapeutics.

Inclusion in our project

The interview with Prof. Hausch has greatly helped us advance our ambition of developing truly drug-like compounds, as it allowed us to compare different peptide types and their pharmacological properties with a leading expert in the field. Based on these insights, we decided to incorporate macrocycles rather than linear peptides as the core scaffold for our platform. Their rigid structure provides high stability, improved bioavailability, and the ability to target protein–protein interactions that are often inaccessible to smaller molecules, making them a powerful class of compounds for antibiotic development. We therefore selected a macrocyclic peptide as the core scaffold, ensuring that our library is built around a structure with strong therapeutic potential.

Prof. Dr. med. Isabelle Bekeredjian-Ding

Institute of Medical Microbiology and Hospital Hygiene, Marburg University Hospital

Reason for contact

We contacted Prof. Isabelle Bekeredjian-Ding because her combined expertise in infectious disease, drug development and regulatory affairs perfectly matched our needs: she understands the key unmet medical needs our research addresses and knows the regulatory authorities and pathways that need to be involved when innovations reach patients. As Director of the Institute of Medical Microbiology and Hospital Hygiene at the Marburg University Hospitaland former Head of the Division of Microbiology at the Paul Ehrlich Institute (PEI), she could guide our project to ensure clinical relevance and navigate regulatory hurdles. She further looks back on extensive experience in public–private partnerships in R&D, such as Chair of the Scientific Committee of the Innovative Medicines Initiative (IMI2), Chair of the advisory board of Infectognostics in Jena and member of the INCATE selection committee.

Key takeaways

Obstacles in going from bench to bedside

In our conversation with Prof. Bekeredjian-Ding, we explored why many academic projects aiming to combat AMR fail to translate into real treatments. Her key message was clear: the lack of a defined clinical goal often prevents promising research from progressing.

“Most academic initiatives end because they have no idea what indications and formulations they’re really aiming at.”

Drawing on her experience as Head of Division of Microbiology at the PEI, she explained that researchers frequently propose projects, such as vaccine candidates, without specifying which patients they aim to treat or what formulation they envision.
Her advice was concise:

“At some point, you need to decide. You say, okay, I’m developing this for this purpose - and you stick to it.”

This insight highlighted a central barrier between research and application: while broad exploration is valuable, translational success requires focus and direction. To move from bench to bedside, projects must evolve from open-ended experimentation to a clear, purpose-driven development path.

Tools to Turn Ideas into Products: The Value of Target Product Profiles (TPPs)

Prof. Bekeredjian-Ding emphasized that academia already has tools to bridge this gap, they’re just underused. One particularly powerful framework is the Target Product Profile (TPP), which defines a product’s intended characteristics such as indication, formulation, and patient group.

“If you want to move in the direction of product development, the next step is to create a Target Product Profile.”

By using TPPs, academic researchers can increase efficiency, align goals, and focus development efforts more effectively. Prof. Bekeredjian-Ding also highlighted that the World Health Organization (WHO) recommends TPPs to improve communication between funders and researchers, helping align innovation with public health priorities and investment opportunities.

Inclusion in our project

Through the interview we uncovered several practical challenges that we had not anticipated. Originally, we thought that TPPs were mainly relevant for industry. However, it has become clear that they should also play a key role in academia to prevent loss of innovation.

To apply what we learned to our project, she mentioned that we could already develop a TPP after the hit identification stage. At first, we were unsure whether to create our own or adopt one from the WHO. She recommended using the WHO TPP. This choice does more than provide structure. It aligns our work with globally recognized public health priorities and ensures that a concrete clinical need and market already exist.

Prof. Dr. Alexander Visekruna

Safety Officer at Biosafety Level 2 Laboratory, University of Marburg

Reason for contact

As part of our mission to create a platform for developing novel antibiotics, our team is committed to testing newly produced peptides against the ESKAPE pathogens, which are widely recognized as major contributors to multidrug resistance in clinical settings. For these assays, access to a biosafety level 2 (BSL-2) laboratory was essential. We reached out to Prof. Bekeredjian-Ding´s group, where Alexander Visekruna serves as safety officer. Given his extensive expertise with BSL-2 organisms and regulatory standards, our goal was to receive expert guidance on laboratory safety and identify key instructions for inclusion in our workflow protocols.

Key takeaways

Safety Instructions for Experimental Protocols

A primary outcome of the interview was clarification of critical safety measures for handling BSL-2 organisms. Prof. Visekruna emphasized the following practices for reliable containment and infection prevention: consistent use of clean benches, appropriate personal protective equipment, thorough waste decontamination, and oversight by trained scientists. He stressed that these standards must be adopted not only in our own activities, but also integrated into any protocols shared with the iGEM community to ensure collective safety. This systematic approach, according to Prof. Visekruna, is frequently overlooked and warrants explicit inclusion in all future project documentation.

Biosafety and Biosecurity Regulatory Considerations

Our discussion extended to the essential legal and procedural safeguards for safe research. In addition to fulfilling all basic BSL-1 requirements, the BSL-2 environment must maintain self-closing doors, permanently closed windows, flagged biohazard signage at entries, and prominent escape route indications. Biosecurity was also discussed in depth: Prof. Visekruna underscored the need to consciously control dissemination of materials and data, referencing the historical misuse seen in incidents like the 2001 anthrax letters. Institutional cooperation with regulatory authorities and the use of material transfer agreements were highlighted as tools for regular assessment and management of biosafety risks.

Risk–Value Assessment

We also sought Prof. Visekruna’s expert opinion on the potential risks our work might pose to humans, animals, and the environment. His conclusion was that - with strict adherence to safety standards - the use of BSL-2 organisms poses a low risk. He contrasted this with research involving BSL-3 or BSL-4 agents, where biosafety and biosecurity concerns are much greater. Prof. Visekruna regarded our project as highly valuable, especially in the urgent context of combatting antimicrobial resistance (AMR), and pointed to the historical example of the penicillin discovery to illustrate the societal benefits of foundational research.

Inclusion in our project

Following the interview, we updated our protocols to incorporate all recommended safety procedures and requirements essential for responsible BSL-2 research. Prof. Visekruna provided direct instruction on facility-specific practices and significantly contributed to our project’s safety training. His shared expertise greatly influenced our biosafety competence and shaped our understanding of biosafety as an integral part of responsible science. We remain committed to maintaining the highest biosafety standards and regularly revising our protocols based on expert feedback.

Dr. Andreas Haldimann

Expert in antibiotics R&D at Roche Switzerland

Reason for contact

We contacted Dr. Andreas Haldimann, a scientist in Research and Early Development at Roche Switzerland. Based at the Roche Innovation Center in Basel within the Department of Immunology, Infectious Disease, and Ophthalmology, he offered a unique perspective on the scientific challenges in antibiotic research and development. Given that Roche remains one of the few companies actively pursuing antibiotic innovation, Dr. Haldimann’s insights are especially valuable for understanding both the scientific hurdles and practical constraints in the field.

Key takeaways

Identifying a Key Problem: The Bacterial Cell Envelope

As one of the main scientific challenges in antibiotic discovery, Dr. Haldimann identified the outer membrane of gram-negative pathogens which forms a formidable permeability barrier that prevents most compounds from entering the bacterial cell. He emphasized that the major difficulty lies in getting molecules across this membrane, rather than in a lack of sufficient binding of antibiotic compounds to their bacterial targets. As a result of this lacking entry into the bacterial cell, high doses of the affected antibiotic compounds need to be administered, which often results in toxicity issues. Dr. Haldimann pronounced that this issue is often a key reason why newly discovered antibiotics fail in further drug development cycles.

"One of the key challenges in antibiotic development, in contrast to oncology or other therapeutic areas, lies in overcoming the additional biological barriers posed by the bacterial cell wall and membrane. The issue isn’t usually the target itself, but rather to get the compound into the bacterial cell. This limited permeability creates a major toxicity challenge: to reach the therapeutic window drugs often must be administered at levels that exceed safe limits for eukaryotic cells. And that is exactly why many antibiotic development programs ultimately failed: due to toxicity-related setbacks.”

The golden age of antibiotics is long past, and identifying new compounds with antibacterial activity has become increasingly difficult. At the same time, he pointed out the potential of macrocyclic peptides, which inherently possess rich 3D structures that make them attractive for expanding accessible chemical space. Their structural diversity offers new opportunities in antibiotic discovery.

