Synthetic biology of non-ribosomal peptide synthetases to generate new peptides

Projektinformationen

SYNPEP

ID Finanzhilfevereinbarung: 835108

DOI

10.3030/835108

Projekt abgeschlossen

EK-Unterschriftsdatum 30 Juli 2019

Startdatum 1 Oktober 2019

Enddatum 30 September 2024

Finanziert unter

• EXCELLENT SCIENCE - European Research Council (ERC)

Gesamtkosten

€ 3 165 788,00

EU-Beitrag

€ 3 165 788,00

Koordiniert durch

JOHANN WOLFGANG GOETHE-UNIVERSITAET FRANKFURT AM MAIN
Germany

Periodic Reporting for period 4 - SYNPEP (Synthetic biology of non-ribosomal peptide synthetases to generate new peptides)

Berichtszeitraum: 2024-04-01 bis 2024-09-30

Zusammenfassung vom Kontext und den Gesamtzielen des Projekts

Natural products and especially peptides with a molecular weight of up to 1500 Da are one cornerstone of our current health system, with several clinically used drugs being derived from them. Among them are important antibiotics (penicillin, vancomycin, or daptomycin), anti-cancer drugs (romidepsin), or immune-suppressive drugs (cyclosporin).
All the mentioned examples and several additional peptidic natural products are derived from non-ribosomal peptide synthetases (NRPS), giant multifunctional and multidomain enzymes found in several bacteria and fungi. While their biochemical principles have been identified more than 40 years ago, no robust or reproducible methods have been developed to engineer these enzymes in a predictable manner in order to generate novel peptides or modify existing ones. While these systems could be modified in some cases, leading to the expected nonribosomal peptides (NRPs), the production titer was often dramatically decreased for these variants compared to that of the parental NRP.
NRPS engineering could be a major game changer in drug development since it allows the (i) generation of a library of small molecule drugs, similar to natural products or macrocycles often showing potent bioactivities, and (ii) the derivatization and modification of existing peptide-drugs in an easy and fast way, and (iii) the biotechnological production of also synthetic peptide drugs is a more sustainable way avoiding organic solvents and fossil resources.

The ERC Advanced Grant SYNPEP is dedicated towards the engineering/modification of non-ribosomal peptide synthetases (NRPS) for the production of novel and bioactive peptides and related natural products (NPs). Within SYNPEP, we apply fast and high-throughput methods to find new NRPS systems in bacteria, activate the underlying biosynthetic gene clusters (BGCs), use the identified NRPS fragments to generate novel peptides based on NRPS engineering approaches, and produce these products in amounts sufficient for bioactivity testing and/or chemical modification.
The latter is facilitated by new-to-nature NRPS systems that accept unusual and chemically reactive building blocks for targeted modifications, which we screen for systematically within SYNPEP.
While we had identified a first approach for NRPS engineering prior to SYNPEP, we wanted to improve it further and reduce its limitations in order to have robust and reliable methods applicable to a broad variety of NRPS systems and organisms and allowing high-throughput approaches to produce thousands (later millions) of novel NRPs. These are then screened for their bioactivity in clinically relevant assays based on microfluidics coupling the bacterial production of these peptides directly to their bioassay.
The ultimate goal is to make NRPS engineering accessible to even non-experts in the field. For commercial application, the idea is to start a company with a focus on NRPS engineering.

Arbeit, die ab Beginn des Projekts bis zum Ende des durch den Bericht erfassten Berichtszeitraums geleistet wurde, und die wichtigsten bis dahin erzielten Ergebnisse

SYNPEP was composed of different parts. One part was the generation of novel natural products, namely NRPs. In total, more than3000 peptides have been produced and screened for bioactivity, leading to the identification of bioactive hits. As the next generation of NRPS engineering, two novel approaches have been developed: (i) The simple combination of 2-3 natural NRPS fragments connected by synthetic docking domains called SYNZIPs that allow a fast and combinatorial generation of new-to-nature peptides. (ii) NRPS engineering based on novel assembly points identified within thiolation (T) domains named XUT approach. Especially, the exceptionally useful XUT-based NRPS engineering approach was developed towards a high-throughput approach, which will also be implemented in standard bioinformatics pipelines, democratizing NRPS engineering also for non-experts in the field. Furthermore, XUT is based on the fusion of NRPS within thiolation (T) domains, the only domain shared with polyketide synthases (PKS), another large class of natural product enzymes, responsible for several additional clinically used drugs. Therefore, hybrids of NRPS and PKS can be generated, as we have shown by the generation of a new-to-nature glidobactin derivative with some selectivity against the human immunoproteasome.

Additionally, Myria Biosciences AG, dedicated to NRPS engineering and high-throughput screening based on methods developed in SYNPEP, was founded in 2021 as a spin-off from scientists of the Goethe Universität Frankfurt and the ETH Zurich.

In summary, the following results were achieved within SYNPEP:
- 30 publications were published, also in top-ranking journals like Angewandte Chemie, Chem, Nature Chemistry, and Science.
- Two new NRPS engineering methods were developed (SYNZIP and XUT), which can be combined with the previous methods found in the Bode lab (XU and XUC).
- XUT even allows the engineering of NRPS/PKS hybrids since it is based on the thiolation (T) domain shared between both megasynthetases.
- A company applying the NRPS engineering approaches was founded in late 2021 (Myria Biosciences AG, Basel).
- Several patents describing these approaches were filed; some of them are already licensed to a company.
- Several talks were given by the PI and SYNPEP group members highlighting the power of the described approaches.

Fortschritte, die über den aktuellen Stand der Technik hinausgehen und voraussichtliche potenzielle Auswirkungen (einschließlich der bis dato erzielten sozioökonomischen Auswirkungen und weiter gefassten gesellschaftlichen Auswirkungen des Projekts)

Originally, the goal of SYNPEP was to apply the already identified NRPS engineering approaches. However, during SYNPEP, the two much superior methods SYNZIP and XUT were developed, patented, and published. Both approaches and their success, also exemplified by the use of other labs, are clearly beyond the state of the art. Both approaches allowed the combination of NRPS parts, not foreseeable initially. Furthermore, the good production titers of some engineered peptides with up to 300 mg/L were absolutely unexpected, as well as the fact that XUT would also allow NRPS/PKS hybrid engineering, and the developed approaches can really be applied in high-throughput.

In summary, SYNPEP was very successful and sets the stage for the application of the developed tools for industrial drug development and compound diversification, as it is currently performed within the founded company Myria Biosciences AG. Furthermore, an ERC PoC project was derived from SYNPEP, applying the identified methods for the development of environmentally friendly insect pest control agents.

Summary Figure introducing NRPS and their potential