Biosynthesis of bacterial pyrrolizidine alkaloids and additional nonribosomal peptides
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Philipps-Universität Marburg
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Nonribosomal peptides (NRPs) are a class of structurally diverse and complex natural products (NPs) that exhibit multiple functions and have been shown to be involved in signal transduction as well as pathogenicity in the producing organism. As with other classes of NPs, these activities of NRPs are being exploited for human use. Many drugs are derived from or inspired by NPs. However, the physiological role of an NRP for the producing organism is often unclear, and its ingestion by humans or animals can lead to intoxication. The enzymes that produce NRPs, so called nonribosomal peptide synthetases (NRPS), are modular, and each module is responsible for incorporating a building block into the growing peptide chain. The modules themselves are composed of domains. A minimal module consists of three domains, the adenylation (A) domain, the thiolation (T) domain, and the condensation (C) domain. When the peptide chain reaches the last module, an offloading domain separates the oligopeptide from the NRPS, either as a linear or cyclic product. NRPs are known for their high structural diversity, which is achieved by modification of the product and incorporation of non-proteinogenic amino acids by the NRPS. Genes encoding NRPS are usually accompanied by several other genes and are grouped into biosynthetic gene clusters (BGCs). These genes typically encode for self-resistance, product decoration, peptide export, and enzymes required for building block synthesis.
NRPs are a promising starting point for the development of new drugs, but in order to improve or modify their bioactivity, they often need to be chemically modified. Great progress has been made in NRPS engineering over the last 25 years. Recently, additional NRPS engineering concepts have been developed in the Bode lab, two of which are relevant to the following projects, namely the concept of eXchange Units (XU) and the concept of eXchange Units between T domains (XUT). Here, an A-T-C (XU) unit or a T C A (XUT) unit is considered as an exchange unit that can be fused with other exchange units to form NRPS hybrids.
The following work is divided into three parts, each focused on a different NRP, namely pyrrolizwilline, pyrrolizixenamide, and FR900359. Two of these, pyrrolizwilline and pyrrolizixenamide, belong to the class of pyrrolizidine alkaloids (PAs). PAs are widespread natural products produced by plants and microorganisms. They are defined by their core structure, a bicyclic aliphatic hydrocarbon consisting of two ortho-fused five-membered rings with a bridgehead nitrogen. To date, about 40 microbial PAs have been identified, but their activities and ecological roles are largely unknown. A common pathway involving an NRPS and a Baeyer-Villiger monooxygenase (BVMO) forms the backbone of microbial PAs. BVMOs are known to convert ketones and lactones into linear and cyclic esters, respectively, by facilitating the insertion of an oxygen atom from molecular oxygen.
The first topic of this thesis was the biosynthesis of pyrrolizwilline, an unusual bacterial pyrrolizidine alkaloid dimer. Pyrrolizwilline produced by Xenorhabdus hominickii has been characterized in a previous work and promoter exchange experiments showed that the BGC xhp encodes the enzymes responsible for its production. However, the biosynthetic pathway of this compound remained unclear. Therefore, the aim of this study was to elucidate the biosynthesis of pyrrolizwilline. For this, heterologous expression of xhpA-G in Escherichia coli was used for the elucidation of the biosynthetic pathway. The experimental results obtained after heterologous expression of the xhp BGC and production of selected combinations of the encoded enzymes, followed by extensive analysis by high-performance liquid chromatography coupled with mass spectrometry (HPLC-MS), allowed postulating the complete biosynthetic route to pyrrolizwilline. This biosynthetic route is the first described to date involving both non-enzymatic and enzymatic reactions leading to the cross-linking of two bacterial pyrrolizidine alkaloid moieties in a natural context. The biosynthesis of pyrrolizwilline involves a key enzyme, the hydrolase XhpG, and a key non-enzymatic step that relies on the intrinsic reactivity of the pyrrolizidine intermediate to react with a biologically available aldehyde.
The second project focused on the derivatization of pyrrolizixenamide. PAs are recognized as privileged structural motifs in small molecule drug discovery and show a broad pharmaceutical potential, therefore it is of great interest to generate new-to-nature PAs. The previously characterized NRPS PxaA, together with the BVMO PxaB, is known to produce the PA pyrrolizixenamide in Xenorhabdus stockiae. In this project, the biosynthetic pathway of pyrrolizixenamide has been exploited with the aim of producing novel molecules. Several strategies were applied: precursor feeding, engineering of the PxaA termination module, and engineering of the PxaA starter module. In total, six out of 46 PxaA hybrids were shown to be functional and five novel peptides were produced.
In the third, and last, project the biosynthesis of the the depsipeptide FR900359 (FR) was investigated by NRPS engineering. The biosynthetic pathway leading to the production of FR is encoded in the BGC frsA-G in Chromobacterium vaccinii MWU205. FR is known as the most potent inhibitor of heterotrimeric Gq family proteins, one of the four major G protein families involved in cell-signaling and therefore of great interest. FR is also well known for its biosynthetic pathway leading to exceptional properties, such as hydroxyleucine, phenyllactic acid, N-methyldehydroalanine, N-methylalanine, or N-,O-dimethylthreonine. However, the presence and dependence of the A domains on their native MbtH-like protein (MLP) FrsB must be considered. MLPs are small proteins (60-70 amino acids) that are often encoded along with NRPS in bacterial BGCs. MLPs have been shown to act as chaperones by affecting the solubility of A domains and are often required for proper folding. In the first part of this project, the FR-producing NRPS was engineered to generate FR derivatives. For this purpose, the NRPS FrsA was chosen, which is responsible for the transfer of the side chain to the heptameric peptide FR-core produced by the NRPS FrsD-G. Engineering of FrsA was successful and is promising to enable FR derivatization. Finally, the biocombinatorial potential of the FrsADEFG NRPS modules was investigated, with special emphasis on their MLP dependency, in order to make these modules available for the diversification of other NRPs. The application of engineering strategies to the NRPS Frs was successful, allowing the use of FR building blocks in other MLP-independent NRPS systems. Furthermore, a tool was developed to study the MLP dependency of A domains, which also led to the conclusion that the native MLP should always be co-produced when using MLP-dependent NRPS modules.
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Created: 2025Issued: 2025-12-01Updated: 2025-05-14
Faculty
Fachbereich Chemie
Publisher
Philipps-Universität Marburg
Language
eng
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DoctoralThesis
DFG-subjects
NRPS engineeringnonribosomal peptidespyrrolizidine alkaloids
DDC-Numbers
540
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Effert, Juliana (M.Sc.): Biosynthesis of bacterial pyrrolizidine alkaloids and additional nonribosomal peptides. : Philipps-Universität Marburg 2025-12-01. DOI: https://doi.org/10.17192/z2025.0117.
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