Scientific Background: Mutually Exclusive Properties

To better understand the described problem, we decided to conduct further literature research. Through this, we have understood, that the problem of antibiotic penetration into gram-negative bacteria is rooted in mutually exclusive physicochemical requirements. To cross the outer membrane, compounds generally need to be small and hydrophilic, allowing them to diffuse through porins. However, to pass through the inner membrane, they must be more hydrophobic to enter the lipid bilayer. Designing molecules that fulfill both properties simultaneously is extremely difficult and even when they do, multidrug resistance (MDR) pumps can still actively expel them. This makes the gram-negative cell envelope an almost perfect filter that exclues both large and hydrophobic compounds, while tightly controlling nutrient uptake through specialized transporters and porins (Li et al., 2015; Lomovskaya & Lewis, 1992). The target-based screening approach that AstraZeneca and GSK conducted, was a high-tech approach combining genomics, combinatorial chemistry, and structure-guided design. This drug discovery and design approach works brilliantly in other therapeutic areas, but for antibiotics, it completely failed. Though not discussed in the corresponding interview text, this aspect was also confirmed by Prof. Felix Hausch, Professor for Rational Structure-based drug design, who stated:

"Target-based drug discovery has been the basis for a lot of non-antibiotic drugs, but work extremely poorly for antibiotic discovery."

Despite large-scale campaigns against dozens of bacterial targets, no viable leads emerged. As later described by Payne et al. (2007) and Tommasi et al. (2015), the reason was clear in hindsight: Synthetic compounds simply could not penetrate the gram-negative cell envelope. This realization effectively ended the era of target-based antibiotic discovery and caused many companies to withdraw from antibiotic R&D altogether.

Historical Context: How This Problem Drove the Industry Away

This exact permeability barrier also explains why the pharmaceutical industry lost confidence in antibiotic discovery after the so-called Waksman era of natural products. In the late 1990s and early 2000s, in the so-called genomics era, companies such as GlaxoSmithKline (GSK) and AstraZeneca launched ambitious target-based antibiotic discovery programs. In contrast to phenotypic screening, where compounds are tested directly on whole cells to observe antibacterial effects, target-based screening isolates a specific bacterial protein, that was previously identified as essential for bacterial survival or growth, and searches for molecules that inhibit it in vitro.

Inclusion in our project

The conversation with Dr. Haldimann had a transformative impact on our project. His emphasis on the Gram-negative permeability problem made us realize that overcoming this barrier is essential for any new antibiotic strategy. As a result, we created a dedicated “Drug Delivery” pillar in our project - focusing on strategies to transport active compounds across bacterial membranes. By directly addressing the challenge that once discouraged the pharmaceutical industry, we connected our iGEM project to one of the most fundamental bottlenecks in antibiotic innovation.

Danini Marin

International PAHO consultant, Pan American Health Organization

Reason for contact

Reflecting on our project, we realized that a full antibiotic pipeline is not the only answer to the problem of antimicrobial resistance (AMR). Simply developing new antibiotics does not address the broader challenges surrounding what has been called the “silent pandemic”. This reflection led us to critically re-evaluate the metaphor of the “pipeline”. The term suggests a linear, technical process such as to discover a compound, to develop it, to deliver it. It implies that filling this pipeline would be a sufficient solution. But as the Covid-19 pandemic as shown us, no single drug, like Paxlovid, can end a pandemic on its own. Preventive measures, monitoring, public health infrastructure and community awareness are equally crucial.

In our research, we came across the One Health Approach which emphasizes the interconnectedness of human, animal and environmental health. To explore this concept further, we interviewed Danini Marin who is a seasoned expert in global AMR policy, having collaborated with the different international organizations (e.g. WHO/PAHO) on AMR policy related activities. In contrast to our interview with Prof. Gradmann, which focused on economic barriers, this conversation helped us understand the systemic and social dimensions needed to combat AMR effectively.

Key takeaways

We began by asking Danini Marin on her experience in addressing AMR using a One Health Approach in the Global South.

In 2015 with the Global Action Plan on AMR developed, it introduced One Health as a key concept. She explained that addressing AMR through a One Health approach requires the involvement of multiple stakeholders not only from the health sector but animal health, environment and food safety. Although these tend to work on the topic in isolation and often unaware of each other’s efforts:

“At the national level, we noticed that there was a vacuum or that we were working in silos.”

This fragmented approach, although the AMR problem had been recognized as a public health threat, meant that it was important to develop a plan that involved stakeholders from the One Health sectors.

She elaborated that an important stakeholder group are healthcare professionals (e.g. doctors, nurses, pharmacists, microbiologists, etc.), who can jointly reinforce the responsible antibiotic stewardship and use. However, in limited resource settings, time constraints and lack of accessible resources often prevent them from developing and implementing best practices in clinical settings. Therefore, context specific and applicable guidelines adapted from international standards are essential. However, there is a need to communicate with other One Health sectors to address the various factors involved in AMR.

Danini Marin stressed the importance of community involvement in tackling AMR, including in governance and policy decisions. While often overlooked,

“At the end of the day, it’s the public that consumes the antimicrobials.”

Therefore, the patients and wider community ought to be engaged in addressing this issue.

Finally, she stressed that good policies require good research, not just in the lab, but across all sectors. While the One Health concept is widely acknowledged, there is still a gap between theory and practice:

“Research is important so that we are guided by evidence.”

Inclusion in our project

This interview profoundly deepened our understanding of what systemic change in the fight against AMR could look like. Inspired by this perspective, we engaged in several science communication initiatives to help bridge the gap between scientific innovation and public awareness. Through these efforts, we aim to contribute to a more sustainable and informed response to the antibiotic crisis. Learn more in our Public Engagement section.

Prof. Dr. Christoph Gradmann

Professor of the History of Medicine, University of Oslo and Head of Department at the Department of Community Medicine and Global Health

Reason for contact

To understand the roots of the current antibiotic crisis, we looked beyond biology and lab research. Scientific challenges are only one piece of the puzzle. Economic, political, and structural factors are deeply intertwined with the decline in antibiotic innovation.

While researching this broader picture, we encountered the question:

"How did the antibiotic pipeline run dry?"

This led us to Prof. Dr. Christoph Gradmann, a renowned historian of medicine and expert in pharmaceutical development. His work investigates the long-term developments and institutional shifts that contributed to today’s crisis. We reached out to gain insights into how historical patterns could inform more sustainable strategies for the future.

Key takeaways

Understanding the Deeper Structural Issues Behind the Antibiotic Innovation Crisis

During our conversation with Prof. Dr. Christoph Gradmann, historian of medicine, he shared a compelling case study that illustrates how systemic challenges have shaped the antibiotic landscape. The case focused on Bayer’s antibiotic program, particularly the rise and fall of Ciprofloxacin.

The Last Blockbuster - Success of Ciprofloxacin ends in slow decline

In 1987, Bayer launched Ciprofloxacin, a breakthrough fluoroquinolone antibiotic. It quickly became a global blockbuster and a major success for the company.

However, its very success set a commercial and scientific benchmark that became nearly impossible to meet again. What began as a triumph ended in a slow decline, reflecting deeper issues in the structure of pharmaceutical innovation.

The “Next Better Thing” - No new family of antibiotics could be established

Bayer tried to build on Ciprofloxacin’s success by developing a family of similar drugs. In 1999, they launched Moxifloxacin, another fluoroquinolone.

While clinically effective, Moxifloxacin failed to match Ciprofloxacin’s commercial success.
As Prof. Gradmann put it:

“As a company, you need a family of those.”

Unfortunately, the family tree never grew, and Bayer struggled to replicate its earlier breakthrough.

From Crisis to Cutbacks - Bayer shifts in “survival mode”

In 2001, Bayer was hit hard by the Lipobay scandal, which caused major financial and reputational damage. The company shifted into what Gradmann described as “survival mode,” forcing it to restructure and reprioritize its entire R&D portfolio.

Promising antibiotic candidates, like Faropenem, were dropped due to technical difficulties and increasingly strict FDA regulations - challenges Bayer could no longer afford to tackle.

Even a collaboration with Paratek Pharmaceuticals in 2003 on a new antibiotic collapsed by 2005, again due to unresolved technical and commercial obstacles.

