Biology subjects

Bozhueyuek, K. A. J.

Publications and source records attributed to Bozhueyuek, K. A. J..

9 recordsLinked to original sources

bioRxiv2026

TopFor: Automated Preparation of AMBER-Compatible Parameters for Noncanonical Amino Acids in Peptides

Peptides containing noncanonical amino acids (ncAAs) are becoming increasingly important in drug discovery, but their molecular dynamics (MD) simulation remains limited by labor-intensive residue parameterization and topology preparation. Here, we present TopFor (Topology and Residue Force-Field Parameter Preparation), an open-source, peptide-oriented workflow that automates the generation of ncAA residue templates and force-field parameter files for MD simulations with AMBER using established charge-derivation methods. TopFor supports single-residue, whole-peptide, and batch parameterization workflows. The workflow was validated using the MDM2-p53 complex by comparing native AMBER residue definitions with residues processed by TopFor using three charge-derivation methods, each evaluated in three independent 100 ns MD simulations. TopFor-generated residue definitions showed structural and conformational behavior comparable to the native AMBER representation. Applicability to more complex peptide architectures was demonstrated using the head-to-tail cyclic peptide cyclosporin A and the cyclic depsipeptide cyclohexylgriselimycin. At library scale, 1,138 of 1,214 cyclohexylgriselimycin derivatives (93.7%) successfully completed TopFor parameterization and the subsequent initial AMBER-based computational workflow across two derivatization campaigns. TopFor was further integrated into a structure-based peptide discovery workflow based on the DnaN-griselimycin complex. Six GM-derived peptides were computationally prioritized and experimentally evaluated, of which four produced SPR responses against Mycobacterium smegmatis (M. smegmatis) DnaN and three showed concentration-dependent responses. TopFor reduces the manual effort required to prepare ncAA-containing peptides for MD simulations and enables their integration into both library-scale computational screening and structure-based peptide discovery workflows. Keywords: noncanonical amino acids; peptide parameterization; molecular dynamics; AMBER; AmberTools; peptide design; high-throughput virtual screening

bioinformatics↗

bioRxiv2026

Generative AI designs functional thiolation domains for reprogramming non-ribosomal peptide synthetases

Large language models and generative protein design promise to accelerate biotechnology, but it remains unclear whether they can engineer dynamic megasynth(et)ases whose activity depends on transient, context-specific domain interfaces. Non-ribosomal peptide synthetases (NRPSs) are an especially demanding target, yet a high-value one because they produce many clinically important natural products and offer a route to analogs that are often difficult or impractical to access by chemical synthesis. Here we integrate pretrained generative models (ESM3, ProteinMPNN and EvoDiff) with design-build-test-learn cycles and data-guided prioritization to generate 76 de novo thiolation (T) domains. We built and tested 578 recombinant NRPS variants in vivo spanning minimal, full-length and hybrid assembly lines. AI-designed T-domains supported product formation across architectures, enabled catalytically active hybrids at recombined junctions and increased yields by up to [~]3-fold relative to NRPSs carrying the native T-domain. A representative design showed improved soluble expression, refolding, and a 12 {degrees}C higher melting temperature, while molecular dynamics simulations indicated preserved global stability but reshaped, state-dependent interdomain contact networks. Together, these results establish generative design as an effective route to context-conditioned optimization and reprogramming of biosynthetic assembly lines.

synthetic biology↗

bioRxiv2025

Split inteins for generating combinatorial non-ribosomal peptide libraries

Engineering Non-Ribosomal Peptide Synthetases (NRPS) is a promising strategy for discovering new bioactive compounds, which can serve as valuable leads for drug development, such as new antibiotics. However, their engineering is hampered by the limited availability of molecular tools for the efficient heterologous expression of their large biosynthetic gene clusters. In fact, a single NRPS gene can already exceed the size limits of standard cloning vectors. In this study, we establish split inteins as a novel tool for NRPS engineering to enable the expression of single, covalently linked NRPS proteins from multiple plasmids and to perform cloning-free module swapping. Using the xenotetrapeptide synthetase as model system, we show that an NRPS can be split into three parts and reconstituted via trans-splicing using two orthogonal inteins at four different engineering sites. Based on this tripartite platform we build a library comprising 21 plasmids and generated 324 hybrid NRPS by combinatorial transformation. More than half were catalytically active, producing over 200 novel peptides. This intein-based technology provides a modular platform for generating natural product-like peptide libraries, expanding biocatalytically accessible chemical space.

synthetic biology↗

bioRxiv2023

Guidelines for Optimizing Type S Non-Ribosomal Peptide Synthetases

Bacterial biosynthetic assembly lines, such as non-ribosomal peptide synthetases (NRPS) and polyketide synthases, are often subject of synthetic biology - because they produce a variety of natural products invaluable for modern pharmacotherapy. Acquiring the ability to engineer these biosynthetic assembly lines allows the production of artificial non-ribosomal peptides (NRP), polyketides, and hybrids thereof with new or improved properties. However, traditional bioengineering approaches have suffered for decades from their very limited applicability and, unlike combinatorial chemistry, are stigmatized as inefficient because they cannot be linked to the high-throughput screening platforms of the pharmaceutical industry. Although combinatorial chemistry can generate new molecules cheaper, faster, and in greater numbers than traditional natural product discovery and bioengineering approaches, it does not meet current medical needs because it covers only a limited biologically relevant chemical space. Hence, methods for high-throughput generation of new natural product-like compound libraries could provide a new avenue towards the identification of new lead compounds. To this end, prior to this work, we introduced an artificial synthetic NRPS type, referred to as type S NRPS, to provide a first-of-its-kind bicombinatorial approach to parallelized high-throughput NRP library generation. However, a bottleneck of these first two generations of type S NRPS was a significant drop in production yields. To address this issue, we applied an iterative optimization process that enabled titer increases of up to 55-fold compared to the non-optimized equivalents, restoring them to wild-type levels and beyond.

