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Max J Cryle - One of the best experts on this subject based on the ideXlab platform.

  • Understanding condensation domain selectivity in Non-Ribosomal Peptide biosynthesis: structural characterization of the acceptor bound state
    2021
    Co-Authors: Max J Cryle, Robert J A Goode, Ralf B Schittenhelm, Thierry Izoré, Joe A. Kaczmarski, Athina Gavriilidou, Ka Chow, David Steer, Julien Tailhades
    Abstract:

    Abstract Non-Ribosomal Peptide synthetases are important enzymes for the assembly of complex Peptide natural products. Within these multi-modular assembly lines, condensation domains perform the central function of chain assembly, typically by forming a Peptide bond between two peptidyl carrier protein (PCP)-bound substrates. In this work, we report the first structural snapshots of a condensation domain in complex with an aminoacyl-PCP acceptor substrate. These structures allow the identification of a mechanism that controls access of acceptor substrates to the active site in condensation domains. The structures of this previously uncharacterized complex also allow us to demonstrate that condensation domain active sites do not contain a distinct pocket to select the side chain of the acceptor substrate during Peptide assembly but that residues within the active site motif can instead serve to tune the selectivity of these central biosynthetic domains.

  • Exploring modular reengineering strategies to redesign the teicoplanin Non-Ribosomal Peptide synthetase
    Chemical Science, 2020
    Co-Authors: Milda Kaniusaite, Robert J A Goode, Julien Tailhades, Ralf B Schittenhelm, Max J Cryle
    Abstract:

    Non-Ribosomal Peptide synthesis is an important biosynthesis pathway in secondary metabolism. In this study we have investigated modularisation and redesign strategies for the glycoPeptide antibiotic teicoplanin. Using the relocation or exchange of domains within the NRPS modules, we have identified how to initiate Peptide biosynthesis and explored the requirements for the functional reengineering of both the condensation/adenylation domain and epimerisation/condensation domain interfaces. We have also demonstrated strategies that ensure communication between isolated NRPS modules, leading to new Peptide assembly pathways. This provides important insights into NRPS reengineering of glycoPeptide antibiotic biosynthesis and has broad implications for the redesign of other NRPS systems.

  • Diversity of nature's assembly lines – recent discoveries in Non-Ribosomal Peptide synthesis
    Molecular bioSystems, 2016
    Co-Authors: Jennifer A.e. Payne, Melanie Schoppet, Mathias Henning Hansen, Max J Cryle
    Abstract:

    The biosynthesis of complex natural products by Non-Ribosomal Peptide synthetases (NRPSs) and the related polyketide synthases (PKSs) represents a major source of important bioactive compounds. These large, multi-domain machineries are able to produce a fascinating range of molecules due to the nature of their modular architectures, which allows natural products to be assembled and tailored in a modular, step-wise fashion. In recent years there has been significant progress in characterising the important domains and underlying mechanisms of Non-Ribosomal Peptide synthesis. More significantly, several studies have uncovered important examples of novel activity in many NRPS domains. These discoveries not only greatly increase the structural diversity of the possible products of NRPS machineries but - possibly more importantly - they improve our understanding of what is a highly important, yet complex, biosynthetic apparatus. In this review, several recent examples of novel NRPS function will be introduced, which highlight the range of previously uncharacterised activities that have now been detected in the biosynthesis of important natural products by these mega-enzyme synthetases.

  • understanding the crucial interactions between cytochrome p450s and non ribosomal Peptide synthetases during glycoPeptide antibiotic biosynthesis
    Current Opinion in Structural Biology, 2016
    Co-Authors: Madeleine Peschke, Max J Cryle, Melanie Gonsior, Roderich D. Süssmuth
    Abstract:

    The importance of Cytochrome P450-catalyzed modifications of natural products produced by Non-Ribosomal Peptide synthetase machineries is most apparent during glycoPeptide antibiotic biosynthesis: specifically, the formation of essential amino acid side chains crosslinks in the Peptide backbone of these clinically relevant antibiotics. These cyclization reactions take place whilst the Peptide substrate remains bound to the Non-Ribosomal Peptide synthetase in a process mediated by a conserved domain of previously unknown function — the X-domain. This review addresses recent advances in understanding P450 recruitment to Non-Ribosomal Peptide synthetase-bound substrates and highlights the importance of both carrier proteins and the X-domain in different P450-catalyzed reactions.