Sacrificing the “Holy Cow” - Bayer ends the in-house antibiotic R&D

Historically, antibiotics were central to Bayer’s pharmaceutical identity. However, by 2006, the company made a pivotal decision: it spun off its anti-infectives division into a separate entity called AiCuris.

With this move, Bayer effectively ended in-house antibiotic R&D. Aside from limited follow-up projects, such as using Moxifloxacin for tuberculosis or developing amikacin-based therapies, the company withdrew from antibiotic innovation.

Economic Pressures & the "Translation Void"

Prof. Gradmann emphasized that promising antibiotic compounds are still being discovered, but most are never developed further. The reason? Market research often deems them unprofitable.

Antibiotics, which are ideally used sparingly, are less attractive than drugs for chronic or high-margin conditions. As a result, companies deprioritize them; not due to lack of scientific potential, but because they don’t fit financial expectations.

This has created a translation void: a gap between discovery and real-world application, driven by economic risk aversion, growing regulatory demands, and outsourcing of early-stage research.

Limitations of Drug Discovery Models

Gradmann also reflected on failed innovation strategies. Traditional mass screenings have become too resource-intensive, while targeted drug development, once popular in boardrooms, often fails in practice.

“Targeted drug development worked in the boardroom, but it didn’t work in reality.”

This makes it clear that successful antibiotic research needs to be designed with both scientific and economic feasibility in mind from the outset.

Patents, Profit, and the Public Interest

When we discussed incentives like patent extensions, Gradmann challenged the logic behind them. While patents are meant to encourage innovation, in the case of antibiotics, they can create perverse incentives: companies are motivated to sell large volumes quickly to recover costs despite the public health need to use antibiotics conservatively.

Instead, he argued for publicly owned, non-commercial drug development, where public health is the priority, not profit. He stressed that tackling antimicrobial resistance also means investing in infection prevention, better sanitation, and fair access to existing treatments, not just chasing new drugs.

Inclusion in our project

Prof. Gradmann’s insights reshaped how we think about our role as an iGEM team. We realized that the antibiotic crisis is not only a scientific problem, it is deeply connected to economic systems, regulatory structures, and political priorities.

This inspired us to go beyond the lab. We began developing ideas for a public licensing model, drawing inspiration from open-source software. In this model, research outputs remain accessible, while licenses for clinical use could support further development. This approach could help keep innovation in the public domain, ensuring transparency, collaboration, and long-term impact.

We also recognized the need to address the translation void by making our work realistic and applicable in future drug development pipelines. Tools like Target Product Profiles (TPPs) - discussed in our interview with Prof. Bekeredjian-Ding - can help define clear development goals from the start.

Our goal is not just to discover new compounds, but to design research frameworks that are scientifically sound, economically viable, and socially responsible. Only by tackling both the discovery void and the translation voidcan we contribute to a future where antibiotic development is sustainable, collaborative, and truly in the public interest.

Prof. Dr. Raphael Reher

Professor for Pharmaceutical Bioanalytics and Natural Product Research at the University of Marburg

Reason for contact

As part of our efforts to translate our project into real-world impact, we explored how our NRPS-derived compounds could progress beyond the lab. We conducted extensive research and dived into the practical challenges of development, aiming to understand where the key bottlenecks lie. In this context, we spoke with Prof. Raphael Reher to gain first-hand insight into the obstacles in advancing NRPS hits toward preclinical candidates.

Key takeaways

One of the most striking points Prof. Reher emphasized was the limited availability of compound material. Many NRPS-derived hits exist only in tiny quantities, often ten less than one milligrams, which allows only rudimentary biological profiling. Without larger amounts, extended biological and chemical characterization cycles, as well as iterative medicinal chemistry programs, cannot take place. This scarcity effectively stalls the translation of hits into viable lead candidates.

The consequences of this bottleneck are significant. Targeted NRP analogs derived from successful engineering often fail to progress simply because there isn’t enough material to structurally and biochemically characterize and optimize them. The limited supply is not just a technical inconvenience; it fundamentally shapes which molecules can advance into preclinical and clinical development.

Inclusion in our project

The conversation with Prof. Reher had direct implications for our own work. Inspired by his insights, we decided to investigate the quantitative profile of our expressed NRP and assess how production limitations might affect further development. We successfully scaled the production of our core scaffold, Chaiyaphumine, to a one-liter culture, purified the expected NRP using HPLC, and quantified yields and production titers. While this represents a proof of principle, we recognize that bridging the gap from lab-scale to relevant preclinical quantities will require further optimization: improving fermentation yields, exploring heterologous hosts, developing robust downstream processing, and securing sustainable funding to support these labor- and resource-intensive steps.

Prof. Dr. Max Crüsemann

Professor of Pharmaceutical Biology at Goethe University Frankfurt

Reason for contact

To strengthen the scientific foundation of our project, we sought guidance from Prof. Dr. Max Crüsemann. His research focuses on natural products, particularly genome engineering approaches to activate underexpressed biosynthetic gene clusters. Due to his expertise and his collaboration with Prof. Raphael Reher, another expert we had previously consulted, we reached out to him to discuss methods of quantifying new-to-nature peptides and to evaluate the reliability of analytical techniques available to us.

Key takeaways

Technical advancements

Prof. Crüsemann highlighted how recent technological progress has revolutionized the field of natural product discovery. With advanced genome sequencing and analysis, researchers can now uncover hidden biosynthetic potential within microorganisms. This has reestablished natural products as a promising source for novel therapeutics, particularly antibiotics, by enabling access to chemical diversity previously overlooked.

Reproducibility

Prof. Crüsemann emphasized:

“Biology can be like a black box.”

This stresses that biological systems are inherently variable. Minor changes in conditions may lead to significant fluctuations in yield. To overcome this, both biological and technical replicates are indispensable in validating experimental results. Reproducibility is not just a technical requirement, but also a fundamental standard to ensure scientific robustness.

Measurements in comparison

A central part of our discussion focused on the quantification of novel peptides. Since our initial experiments already demonstrated peptide production detectable via LC-MS/MS, we asked Prof. Crüsemann about the most suitable analytical approaches. He strongly recommended LC-MS/MS as the “state-of-the-art” method due to its sensitivity, accuracy, and minimal material requirements, an especially critical advantage when working with low-yield compounds. Other methods, such as NMR or UV absorption, were described as less suitable for our context. For example, NMR requires purified compounds and relatively large quantities, making it impractical for early-stage studies on crude extracts. UV absorption, while simple, lacks the specificity and sensitivity needed for accurate quantification in complex mixtures. In contrast, LC-MS/MS combines fractionation with precise detection, enabling the analysis of compounds even in complex backgrounds. Prof. Crüsemann further noted that all quantification methods, whether LC-MA/MS, NMR, or UV absorption, rely on internal standards and calibration curves. For our new-to-nature peptides, this would require prior purification, a time-intensive process that limits rapid quantification efforts. Nevertheless, he emphasized that generating such standards is an essential step toward reliable yield measurements and meaningful comparisons to known antibiotics via minimal inhibitory concentration.

Inclusion in our project

This conversation validated our choice of LC-MS/MS as the primary method for analyzing peptide yields, confirming that we are working in line with the highest standards of natural product research. Moreover, it provided us with an outlook on the future that once purification is achieved, calibration curves can be established to allow accurate quantification. Looking ahead, we plan to integrate Prof. Crüsemann’s advice into the next phase of our project. Specifically, for peptides with promising bioactivity, we aim to perform MIC (Minimum Inhibitory Concentration) assays and compare their effectiveness against ESKAPE pathogens to existing antibiotics. By building on the expert guidance we received, we ensure that our approach is both scientifically robust and relevant to real-world biomedical challenges.

Prof. Dr. Mark Brönstrup

Chemical Biology Research Group, Helmholtz Centre for Infection Research

Reason for contact

Our interview with Prof. Mark Brönstrup was prompted by our conversation with Dr. Andreas Haldimann , where we developed an initial understanding of the importance of drug delivery questions in the drug discovery process. This issue especially applies to Gram-negative bacteria which have a robust outer membrane that adds an additional permeability barrier. After gaining these insights from Dr. Haldimann, we had decided to implement a dedicated strategy to address this pressing challenge of antibiotic delivery to Gram-negative bacteria.