synthetic biology↗

bioRxiv2022

Evolutionary Inspired Engineering of Megasynthetases

Many clinically used drugs are derived from or inspired by bacterial natural products that often are biosynthesised via non-ribosomal peptide synthetases (NRPS), giant megasynthases that activate and join individual amino acids in an assembly line fashion. Since NRPS are not limited to the incorporation of the 20 proteinogenic amino acids, their efficient manipulation would allow the biotechnological generation of complex peptides including linear, cyclic and further modified natural product analogues, e.g. to optimise natural product leads. Here we describe a detailed phylogenetic analysis of several bacterial NRPS that led to the identification of a new recombination breakpoint within the thiolation (T) domain that is important for natural NRPS evolution. From this, an evolution-inspired eXchange Unit between T domains (XUT) approach was developed which allows the assembly of NRPS fragments over a broad range of GC contents, protein similarities, and extender unit specificities, as demonstrated for the specific production of a proteasome inhibitor designed and assembled from five different NRPS fragments.

synthetic biology↗

bioRxiv2021

Type S Non Ribosomal Peptide Synthetases for the rapid generation of tailor-made peptide libraries

Bacterial natural products in general, and non-ribosomally synthesized peptides in particular, are structurally diverse and provide us with a broad range of pharmaceutically relevant bioactivities. Yet, traditional natural product research suffers from rediscovering the same scaffolds and has been stigmatized as inefficient, time-, labour- and cost-intensive. Combinatorial chemistry, on the other hand, can produce new molecules in greater numbers, cheaper and in less time than traditional natural product discovery, but also fails to meet current medical needs due to the limited biologically relevant chemical space that can be addressed. Consequently, methods for the high throughput generation of new-to-nature natural products would offer a new approach to identifying novel bioactive chemical entities for the hit to lead phase of drug discovery programms. As a follow-up to our previously published proof-of-principle study on generating bipartite type S non-ribosomal peptide synthetases (NRPSs), we now envisaged the de novo generation of non-ribosomal peptides (NRPs) on an unreached scale. Using synthetic zippers, we split NRPS in up to three subunits and rapidly generated different bi- and tripartite NRPS libraries to produce 49 peptides, peptide derivatives, and de novo peptides at good titres up to 145 mgL-1. A further advantage of type S NRPSs not only is the possibility to easily expand the created libraries by re-using previously created type S NRPS, but that functions of individual domains as well as domain-domain interactions can be studied and assigned rapidly.

bioengineering↗

bioRxiv2021

Influence of condensation domains on activity and specificity of adenylation domains

Many clinically used natural products are produced by non-ribosomal peptide synthetases (NRPSs), which due to their modular nature should be accessible to modification and engineering approaches. While the adenylation domain (A) plays the key role in substrate recognition and activation, the condensation domain (C) which is responsible for substrate linkage and stereochemical filtering recently became the subject of debate - with its attributed role as a "gatekeeper" being called into question. Since we have thoroughly investigated different combinations of C-A didomains in a series of in vitro, in vivo, and in situ experiments suggesting an important role to the C-A interface for the activity and specificity of the downstream A domain and not the C domain as such, we would like to contribute to this discussion. The role of the C-A interface, termed extended gatekeeping, due to structural features of the C domains, can also be transferred to other NRPSs by engineering, was finally investigated and characterised in an in silico approach on 30 wild-type and recombinant C-A interfaces. With these data, we not only would like to offer a new perspective on the specificity of C domains, but also to revise our previously established NRPS engineering and construction rules.

bioengineering↗

bioRxiv2020

Synthetic Zippers as an Enabling Tool for Engineering of Non-Ribosomal Peptide Synthetases

Non-ribosomal peptide synthetases (NRPSs) are the origin of a wide range of natural products, including many clinically used drugs. Engineering of these often giant biosynthetic machineries to produce novel non-ribosomal peptides (NRPs) at high titre is an ongoing challenge. Here we describe a strategy to functionally combine NRPS fragments of Gram-negative and -positive origin, synthesising novel peptides at titres up to 290 mg l-1. Extending from the recently introduced definition of eXchange Units (XUs), we inserted synthetic zippers (SZs) to split single protein NRPSs into up to three independently expressed and translated polypeptide chains. These synthetic type of NRPS (type S) enables easier access to engineering, overcomes cloning limitations, and provides a simple and rapid approach to building peptide libraries via the combination of different NRPS subunits. One Sentence SummaryDivide and Conquer: A molecular tool kit to reprogram the biosynthesis of non-ribosomal peptides.

synthetic biology↗

bioRxiv2018

Modification and de novo design of non-ribosomal peptide synthetases (NRPS) using specific assembly points within condensation domains

Many important natural products are produced by non-ribosomal peptide synthetases (NRPSs) 1.These giant enzyme machines activate amino acids in an assembly line fashion in which a set of catalytically active domains is responsible for the section, activation, covalent binding and connection of a specific amino acid to the growing peptide chain 1,2. Since NRPS are not restricted to the incorporation of the 20 proteinogenic amino acids, their efficient manipulation would give access to a diverse range of peptides available biotechnologically. Here we describe a new fusion point inside condensation (C) domains of NRPSs that enables the efficient production of peptides, even containing non-natural amino acids, in yields higher than 280 mg/L. The technology called eXchange Unit 2.0 (XU2.0) also allows the generation of targeted peptide libraries and therefore might be suitable for the future identification of bioactive peptide derivatives for pharmaceutical and other applications.

synthetic biology↗