  • Probing the selectivity of β-hydroxylation reactions in Non-Ribosomal Peptide synthesis using analytical ultracentrifugation
    Analytical biochemistry, 2015
    Co-Authors: Bashkim Kokona, Max J Cryle, Emily S. Winesett, A. Nikolai Von Krusenstiern, Robert Fairman, Louise K. Charkoudian
    Abstract:

    Bacteria and fungi use Non-Ribosomal Peptide synthetases (NRPSs) to produce Peptides of broad structural diversity and biological activity, many of which have proven to be of great importance for human health. The impressive diversity of Non-Ribosomal Peptides originates in part from the action of tailoring enzymes that modify the structures of single amino acids and/or the mature Peptide. Studying the interplay between tailoring enzymes and the peptidyl carrier proteins (PCPs) that anchor the substrates is challenging owing to the transient and complex nature of the protein-protein interactions. Using sedimentation velocity (SV) methods, we studied the collaboration between the PCPs and cytochrome P450 enzyme that results in the installation of β-hydroxylated amino acid precursors in the biosynthesis of the depsiPeptide skyllamycin. We show that SV methods developed for the analytical ultracentrifuge are ideally suited for a quantitative exploration of PCP-enzyme equilibrium interactions. Our results suggest that the PCP itself and the presence of substrate covalently tethered to the PCP together facilitate productive PCP-P450 interactions, thereby revealing one of nature's intricate strategies for installing interesting functionalities using natural product synthetases.

Jens Nielsen - One of the best experts on this subject based on the ideXlab platform.

  • Implementation of communication-mediating domains for Non-Ribosomal Peptide production in Saccharomyces cerevisiae.
    Biotechnology and bioengineering, 2010
    Co-Authors: Verena Siewers, Rita San-bento, Jens Nielsen
    Abstract:

    Saccharomyces cerevisiae has in several cases been proven to be a suitable host for the production of natural products and was recently exploited for the production of Non-Ribosomal Peptides. Synthesis of Non-Ribosomal Peptides (NRPs) is mediated by NRP synthetases (NRPSs), modular enzymes, which are often organized in enzyme complexes. In these complexes, partner NRPSs interact via communication-mediating domains (COM domains). In order to test whether functional interaction between separate NRPS modules is possible in yeast we constructed a yeast strain expressing two modules with compatible COM domains from two plasmids. Successful production as well as secretion of the expected diPeptide was detected. This opens the possibility of using yeast as a eukaryotic platform for fast assessment of new module combinations for the development of novel NRP compounds.

  • Heterologous production of Non-Ribosomal Peptide LLD-ACV in Saccharomyces cerevisiae
    Metabolic engineering, 2009
    Co-Authors: Verena Siewers, Xiao Chen, Le Huang, Jie Zhang, Jens Nielsen
    Abstract:

    Non-Ribosomal Peptides (NRPs) are a diverse family of secondary metabolites with a broad range of biological activities. We started to develop an eukaryotic microbial platform based on the yeast Saccharomyces cerevisiae for heterologous production of NRPs using delta-(L-alpha-aminoadipyl)-L-cysteinyl-D- valine (ACV) as a model NRP. The Penicillium chrysogenum gene pcbAB encoding ACV synthetase was expressed in S. cerevisiae from a high-copy plasmid together with phosphopantetheinyl transferase (PPTase) encoding genes from Aspergillus nidulans, P. chrysogenum and Bacillus subtilis, and in all the three cases production of ACV was observed. To improve ACV synthesis, several factors were investigated. Codon optimization of the 50 end of pcbAB did not significantly increase ACV production. However, a 30-fold enhancement was achieved by lowering the cultivation temperature from 30 to 20 degrees C. When ACVS and PPT a seen coding genes were integrated into the yeast genome, a 6-fold decrease in ACV production was observed indicating that gene copy number was one of the rate-limiting factors for ACV production in yeast. (C) 2009 Elsevier Inc. All rights reserved.

David F. Ackerley - One of the best experts on this subject based on the ideXlab platform.