To realize our ambitions, we began to conduct research on current strategies to address this problem and explored the latest innovations in antibiotic design, especially in relation to natural products. During this process, we came across Cefiderocol (Fig 1). Cefiderocol is a so-called “Trojan horse” antibiotic which is designed to target gram-negative bacteria. It combines a β-lactam antibiotic (similar to ceftazidime) with a siderophore, a molecule that binds iron and is actively transported into bacterial cells. By using the bacteria’s iron uptake systems, Cefiderocol can enter the periplasmic space, thereby sneaking inside the bacteria. Once inside, the β-lactam component inhibits penicillin-binding proteins (PBPs), which leads to cell wall synthesis inhibition

🔍

This discovery led us to wonder:

"Could we apply a similar principle to our non-ribosomal peptides (NRPs)?"

To explore this idea further, we looked for experts who specialize in combining natural products with Trojan Horse-based delivery systems and came across Prof. Dr. Mark Brönstrup, Head of the Chemical Biology Research Group at the Helmholtz Centre for Infection Research.

To deepen our understanding and evaluate how this approach might be integrated into our project, we reached out to him and were thrilled that he agreed to share his knowledge in that scientific niche with us.

Key takeaways

Breaking Barriers: Trojan Horse Delivery for Large Molecules

One of our first questions focused on Cefiderocol, a cephalosporin antibiotic using the Trojan Horse strategy, and whether this approach could be extended to larger molecules. Prof. Brönstrup shared promising insights:
He mentioned that daptomycin, a much larger molecule, had successfully been conjugated using a similar strategy and notably showed activity against Gram-negative bacteria after conjugation (Fig.2). Prof. Brönstrup remarked confidently:

“I do not see a problem with becoming larger than the cephalosporin.”

🔍

He also gave us a sneak peek into ongoing work with Peptide Nucleic Acids (PNAs), though unpublished so far, where similarly large molecules are conjugated to siderophores. These insights supported our idea to apply the strategy to NRPS.

Current state of the art for conjugation methods

We also discussed various conjugation methods and the current state of the art:

We ultimately chose click chemistry as our conjugation strategy - not only because it offers high selectivity (a key quality criterion), but also because it is simple to implement and thus accessible for future iGEM teams aiming to build on our approach, a conclusion fully supported by Prof. Brönstrup.

Optimizing Trojan Horse Approaches to Delay Resistance

Our approach uses a monocatechol-based siderophore, which is relatively unspecific and can be taken up via multiple transport channels. This design gave us confidence that resistance development could be delayed or minimized. In our discussion with Prof. Brönstrup, this choice was strongly validated: he confirmed that resistance against Trojan Horse antibiotics can occur, but stressed that the key lies in optimization. He highlighted a promising strategy - designing siderophores that exploit more than one uptake pathway:

“It is good to rely on more than one uptake receptor.”

These insights further confirmed that our design is well suited to minimize resistance development and to achieve a truly sustainable therapeutic impact.

Inclusion in our project

After our inspiring conversation with Prof. Brönstrup, we were eager to put the Trojan Horse strategy directly into practice within our project.

Among the three state-of-the-art conjugation methods we explored, click chemistry immediately stood out as the most exciting and promising approach for several reasons:

We were especially impressed by its bio-orthogonality, meaning the reaction is highly selective and doesn’t interfere with other functional groups in the molecule. This makes click chemistry not only efficient but also very gentle, which is crucial when working with complex natural products.

Looking at previous iGEM projects, we discovered that several teams had successfully combined click chemistry with proteins, demonstrating the method’s robustness and versatility. iGEM Hamburg 2023, for instance, used click chemistry to site-specifically modify proteins, showing how this method enables precise and efficient functionalization without disturbing protein activity. Other teams, such as iGEM Tübingen 2018 and iGEM Eindhoven 2014, also made use of click chemistry in their projects, further highlighting its broad applicability. What stood out to us across these examples was the widespread adoption and success of click chemistry in iGEM, reinforcing its reliability and effectiveness. Combined with protocols introduced from other iGEM teams, this could represent a very promising approach for our own work.

However, we noticed a significant gap:

So far, click chemistry has not been applied within iGEM to the field of non-ribosomal peptides (NRPs).

This realization motivated us even more to engage this approach. We believe that linking click chemistry with NRPs could unlock new possibilities in natural product engineering especially to facilitate drug delivery, taking advantage of the high selectivity and versatility of this method. Inspired by this gap, we planned to reach out to Prof. Hajo Kries, whose groundbreaking work on reprogramming NRPS adenylation domains perfectly matches our vision, to explore how click chemistry and NRPs could be combined - opening up exciting new possibilities for drug delivery and natural product engineering.

Prof. Dr. Hajo Kries

Technical Biochemistry Department, Stuttgart University

Reason for contact

Following an exciting conversation with Prof. Mark Brönstrup (DZIF) about conjugating non-ribosomal peptides (NRPs) to siderophores, we were eager to explore how we could apply the Trojan Horse drug delivery strategy to NRPs in particular. One method stood out in particular: bioorthogonal click chemistry. Its incredible selectivity, efficiency, and compatibility with a wide range of functional groups made it especially promising for use with complex natural products like NRPs.

While previous iGEM teams, like Hamburg 2023, Tübingen 2018, and Eindhoven 2014, successfully used click chemistry with proteins, we couldn’t find any example involving NRPs. That got us thinking:

"Could we be the first to combine these two powerful systems?"

During our literature deep-dive, we discovered the groundbreaking work of Prof. Hajo Kries, who developed a novel approach for reprogramming the substrate specificity of NRPS adenylation domains. His research immediately sparked our interest. We were incredibly grateful that he took the time to share his insights with us!

Key takeaways

The State of NRPS Engineering Before

Prof. Kries helped us understand the limitations that existed before his research. Back then, changing NRPS specificity was mostly limited to very similar amino acids, think aspartic acid to asparagine, using traditional mutagenesis methods. But anything beyond closely related substrates? Pretty much off-limits. With specificity changes by up to five orders of magnitude (10⁵!), his approach marked a true turning point in NRPS engineering and opened up many new possibilities.

How It Works: The Science Behind the Breakthrough

Prof. Kries walked us through the structural logic behind his strategy. By replacing bulky tryptophan residues with smaller serine residues in the substrate-binding pocket of the A-domain, more space is created. This allows larger or differently functionalized amino acids, like tyrosine with its additional OH group, but also azides and alkines to bind more efficiently.

One of our biggest questions was:

"Will the enzyme still be active after mutation?"

Enzyme activity is often the bottleneck in NRPS engineering, so we were a bit skeptical at first. But Prof. Kries confirmed to us that the catalytic activity of the mutated A-domains was preserved which is a major win for integrating them into functional assembly lines.

Inclusion in our project

Encouraged by Prof. Kries’ validation of our approach, we got to work. We integrated his engineered A-domain into our modular NRPS toolbox. The A-domain Prof. Kries engineered came from Bacillus brevis, but our chassis organisms (Xenorhabdus and Photorhabdus) are phylogenetically closer to E. coli. Since NRPS from different origin species often show compatibility issues, we wanted to test whether his modified A domain was compatible with our NRPS clusters.

We are incredibly grateful for Prof. Kries’ openness, insights, and enthusiasm. His work inspired a key component of our project and showed us what’s possible when cutting-edge science meets synthetic biology creativity.

Dr. Barbara Terlouw

Bioinformatician at Wageningen University and involved in the development of antiSMASH and PARAS(ECT)

Reason for contact

In the field of synthetic biology, Nonribosomal Peptide Synthetases (NRPS) hold great promise, but their engineering is still limited by one major challenge: the unpredictable compatibility between modules. After learning about recent technical advances in natural product research from Prof. Raphael Reher, we sought to better understand what is still missing to push NRPS engineering forward. Prof. Reher connected us to Dr. Barbara Terlouw, one of the leading experts in the field of NRPS bioinformatics and researcher at Wageningen University. Speaking with Dr. Terlouw offered us the opportunity to critically reflect on our approach and align our project more closely with the needs and realities of the field.

Key takeaways

Sequence vs. Structure

Before the interview, our design relied heavily on the assumption that protein 3D structural data was the most important factor in predicting NRPS module compatibility. Dr. Terlouw challenged this view, explaining that structure-based predictions often underperform compared to sequence-based approaches. Her reasoning was that structures are static, while proteins are inherently flexible. A structural model might miss key aspects of how modules interact dynamically. In contrast, sequence data retains this flexibility and therefore often provides more reliable input.

