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Establishing new tools for engineering nonribosomal peptide synthetases

Year: 2025
Type: article
Abstract: Natural products derived from bacteria and fungi are of great interest due to them having a range of bioactivities leading to an important role as therapeutics. One important class of natural products are nonribosomal peptides (NRPs) which are biosynthesized by modular megaenzymes referred to as nonribosomal peptide synthetases (NRPS). NRPS consist of a series of modules, which incorporate different amino acid building blocks into a peptide chain before it is released to produce linear or cyclic peptides. In their most basic form modules consist of an adenylation (A) domain, thiolation (T) domain and a condensation (C) domain. The A domain is responsible for selecting the amino acid substrate, while the role of the T domain is to transfer the activated substrates between domains while bound as a thioester and the C domain is responsible for the formation of the peptide bond between the substrates incorporated by the NRPS. In order for the peptide to be released from the assembly line a release mechanism is employed either by cyclisation of hydrolysis, which is often catalysed by a thioesterase (TE) domain. One key reason that NRPS are responsible for the production of bioactive compounds is that they can incorporate a variety of different substrates as well being able to modify these by either Cis or Trans acting enzymes, where modifications such as N-methylations, cyclisations, heterocylisations, epimerisations and acylations can commonly be found to have been implemented in the final NRP structure. Due to their modular structure NRPS are considered a potential biotechnological method for producing peptide-based compounds of interest. The emergence of synthetic biology strategies has offered the potential for engineering these megasynthetases in order to produce tailor made peptide based products in a more sustainable way to traditional chemical synthesis techniques. In recent years engineering strategies of NRPS have advanced significantly in particular due engineering concepts such as the eXchange Units (XU), eXchange Unit condensation domain (XUC) and eXchange Units between the T domains (XUT) concepts. In this work these concepts were further built upon with the use of split inteins in order to develop combinatorial libraries of hybrid NRPS and thus produce a library of novel NRPs. Initially the xenotetrapeptide synthetase (XtpS) was taken as a model system,Summary 11 which produces a cyclic tetratpeptide referred to as xenotetrapeptide (XTP). XtpS was split at the XUTI, XUTII, XUC and XU engineering sites in a single split system, using five different pairs of split inteins and assessed for whether it could be reconstituted and produce cyclic XTP. The amount of cyclic XTP was then compared to that of the wild type (WT) XtpS and assessed for its compatibility with the use of each pair of split inteins to reconstitute the production of cyclic XTP. After it was established that split inteins were functional in a NRPS, a tripartite system was established. In a tripartite system the NRPS is split into three separate subunits and two separate pairs of orthogonal inteins must be used to reconstitute the protein. The tripartite system was established again for all engineering sites, with the overall goal being to use this system to build a combinatorial library of hybrid NRPS. A hybrid NRPS library was then established whereby 324 combinations of chimeric NRPS were evaluated for their production of different NRPs. Over 50 % of the combinations tested were successful in producing the predicted NRP, establishing this split intein methodology as an additional tool kit to the NRPS engineering arsenal. The second topic described in this work describes a newly found protein-protein interaction occurring in NRPS. It was shown that when NRPS are split into separate subunits at the XUC fusion site it was observed that natural docking domains or artificial docking domains are not required in order to re-establish production of the NRP. This was tested for two separate NRPS, being XtpS and the GameXPeptide synthetase (GxpS). In addition, it was further demonstrated that even chimeric NRPS could be assembled in a tripartite system using this split C and dual C/E methodology. However, when comparing th tripartite split XtpS system production of cyclic XTP to the single split and wild type XtpS a decrease in production was observed. A lack of orthogonality was observed between dual split C/E and split LCL domains, which is thought to be a reason for the diminished titer of the final NRP. Finally, a di-modular system using the XUC split method was established so that the interaction between the C domain lobes could be studied further by gaining structural insights. Through a collaboration with the lab of Prof. Dr. Michael D. Burkart at the University of California a cross-linked di-modular GxpS system has been established. The Burkart lab have pioneered a NRPS cross-linking methodology for Cryo-EM analysis of NRPS and now the aim is to obtain structural insights into the di-modular GxpS system.Summary 12 In the final section of this thesis a prodrug mechanism was developed for inhibiting the growth of an E. coli strain expressing clbP. clbP is a gene encoding for a periplasmic peptidase, ClbP, which is involved in cleaving the inactive precursor of colibactin referred to as precolibactin. Colibactin has been linked to colon cancer and so it is of interest in order to develop methodologies for preventing its production. Precolibactin is produced by a NRPS-PKS hybrid similarly to that of prexenocoumacin A (PXCN-A). PXCN-A is a precursor compound to xenocoumacin I, which has been shown to have antibiotic activity against both Gram-positive and Gram-negative strains. In this work PXCN-A was shown that when fed to an E. coli strain expressing clbP the growth of the strain could be inhibited. Furthermore, it was tested if PXCN-A could be specific to inhibiting the growth of strains expressing clbP in a mixed population of E. coli strains. In this part of the work a flow cytometry workflow was utilized to show that only strain expressing clbP had their growth inhibited in a mix population when PXCN-A was fed to a co-culture.
Source: data_UMR
DOI: 10.17192/z2025.0521
Language: English
Subfield: Pharmacology
Field: Medicine
Open Access status: green
OpenAlex ID: W7124142358
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