  • Efficient rational modification of Non-Ribosomal Peptides by adenylation domain substitution.
    Nature communications, 2020
    Co-Authors: Mark J. Calcott, Jeremy G. Owen, David F. Ackerley
    Abstract:

    Non-Ribosomal Peptide synthetase (NRPS) enzymes form modular assembly-lines, wherein each module governs the incorporation of a specific monomer into a short Peptide product. Modules are comprised of one or more key domains, including adenylation (A) domains, which recognise and activate the monomer substrate; condensation (C) domains, which catalyse amide bond formation; and thiolation (T) domains, which shuttle reaction intermediates between catalytic domains. This arrangement offers prospects for rational Peptide modification via substitution of substrate-specifying domains. For over 20 years, it has been considered that C domains play key roles in proof-reading the substrate; a presumption that has greatly complicated rational NRPS redesign. Here we present evidence from both directed and natural evolution studies that any substrate-specifying role for C domains is likely to be the exception rather than the rule, and that novel Non-Ribosomal Peptides can be generated by substitution of A domains alone. We identify permissive A domain recombination boundaries and show that these allow us to efficiently generate modified pyoverdine Peptides at high yields. We further demonstrate the transferability of our approach in the PheATE-ProCAT model system originally used to infer C domain substrate specificity, generating modified diPeptide products at yields that are inconsistent with the prevailing dogma. Non-Ribosomal Peptide synthases are multimodular enzymes comprised of adenylation (A), condensation (C) and thiolation domains. Here, the authors show that Non-Ribosomal Peptides can be generated solely by A domain substitutions, providing evidence that the postulated substrate specifying role of C-domains may be rare in nature.

  • Structural, functional and evolutionary perspectives on effective re-engineering of Non-Ribosomal Peptide synthetase assembly lines.
    Natural product reports, 2018
    Co-Authors: Alistair S. Brown, Mark J. Calcott, Jeremy G. Owen, David F. Ackerley
    Abstract:

    Covering: up to May 2018 Non-Ribosomal Peptide synthetases (NRPSs) are mega-enzymes that form modular templates to assemble specific Peptide products, independent of the ribosome. The autonomous nature of the modules in the template offers prospects for re-engineering NRPS enzymes to generate modified Peptide products. Although this has clearly been a primary mechanism of natural product diversification throughout evolution, equivalent strategies have proven challenging to implement in the laboratory. In this review we examine key examples of successful and less-successful re-engineering of NRPS templates to generate novel Peptides, with the aim of extracting practical guidelines to inform future efforts. We emphasise the importance of maintaining effective protein–protein interactions in recombinant NRPS templates, and identify strengths and limitations of diverse strategies for achieving different engineering outcomes.

  • portability of the thiolation domain in recombinant pyoverdine non ribosomal Peptide synthetases
    BMC Microbiology, 2015
    Co-Authors: Mark J. Calcott, David F. Ackerley
    Abstract:

    Background Non-Ribosomal Peptide synthetase (NRPS) enzymes govern the assembly of amino acids and related monomers into Peptide-like natural products. A key goal of the field is to develop methods to effective recombine NRPS domains or modules, and thereby generate modified or entirely novel products. We previously showed that substitution of the condensation (C) and adenylation (A) domains in module 2 of the pyoverdine synthetase PvdD from Pseudomonas aeruginosa led to synthesis of modified pyoverdines in a minority of cases, but that more often the recombinant enzymes were non-functional. One possible explanation was that the majority of introduced C domains were unable to effectively communicate with the thiolation (T) domain immediately upstream, in the first module of PvdD.

  • Genetic manipulation of Non-Ribosomal Peptide synthetases to generate novel bioactive Peptide products
    Biotechnology Letters, 2014
    Co-Authors: Mark J. Calcott, David F. Ackerley
    Abstract:

    Non-Ribosomal Peptide synthetases (NRPS) are large modular enzymes that govern the synthesis of numerous biotechnologically relevant products. Their mode of action is frequently compared to an assembly line, in which each module acts in a semi-autonomous but coordinated manner to add a specific monomer to a growing Peptide chain, unfettered by ribosomal constraints. The modular nature of these systems offers tantalising prospects for synthetic biology, wherein the assembly line is re-engineered at a genetic level to generate a specific or combinatorial modified product. However, despite some success stories, a "one size fits all" approach to NRPS synthetic biology remains elusive. This review examines both rational and random mutagenesis strategies that have been employed to modify NRPS function, in an attempt to highlight key points that should be considered when seeking to re-engineer an NRPS biosynthetic template.

Gregory L. Challis - One of the best experts on this subject based on the ideXlab platform.