She also pointed out a way for us to combine sequence-based and structure-based predictions - using the structures to determine which residues are close to the active site and to the domain interfaces of condensation complexes and then using those residues only as part of a sequence-based model. This is similar to the approach taken by NRPSPredictor2, the A-domain specificity predictor that is incorporated in antiSMASH.

Building a pipeline

Another major part of our discussion focused on the pipeline we envisioned for generating 3D structures of NRPS condensation complexes. Initially, we planned to integrate tools such as AntiSMASH, ChaiDiscovery, and GetContacts. Dr. Terlouw highlighted that the accuracy of such a pipeline is limited by the performance of each individual tool and suggested we incorporate PARAS, a tool with superior accuracy for predicting A-domain substrate specificity.

Designing for multiple users

We had initially imagined our tool as a way to reduce trial-and-error in laboratory NRPS engineering. However, Dr. Terlouw encouraged us to think more broadly. For bioinformaticians, intermediate outputs and parts of our code might be as useful as the final prediction. She even expressed interest in using elements of our pipeline for her own research.

Inclusion in our project

The insights from this interview directly shaped the direction of our project. Most importantly, we integrated PARAS into our pipeline, following Dr. Terlouw’s guidance. We emphasized user-oriented design, planning separate interfaces for different types of users. Furthermore, we decided to package our software tools in a wetlab-user-friendly Docker file while also providing a developer’s guide on our gitlab page to aid bioinformaticians who want to customize and repurpose parts of our software.

Dr. David Kneis

Microbiologist at Dresden University of Technology

Reason for contact

Human Practices at iGEM is all about asking:

"How does our project influence the world beyond the lab?"

Our platform enables the production of novel antibiotics. After speaking with Danini Marin and learning about the importance of the One Health approach, we began to consider the scale of antimicrobial resistance (AMR) across its three interconnected sectors: human health, animal health, and the environment. That led us to a critical question:

What happens when these antibiotics leave controlled environments and enter ecosystems?
What role does this play in the development of AMR?

To explore these questions, we spoke with Dr. David Kneis, an environmental microbiologist who studies the environmental dynamics of resistance. His insights helped us understand both the risks and the complexities of antibiotic pollution.

Key takeaways

From Micro- to Macroscale: Pollution as a Driver of Resistance

Dr. Kneis explained that antibiotic resistance genes (ARGs) have existed in nature long before the first antibiotic was synthesized. Microorganisms for example have always competed using antimicrobial compounds. However, the rapid spread of resistance across species and ecosystems is a phenomenon driven by human activity, especially through the widespread use and release of antibiotics.

While naturally produced antibiotics act on a microscale, quickly diluted around the producing microbes, man-made antibiotics operate on a macroscale. Their presence in the environment leads to selective pressure across entire microbial communities.

How Antibiotics Enter the Environment

Dr. Kneis outlined several major pathways:

• Excretion from treated humans and animals (e.g., in the form of wastewater or manure)

• Direct contamination (e.g., waste streams of pharmaceutical manufacturing, improper disposal of drugs, drug application in aquaculture)

Each pathway represents a point of intervention. If we don’t address these emissions, even the most innovative antibiotic research risks being undermined.

Measuring Environmental Impact

We explored how much antibiotic pollution is actually present in the environment - and whether it poses a real risk for resistance development.

Environmental concentrations are typically well below the Minimum Selective Concentrations (MSC). However, localized hotspots, as for example hospitals, can exceed these thresholds as Dr. Kneis stressed.

Agriculture, Manure, and Gene Exchange

After our conversation with Prof. Kreienbrock on antimicrobial resistance (AMR) in the veterinary sector, we wanted to deepen our understanding of how animals impact the environment about AMR. Dr. Kneis explained that manure from antibiotic-treated animals introduces both resistant bacteria and antibiotic residues into the environment, which increases the likelihood of horizontal gene transfer between gut microbes and environmental bacteria. Although directly confirming the development of resistance in soil is challenging, these environments facilitate genetic exchange that may contribute to the evolution of resistance over time.

Inclusion in our project

Even though the evidence for environmental on-site selectionremains limited and complex, we believe the risk is serious enough to act on. As antibiotic developers, we must think not only about molecular design, but also about the afterlife of our compounds. What happens post-use through excretion, disposal, or waste matters just as much as what happens in the petri dish.

Dr. Kneis’s insights highlighted the importance of context and caution. Antibiotic resistance is a complex, multifactorial issue and environmental release is one piece of that puzzle.

Dr. Christian Lanckohr

Münster University Hospital

Reason for contact

As part of our Human Practices work, we wanted to understand how antimicrobial resistance (AMR) impacts patient care on the ground. To gain real-world clinical insights, we interviewed Dr. Christian Lanckohr, an anesthesiologist, intensive care specialist, antibiotic stewardship expert, and hospital hygiene officer at Münster University Hospital. Our focus was especially on antibiotic stewardship - promoting responsible and rational antibiotic use to optimize patient outcomes. Additionally, since Danini Marin highlighted in her interview that healthcare professionals are often too stressed and time-constrained to follow complex protocols, we aimed to learn more about how guidelines for antibiotic use are designed to be simple, accessible, and practical for clinicians working in high-pressure environments.

Key takeaways

The Reality of AMR in Hospitals

Dr. Lanckohr emphasized that AMR is not a future threat, but a current global health challenge. It already contributes to patient mortality and puts increasing pressure on healthcare systems. While Germany is not among the most severely affected countries, AMR remains a significant concern in clinical practice. Regional differences are noticeable: hospitals in Southern and Eastern Europe face much higher AMR burdens than those in Germany.

Even within Germany, local variability is substantial. For example, university and oncology hospitals often face different microbial challenges than general or district hospitals, requiring tailored antibiotic strategies.

Novel antibiotics are essential tools for patient care

Although novel antibiotics cannot solve the ecological problem of resistance, they are essential tools for managing the clinical consequences of AMR. Dr. Lanckohr highlighted their importance in treating multidrug-resistant infections, especially when other therapeutic options have failed.

“Finding a substance that offers therapeutic possibilities is the highest achievement you can attain from a medical perspective. You enable someone to receive treatment.”

This insight reinforced our motivation to contribute to the discovery of novel antimicrobial agents.

Antibiotic Use and Monitoring: Widespread Knowledge Gaps Lead to Suboptimal Prescriptions

Dr. Lanckohr pointed out that antibiotic prescribing is a broad medical responsibility, not limited to infectious disease specialists. However, knowledge gaps are widespread. Many physicians lack comprehensive training in infection management, antibiotic pharmacology, and resistance patterns.

As a result, it is estimated that 30-50% of antibiotic prescriptions are suboptimal, ranging from incorrect dosages to unnecessary use. In Germany, the Infection Protection Act mandates documentation of antibiotic use in hospitals, but enforcement is limited, and the out-patient sector (where 20-25% of antibiotics are prescribed) often lacks diagnostic support due to time and cost constraints.

Antibiotic Stewardship (ABS): A Practical Approach to Optimize Antibiotic Use

After outlining the challenges in current antibiotic prescription practices, Dr. Lanckohr emphasized that Antibiotic Stewardship (ABS) can substantially reduce antibiotic use - without compromising patient outcomes. ABS is a comprehensive strategy that includes education, consultation, structured monitoring, and clear clinical guidelines, aimed at optimizing antibiotic use across all levels of care. According to Dr. Lanckohr, two-thirds of inappropriate antibiotic use stem from educational gaps, while one-third results from outdated practices. He also highlighted the importance of using narrow-spectrum antibiotics to target specific pathogens.

ABS is a collaborative, multidisciplinary effort involving microbiologists, pharmacists, infectious disease specialists, and clinicians. Importantly, ABS is designed to support rather than punish; it focuses on coaching, not control, and guidance, not enforcement. This approach directly addresses the challenges raised earlier by Danini Marin: by providing accessible, practical supporttailored to busy clinical environments, ABS helps healthcare professionals improve prescribing practices without adding stress. As Dr. Lanckohr concluded:

“We need to establish better practices in antibiotic prescribing through education, training, and monitoring.”

Inclusion in our project

Our conversation with Dr. Lanckohr confirmed our decision to focus on Xenorhabdus and Photorhabdus strains for our cluster selection. These competitive microbes live in symbiosis with nematodes, producing antimicrobial compounds that help them eliminate microbial rivals inside insect hosts. Their natural target specificity aligns with Dr. Lanckohr’s call for more narrow-spectrum antibiotics, a need we aim to address through exploring these finely tuned ecosystems.

Although the link between microbial competition and the production of narrow-spectrum antibiotics is not yet fully proven, current evidence is highly promising. By harnessing these natural mechanisms, we strive to design antibiotics that are precise, effective, and microbiome-friendly, advancing a more sustainable use of antimicrobials.

Dr. Lanckohr’s clinical insights also emphasized the urgency of developing treatments for multidrug-resistant gram-negative pathogens, especially the ESKAPE group, which cause many of the most dangerous hospital infections. This reinforced our strategy of screening our novel peptides against these high-priority targets, ensuring our research directly meets clinical needs.

While we cannot end antimicrobial resistance overnight, every effective new compound can save lives. As Dr. Lanckohr put it:

“We may not solve global antimicrobial resistance, but we can save patients with multidrug-resistant infections.”

With our approach, we are doing exactly that: addressing the crisis we face today while creating something rare in global health: time - time to treat, time to innovate, and time to build the sustainable solutions of tomorrow.

Prof. Dr. Georg Hochberg

Expert in Evolutionary Biochemistry at the Max Planck Institute for Terrestrial Microbiology

Reason for contact

Upon completing our second DBTL cycle, we critically evaluated our results and considered strategies to address the observed low engineering success rates. Reflecting on our initial approach of utilizing Xenorhabdus and Photorhabdus genomes, which we had selected due to their evolutionary proximity to our model organism E. coli, we hypothesized that a similar rationale could be applied to guide the selection of our XUT donor modules. To explore this possibility further, we consulted Prof. Dr. Georg Hochberg, an expert in Evolutionary Biochemistry. This interview aimed to investigate whether and how phylogenetic relationships between donor modules and acceptor clusters could provide a systematic framework for more reliable NRPS engineering.

Key takeaways

To test our idea, we realized that we first needed to compare phylogenetic relationships of NRPS clusters from different source organisms. However, we were uncertain how to proceed, since NRPSs undergo frequent recombination and therefore lack a clear common evolutionary history. Therefore, we came up with the idea to employ the thioesterase (TE) domain as a phylogenetic marker due to its uniqueness in the NRPS cluster.

To validate our idea, we consulted Prof. Dr. Hochberg. He confirmed that the TE domain is indeed suitable as in contrast to domains such as adenylation (A) domain, the TE domain typically occurs only once per cluster. The absence of TE duplication within the same cluster enables direct comparison between different clusters.

Inclusion in our project

Based on this interview, we could confirm our systematic workflow for donor module selection. To test this concept, we identified 45 donor modules using this workflow. Of these, 35 donors were successfully cloned into donor plasmids. Within the scope of our library characterization we tested each module in three different XUT positions within the NRPS revealing a much higher success rate of catalytically active hybrid NRPS enzymes compared to those engineered with randomly selected donor modules.

The implementation of our workflow proved ground-breaking for our NRPS engineering strategy. For the first time, we established a scientifically rational framework for donor module selection. Incorporating phylogenetically selected XUT modules significantly improved engineering success rate.

Rachel Cattois

D’Youville University, Buffalo

Reason for contact

Following our discussion with Danini Marin on the importance of the One Health approach, we sought to better understand how antimicrobial resistance (AMR) manifests in human healthcare settings. To gain insights into clinical practice and the social dynamics of AMR, we interviewed Rachel Cattois, a senior nurse at D’Youville University in Buffalo, New York, with extensive experience in medical-surgical nursing.

Key takeaways

Antibiotic Use in Healthcare

Rachel Cattois shared that broad-spectrum antibiotics like vancomycin and azithromycin are commonly used in emergency departments, often empirically before the pathogen is known.

“Our main goal is to administer broad-spectrum antibiotics as quickly as possible to avoid sepsis or reduce septic shock.”

While lifesaving in urgent cases, this practice contributes to AMR. Many patients arrive after failed outpatient antibiotic therapy, often due to incorrect or ineffective prescriptions. Sepsis protocols, regulated by the CDC and WHO, guide much of this treatment. Cattois emphasized that antibiotics are often prescribed without clear pathogen identification, contributing to resistance.

“You might have some form of infection - so here’s an antibiotic. I feel like that’s an inappropriate way to care for patients.”

Importance of Education

Cattois highlighted the need for standardized protocols, interdisciplinary collaboration, and especially education, both for healthcare providers and patients.

“A lot of people aren’t aware of what AMR actually is, or what it can do.”

There are ongoing training requirements for healthcare staff, but patient education remains a gap. Cattois emphasized that information should be clear, accessible, and stress the responsible use of antibiotics.

“Educating the public is very important [...] Patients need to understand why and how to take antibiotics correctly.”

Inclusion in our project

The interview illustrated key AMR challenges in human healthcare, especially the overuse and misuse of broad-spectrum antibiotics and limited public awareness.

In response, we integrated a science communication component into our project, aiming to educate the public. To reach a broad audience, we participated in events such as Chemikum, the Summer Festival, Excellence Cluster Celebration, ScienceBox, Hessen Trade & Invest, and CSL Behring. These initiatives promote awareness, improve understanding, and encourage responsible antibiotic use across all sectors.

Prof. Dr. Lothar Kreienbrock

Veterinary epidemiologist, University of Veterinary Medicine Hannover

Reason for contact

As part of our effort to apply the One Health concept to our Human Practices work, we aimed to understand how antimicrobial resistance (AMR) develops and spreads within the animal health sector and especially how this connects to human health. Following interviews focused on environmental and clinical dimensions of AMR, we completed the triangle by speaking with Prof. Dr. Lothar Kreienbrock, a veterinary epidemiologist at the University of Veterinary Medicine Hannover. His research focuses on epidemiological surveillance, antibiotic use, and risk assessment in veterinary contexts.

Key takeaways

AMR and the Human-Animal Connection

During our conversation, Prof. Kreienbrock emphasized that AMR does not respect species boundaries. Bacteria carrying resistance genes can spread from animals to humans, particularly through the food chain. Pathogens such as Salmonella and Campylobacter, often found in poultry and swine, can enter food products during slaughter and processing. While these bacteria may not cause illness in animals, they can lead to difficult-to-treat infections in humans, especially when they carry resistance genes.

He also pointed out the increasing role of livestock-associated MRSA (livestock-associated methicillin-resistant staphylococcus aureus) in hospital-acquired infections, reinforcing that AMR is a shared threat that links farm, fork, and hospital bed.

Food Chain as a Transmission Route

One particularly relevant insight was the role of commensal bacteria like Escherichia coli, which are not only ubiquitous but also capable of transferring resistance genes via plasmids, even to other pathogenic species. Although these E. coli strains may not cause disease themselves, they can act as vehicles of resistance. During slaughter, these bacteria may contaminate meat, survive in food products, and eventually reach consumers, becoming part of the larger resistance problem in human health.

Prof. Kreienbrock stressed that AMR can arise even if bacteria are not the primary target of antibiotic treatment - a key challenge in controlling its spread across the food chain.

Importance of Hygiene in AMR Prevention

A central takeaway from the interview was that hygiene is the most effective long-term strategy for reducing antibiotic use in animal farming. According to Prof. Kreienbrock, it’s not only about having high-tech infrastructure, but also about how consistently and correctly hygiene measures from barn ventilation to feed management and animal monitoring are implemented.

“The hygiene control dial is our main tool for moving forward - both in animal and human health.”

Preventing infections before they occur minimizes the need for antibiotics in the first place. Given the slow rate at which resistance declines, even when antibiotic use is reduced, this is an essential part of combatting AMR.

Inclusion in our project

The insights gained from Prof. Kreienbrock underscored the vital importance of the One Health approach, highlighting the deep interconnection between human health, veterinary medicine, and the environment. In particular, the link between foodborne transmission of resistance and the lack of new antimicrobials in veterinary medicine reinforced the urgency of developing novel active compounds to reduce reliance on existing antibiotics in both human and animal health. Additionally, our conversation emphasized the need for diagnostic tools and rapid screening systems to identify effective alternatives, especially against Gram-negative pathogens frequently found in livestock and food products.