  • the insect pathogen serratia marcescens db10 uses a hybrid non ribosomal Peptide synthetase polyketide synthase to produce the antibiotic althiomycin
    PLOS ONE, 2012
    Co-Authors: Amy J Gerc, Gregory L. Challis, Lijiang Song, Nicola R Stanleywall, Sarah J Coulthurst
    Abstract:

    There is a continuing need to discover new bioactive natural products, such as antibiotics, in genetically-amenable micro-organisms. We observed that the enteric insect pathogen, Serratia marcescens Db10, produced a diffusible compound that inhibited the growth of Bacillis subtilis and Staphyloccocus aureus. Mapping the genetic locus required for this activity revealed a putative natural product biosynthetic gene cluster, further defined to a six-gene operon named alb1-alb6. Bioinformatic analysis of the proteins encoded by alb1-6 predicted a hybrid Non-Ribosomal Peptide synthetase-polyketide synthase (NRPS-PKS) assembly line (Alb4/5/6), tailoring enzymes (Alb2/3) and an export/resistance protein (Alb1), and suggested that the machinery assembled althiomycin or a related molecule. Althiomycin is a ribosome-inhibiting antibiotic whose biosynthetic machinery had been elusive for decades. Chromatographic and spectroscopic analyses confirmed that wild type S. marcescens produced althiomycin and that production was eliminated on disruption of the alb gene cluster. Construction of mutants with in-frame deletions of specific alb genes demonstrated that Alb2-Alb5 were essential for althiomycin production, whereas Alb6 was required for maximal production of the antibiotic. A phosphopantetheinyl transferase enzyme required for althiomycin biosynthesis was also identified. Expression of Alb1, a predicted major facilitator superfamily efflux pump, conferred althiomycin resistance on another, sensitive, strain of S. marcescens. This is the first report of althiomycin production outside of the Myxobacteria or Streptomyces and paves the way for future exploitation of the biosynthetic machinery, since S. marcescens represents a convenient and tractable producing organism.

  • mbth like protein mediated cross talk between non ribosomal Peptide antibiotic and siderophore biosynthetic pathways in streptomyces coelicolor m145
    Microbiology, 2007
    Co-Authors: Sylvie Lautru, Daniel Ovescostales, Jeanluc Pernodet, Gregory L. Challis
    Abstract:

    MbtH-like proteins are a family of small proteins encoded by genes found in many, but not all, Non-Ribosomal Peptide synthetase-encoding gene clusters that direct the biosynthesis of Peptide antibiotics and siderophores. Studies published to date have not elucidated the function of MbtH-like proteins, nor have they clarified whether they are required for metabolite biosynthesis. Here it is shown that only one of two genes (cdaX or cchK) encoding MbtH-like proteins in Streptomyces coelicolor is required for biosynthesis of the Peptide siderophore coelichelin and the calcium-dependent Peptide antibiotic (CDA). The cdaX and cchK genes can functionally complement each other in trans, suggesting that CdaX and CchK can cross-talk with the coelichelin and CDA biosynthetic pathways, respectively. Transcriptional analyses of wild-type S. coelicolor and a double cchK/cdaX replacement mutant indicate that CchK and CdaX may not be involved in transcriptional regulation of coelichelin and CDA biosynthetic gene clusters.

  • Structural aspects of Non-Ribosomal Peptide biosynthesis
    Current opinion in structural biology, 2004
    Co-Authors: Gregory L. Challis, James H. Naismith
    Abstract:

    Small Peptides have powerful biological activities ranging from antibiotic to immune suppression. These Peptides are synthesized by Non-Ribosomal Peptide synthetases (NRPS). Structural understanding of NRPS took a huge leap forward in 2002; this information has led to several detailed biochemical studies and further structural studies. NRPS are complex molecular machines composed of multiple modules and each module contains several autonomously folded catalytic domains. Structural studies have largely focused on individual domains, isolated from the context of the multienzyme. Biochemical studies have looked at individual domains, isolated whole modules and intact NRPS, and the combined data begin to allow us to visualize the process of Peptide assembly by NRPS.

  • coelichelin a new Peptide siderophore encoded by the streptomyces coelicolor genome structure prediction from the sequence of its non ribosomal Peptide synthetase
    Fems Microbiology Letters, 2000
    Co-Authors: Gregory L. Challis, Jacques Ravel
    Abstract:

    A gene cluster for the Non-Ribosomal synthesis of a Peptide of unknown structure has been identified in the partial genome sequence of Streptomyces coelicolor. Using molecular and computational analyses, the total structure of a triPeptide siderophore synthesized by the Non-Ribosomal Peptide synthetase within the cluster has been deduced from the translated sequence of its encoding gene. This represents a novel method for the structural assignment of natural products from genome sequence data.

Jacques Ravel - One of the best experts on this subject based on the ideXlab platform.