Dr. Otto Quintus Russe

Managing Director, House of Pharma & Healthcare

Reason for contact

In our research concerning new antibiotics, we came across a striking statistic: only one in 25 preclinical substances reaches market approval – and of those that enter clinical trials, only one in ten succeeds. This high failure rate highlights a major challenge: it is not only difficult to discover a promising hit – something our platform aims to address – but even more so to translate that hit into a finished drug. Many promising compounds remain in the lab and never reach patients.

To better understand the hurdles between hit generation and market entry, we wanted to examine both the compound and the technology levels. The same article that drew our attention to these translational losses also introduced us to the House of Pharma, a cluster that connects politics, academia, and industry to strengthen translation. We were therefore thrilled to interview Dr. Otto Quintus Russe, Managing Director of the House of Pharma, to gain insights into the challenges of translation.

Key takeaways

The Valley of Death – the Most Critical Phase

Dr. Russe emphasized that the most vulnerable stage is the transition from preclinical research to Phase 1 clinical trials. This so-called “Valley of Death” is where most projects fail: costs for toxicology and early studies are high, while industry interest is still low. Success in pharmaceutical research, he stressed, requires true leap innovations – novel mechanisms of action rather than "me-too drugs" (slightly modified versions of existing medicines that offer little therapeutic improvement but compete commercially).

Financing and Structural Challenges

The central question is:

“Who pays what, and when?”

Academic funding typically covers only the earliest stages, leaving a financing gap that causes many projects to fail. Small research groups often lack the critical mass of infrastructure, expertise, and personnel needed to push innovations forward.

Patents as a Prerequisite

According to Russe, successful translation is only possible with timely patenting: “First patent, then publish” is the golden rule. A patent only becomes economically valuable when supported by a proof of concept. Without it, even strong scientific ideas struggle to attract industry interest.

Platform Technologies and BioNTech

Since we are not only searching for a hit, but also developing a discovery platform, we asked whether both could be valuable for industry. Dr. Russe emphasized that not only individual compounds, but also the underlying technologies, can be attractive to industry if they are properly protected. Platform technologies – such as BioNTech’s mRNA – hold particular promise, as they can be applied to multiple indications. However, they require extensive legal protection:

“For something like this, you don’t just need one patent – you need an entire patent family.”

He explained how BioNTech strategically secured patents worldwide and even resolved long-standing disputes (for example, with CureVac) through acquisition. For Russe, this demonstrates how strong patent strategies can turn a platform idea into a global success story.

Pathways to Translation

To overcome the "Valley of Death", Russe sees great potential in public-private partnerships and proof-of-concept initiatives that help projects reach early clinical stages. Beyond founding start-ups, licensing technology to pharmaceutical companies is another viable route. Politics, he emphasized, plays a crucial role by shaping funding programs and regulatory frameworks that make translation possible.

Inclusion in our project

Our conversation with Dr. Russe highlighted the importance of thinking about translation early. His insights reinforced the need to secure patents and showed us that both our compound and our platform technology could be attractive to industry – if properly protected. This aligns with what we learned from Prof. Gradmann: that public funding models and licensing systems are key to overcoming systemic barriers. Inspired by this, we explored the development pathway of our compound in more detail and analyzed how our technology could stand out compared to solid-phase peptide synthesis (SPPS).

Dr. Iris Hunger & Dr. Anja Blasse

Dual-use research concerns Officer and Deputy at Robert-Koch-Institute (RKI)

Reason for contact

Recognizing the importance of rigorous biosecurity assessment, our team engaged with the Robert Koch Institute (RKI), Germany’s federal agency for disease control and prevention. To obtain expert insight into biosecurity and dual-use research concerns (DURC) relevant to our work, we consulted with Dr. Iris Hunger, DURC-officer at RKI and Dr. Anja Blasse, her DURC-deputy.

Key takeaways

Assessment of Project Biosecurity

Following a detailed project overview, we asked Dr. Hunger and Dr. Blasse to review our work for potential dual-use risks. They concluded that, as long as our peptides do not correspond to known toxins or toxin-like compounds, the biosecurity risk remains low. To ensure this, they recommended comparing our target structures against toxin databases. After evaluating available options that support SMILES-based searches, we selected the ChEMBL database as the most suitable resource.

Dual Use Risks in NRPS Engineering

During our conversation, we discussed how most DNA synthesis companies screen submitted sequences against toxin and pathogen databases to mitigate bioweapons risks. However, this approach presents only a partial safeguard when considering nonribosomal peptide synthetases (NRPS). Because NRPS genes encode modular enzymes rather than toxins directly, these screening methods do not prevent the synthesis of NRPSs capable of assembling toxic peptides post-translational. Through recombination or engineering of NRPS modules, it is theoretically possible to reconstruct or create toxins without the DNA sequence of the toxin itself, thus circumventing sequence-based biosecurity protocols. As Dr. Hunger and Dr. Blasse emphasized, this underscores the need for careful risk assessment in synthetic biology. At the same time, they highlighted the need for publication of produced data as an essential part of good scientific practice research, and encouraged us to continue sharing our results openly. Their position reflects the balance between scientific transparency and security, openly publishing engineered NRPS data allows the scientific community to objectively assess risks and benefits, while keeping dual-use concerns in mind during every step of the research process.

General Approaches to Biosecurity

Dr. Hunger and Dr. Blasse outlined a series of measures to responsibly manage biosecurity in research, many of which are established practice within the RKI. These include: strict control of laboratory and data access; regular, expert-led reassessment of dual-use risks; and the willingness to halt projects if risk profiles change unfavorably. They also emphasized that effective biosecurity management must be balanced with maintaining scientific freedom.

DURC and Antimicrobial Resistance

We further explored dual-use risks associated with antimicrobial resistance (AMR) research. The experts noted that the most significant danger lies in research on resistance mechanisms, as knowledge of such mechanisms can be exploited to engineer multi-resistant pathogens. They emphasized the critical importance of responsible management of all AMR-related data by controlling access to data with high dual use potential. Since our project does not focus on resistance mechanisms, these risks were deemed less directly relevant.

Inclusion in our project

Our discussion with the RKI reinforced the importance of maintaining vigilance regarding dual-use risks throughout all stages of research. In our project, this is reflected in the systematic screening of all novel peptides against the ChEMBL database, which contains over 600,000 compounds with bioactivity data, using a threshold of 70% SMILES similarity to identify potentially concerning matches. Laboratory access is restricted to trained students only, ensuring appropriate handling of material. In addition, we are committed to periodically reviewing our risk assessments and to remaining in dialogue with experts so we can continuously update and adapt our biosecurity strategy as the project evolves.

Marcel Ishimwe

AMR Initiative Rwanda

Reason for contact

In our exploration of antimicrobial resistance (AMR) as a global challenge, we recognized that its impacts are not equally distributed across regions. While learning about this imbalance from our discussion with Danini Marin, we became aware that countries with lower economic resources are disproportionately affected by AMR yet often lack the infrastructure to respond effectively. To gain a deeper understanding of these dynamics, we contacted Marcel Ishimwe, a Rwandan pharmacist, public health advocate, and founder of the AMR Initiative Rwanda. His expertise and work in the field positioned him as an ideal interview partner to provide first-hand insights into the challenges of AMR in low- and middle-income countries (LMICs).

Key takeaways

Inequality in AMR regulations

In low- and high-income countries, an opposing trend can be observed: while the consequences of AMR are felt most severely in low-income countries, effective measures and structures to contain it are primarily established in high-income countries. Ishimwe told us:

"[This is mostly due to] weak regulatory enforcement in many regions, especially in low and middle income countries, worsened by poor sanitation and lack of access to quality diagnostics.”

These structural limitations not only exacerbate the health consequences of AMR but also hinder effective mitigation. He stressed the urgent need for raising public awareness and reinforcing regulation as essential steps toward tackling resistance. Through the AMR Initiative Rwanda, Ishimwe actively works on these challenges, striving to strengthen awareness and capacity. He also highlighted that AMR should not be regarded as a purely medical problem, but rather as a societal challenge requiring broad, multisectoral collaboration.

Inclusion in our project

By engaging directly with a practitioner working in Rwanda, we were able to situate our project within a wider global context and to appreciate the necessity of international collaboration and standardized regulatory frameworks. Ishimwe’s insights emphasized the importance of amplifying voices from those regions that face the heaviest burden, yet have the least access to robust infrastructures. The interview inspired us to embed this perspective of inequality and collaboration into our own work. Recognizing the value of unifying efforts across regions, we decided to develop the One Policy, a publication compiling expert perspectives we gathered through our interviews. In contrast to the established One Health approach, our policy proposes an economic framework to address the discovery void in antibiotic development. To ensure accessibility and inclusivity, we thought of presenting the content not only as a written report, but also with infographics, a simplified language version, and a recorded audio summary. Reflecting Ishimwe’s emphasis on cooperation, we would like to share the publication in collaboration with institutions such as the AMR Initiative Rwanda, thereby contributing to a more collective and globally conscious fight against AMR. Due to time constraints, we could not implement this plan, unfortunately.

Prof. Dr. Yousong Ding

Professor for Medicinal Chemistry, Center for Natural Products, Drug Discovery and Development, University of Florida

Reason for contact

To evaluate our biocatalytic approach for peptide synthesis in comparison to established methods, we wanted to contrast it with Solid Phase Peptide Synthesis (SPPS), the current gold standard in the field. For this, we interviewed one of the leading experts in natural product research and peptide chemistry: Dr. Yousong Ding. He is well known for his work on natural product–based drug discovery and for his contributions to enzymatic synthesis approaches and we were happy that he agreed to speak to us.

Key takeaways

SPPS as the Established Standard

Dr. Ding emphasized that SPPS has been the standard method for peptide synthesis for decades. It works especially well for ribosomal peptides (RIPs), since they only consist of the 20 natural proteinogenic amino acids, which are well-characterized and widely available. In contrast, non-ribosomal peptides (NRPs), like the ones we work with in our project, pose major challenges: they often contain rare or non-commercially available amino acids, which must either be synthesized laboriously or purchased at high costs.

The Challenge of Cyclization

Another major topic was cyclization, which Dr. Ding pointed out is particularly complex in SPPS. Peptides are usually synthesized in linear form and then require an additional cyclization reaction. This step is difficult to control, strongly influenced by stereochemistry, and further complicated by reactive functional groups.

“Cyclization is a challenge. Especially when it’s not a typical terminal cyclization – that is very hard to achieve in SPPS.”

Advantages of the Biocatalytic Approach

In contrast, he confirmed the significant advantage of our biocatalytic approach: peptides can be directly cyclized during biosynthesis, avoiding error-prone and complicated additional reaction steps. Moreover, Dr. Ding stressed that our platform has enormous potential to generate a wide variety of heavily modified and novel peptides that are extremely difficult, if not impossible, to access through SPPS:

“The advantage of biocatalysis is you could use enzymes to synthesize heavily modified peptides, which are difficult to access by SPPS.”

Inclusion in our project

Dr. Ding’s insights were particularly valuable for us because they aligned with our own lab experiences. When attempting to reproduce our target peptides via SPPS as reference samples for our quantitative analysis, cyclization failed. This confirmed that our approach is not only theoretically promising but also offers a clear practical advantage: the efficient production of new-to-nature peptides that cannot be obtained through classical synthesis methods.

Talking to Dr. Ding gave us valuable scientific validation for our project and showed us that our platform could significantly advance peptide synthesis.

Dr. Friederike Danneberg

Expert in Chemical Regulation and REACH Consultant at knoell

Reason for contact

With our platform, we are developing a biotechnological method for peptide synthesis that offers an alternative to the classical chemical solid-phase peptide synthesis (SPPS). In a previous interview with Prof. Yousong Ding, we already discussed the differences between our method and SPPS from a laboratory perspective - especially regarding efficiency, reaction conditions, and practical challenges.

Our next step was to specifically examine the chemicals used, their regulatory classification, and the impact on industry and society. Our research led us to the REACH regulation, the central EU-wide framework for the safe use of chemicals. REACH clearly states that no chemical substance may be placed on the market without sufficient data. This regulation not only protects human and environmental health but also aligns closely with SDG 12 (Responsible Consumption and Production), as it ensures that chemicals are produced, used, and disposed of safely. By promoting accountability, transparency, and innovation in chemical management, REACH actively supports sustainable production practices and responsible industrial development.

During this research, we came across Dr. Friederike Danneberg, a renowned expert in industrial chemicals and chemical regulation, as well as a Regulatory Affairs Project Manager at knoell.

Key takeaways

Regulation of DMF

A central topic of the interview was dimethylformamide (DMF) as a widely used solvent in SPPS. Dr. Danneberg confirmed that DMF is classified as reproductively toxic and has been repeatedly assessed under REACH, leading to significant restrictions on its use. This underlines the urgency of developing alternative peptide synthesis methods without DMF - exactly what we pursue with our biocatalytic approach.

While there are already emerging aqueous-based peptide synthesis systems that aim to reduce solvent toxicity, our approach goes a step further: by relying entirely on enzymatic, water-based biosynthesis, it not only eliminates hazardous solvents but also enables the direct generation of complex, non-natural peptides under mild and sustainable conditions. This positions our system as a safer and more versatile alternative to both conventional and currently evolving methods.

Beyond Synthesis: Purification and Extraction

Even before the interview, we considered that not only the synthesis process but also subsequent steps such as extraction and purification should be critically examined. This was confirmed by Dr. Danneberg. Our process also uses methanol and acetonitrile for these steps. Dr. Danneberg emphasized that these substances are less toxic with regard to CMR (carcinogenic, mutagenic, toxic to reproduction) properties - which often trigger regulatory action - than DMF and are not subject to comparable REACH restrictions. However, they are still health-relevant and should be handled carefully.

Regulation of TFA

Trifluoroacetic acid (TFA) is commonly used in SPPS as a cleavage reagent to release the synthesized peptide from the solid support and to remove protecting groups. In the context of ongoing PFAS (per- and polyfluorinated alkyl substances) debates, fluorinated compounds like TFA are increasingly under regulatory scrutiny due to their persistence and environmental concerns. Avoiding DMF and TFA, as we do in our aqueous synthesis system, offers clear advantages in terms of occupational safety for people directly involved in synthesis.

Healthier Outcomes for Everyone

Dr. Danneberg emphasized, however, that the benefits of a biotechnological method go beyond occupational safety:

“For workers it’s good if they have less contact, but also for the final product [...] there might be still impurities from the production process [...] it’s always better if less hazardous substances are included, because you’re not always able to completely get rid of them.”

The use of toxic chemicals in drug synthesis can thus also pose risks for end consumers if residues cannot be fully removed. Our research confirmed that this risk is not just theoretical: the Valsartan scandal, where carcinogenic impurities were found in a widely used blood pressure medication, highlights the relevance of this issue. This insight strongly influenced our decision to choose an aqueous, biocatalytic approach over classical SPPS, as it minimizes the use of hazardous substances from the start. By replacing toxic solvents with water-based systems, we reduce risks for both lab personnel and end consumers, directly contributing to SDG 3 (Good Health and Well-Being).

Challenges for Industry: Process Predictability

Another key point in the interview was the predictability of chemical processes in industry. The constant re-evaluation of chemicals under REACH presents major challenges for companies. Dr. Danneberg pointed out:

“Using chemicals with critical classification is not very sustainable for the companies because they constantly have to check what’s ongoing in the regulatory world with these substances – and they can’t plan ahead very well.”

Regulatory uncertainty means that companies must adapt processes, potentially rebuild production facilities, and implement additional safety measures, resulting in increased resource demands.

Inclusion in our project

The conversation with Dr. Danneberg was both inspiring and confirming. It reinforced that classical SPPS, especially due to the use of DMF and TFA, has problematic aspects that we can avoid with our biocatalytic alternative. We were glad to learn that what we are already doing represents the most sustainable and forward-looking choice in this context.

At the same time, the interview made clear that sustainability must not be limited to the synthesis itself: purification and extraction steps must also be considered. While we do use solvents there, they are less toxic with regard to CMR toxicity and less strictly regulated, which makes our approach more sustainable in these aspects as well.

This approach ensures that our platform is safer for lab personnel, reduces potential risks for end consumers, and aligns with SDGs 3 (Good Health and Well-Being) and 12 (Responsible Consumption and Production). In doing so, our work not only provides a greener and safer alternative to SPPS but also shows how biotechnological innovation can meet regulatory standards and contribute to global sustainability goals.