Author
Kenan A. J. Bozhueyuek
Other affiliations: Max Planck Society, Merck & Co.
Bio: Kenan A. J. Bozhueyuek is an academic researcher from Goethe University Frankfurt. The author has contributed to research in topics: Peptide & Amino acid. The author has an hindex of 2, co-authored 6 publications receiving 65 citations. Previous affiliations of Kenan A. J. Bozhueyuek include Max Planck Society & Merck & Co..
Papers
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TL;DR: A new fusion point inside the condensation domains of NRPSs is described that results in the development of the exchange unit condensation domain (XUC) concept, which enables the efficient production of peptides, even containing non-natural amino acids, in yields up to 280 mg l−1.
Abstract: Non-ribosomal peptide synthetases (NRPSs) are giant enzyme machines that activate amino acids in an assembly line fashion. As NRPSs are not restricted to the incorporation of the 20 proteinogenic amino acids, their efficient manipulation would enable microbial production of a diverse range of peptides; however, the structural requirements for reprogramming NRPSs to facilitate the production of new peptides are not clear. Here we describe a new fusion point inside the condensation domains of NRPSs that results in the development of the exchange unit condensation domain (XUC) concept, which enables the efficient production of peptides, even containing non-natural amino acids, in yields up to 280 mg l−1. This allows the generation of more specific NRPSs, reducing the number of unwanted peptide derivatives, but also the generation of peptide libraries. The XUC might therefore be suitable for the future optimization of peptide production and the identification of bioactive peptide derivatives for pharmaceutical and other applications. Non-ribosomal peptide synthetases have now been modified and de novo non-ribosomal peptide synthetases constructed using new assembly points within condensation domains. This approach enabled the production of new-to-nature peptides, including some carrying synthetic amino acids, as well as the generation of peptide libraries.
104 citations
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TL;DR: This work describes a strategy to functionally combine NRPS fragments of Gram-negative and -positive origin, synthesising novel peptides at titres up to 290 mg l-1, and inserts synthetic zippers to split single protein NRPSs into up to three independently expressed and translated polypeptide chains.
Abstract: 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 Summary Divide and Conquer: A molecular tool kit to reprogram the biosynthesis of non-ribosomal peptides.
15 citations
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TL;DR: A new fusion point inside condensation (C) domains of NRPSs is described that enables the efficient production of peptides, even containing non-natural amino acids, in yields higher than 280 mg/L.
Abstract: Many important natural products are produced by non-ribosomal peptide synthetases (NRPSs). 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. 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.
4 citations
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06 Jul 2016TL;DR: In this paper, a method for the modification and/or custom-made design of artificial non-ribosomal peptide synthetases (NRPSs) from naturally available NRPSs is presented.
Abstract: The present invention concerns a novel method for the modification and/or custom-made design of artificial non-ribosomal peptide synthetases (NRPSs) from naturally available NRPSs. The artificial NRPSs are of predetermined length and amino acid composition and sequence. Via fusion of well-defined NRPS units (so-called "exchange units") in a certain manner, using a specific sequence motif in the linker areas it is possible to construct artificial and/or modified NRPS assembly lines, which have the ability of synthesizing peptides of a desired structure.
1 citations
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TL;DR: In this article, the authors summarize recent technological developments that are enabling natural product-based drug discovery, highlight selected applications and discuss key opportunities, and discuss the potential of using natural products as drug leads.
Abstract: Natural products and their structural analogues have historically made a major contribution to pharmacotherapy, especially for cancer and infectious diseases. Nevertheless, natural products also present challenges for drug discovery, such as technical barriers to screening, isolation, characterization and optimization, which contributed to a decline in their pursuit by the pharmaceutical industry from the 1990s onwards. In recent years, several technological and scientific developments — including improved analytical tools, genome mining and engineering strategies, and microbial culturing advances — are addressing such challenges and opening up new opportunities. Consequently, interest in natural products as drug leads is being revitalized, particularly for tackling antimicrobial resistance. Here, we summarize recent technological developments that are enabling natural product-based drug discovery, highlight selected applications and discuss key opportunities. Natural products have historically made a major contribution to pharmacotherapy, but also present challenges for drug discovery, such as technical barriers to screening, isolation, characterization and optimization. This Review discusses recent technological developments — including improved analytical tools, genome mining and engineering strategies, and microbial culturing advances — that are enabling a revitalization of natural product-based drug discovery.
1,297 citations
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Saarland University1, University of Parma2, Technical University of Denmark3, University of Giessen4, Pasteur Institute5, University of Lorraine6, Goethe University Frankfurt7, Max Planck Society8, University of Lisbon9, National Museum of Natural History10, Wageningen University and Research Centre11, University of Paris12, John Innes Centre13, University of Manchester14, University of Perugia15, University of Tübingen16, University of Strasbourg17, Jacobs University Bremen18, University Hospital Bonn19, University of Bristol20, Uppsala University21, University of Ljubljana22, Drugs for Neglected Diseases Initiative23, University of Dundee24, Novartis25
TL;DR: In this paper, the authors present a strategic blueprint to substantially improve our ability to discover and develop new antibiotics, and propose both short-term and long-term solutions to overcome the most urgent limitations in the various sectors of research and funding.
Abstract: An ever-increasing demand for novel antimicrobials to treat life-threatening infections caused by the global spread of multidrug-resistant bacterial pathogens stands in stark contrast to the current level of investment in their development, particularly in the fields of natural-product-derived and synthetic small molecules. New agents displaying innovative chemistry and modes of action are desperately needed worldwide to tackle the public health menace posed by antimicrobial resistance. Here, our consortium presents a strategic blueprint to substantially improve our ability to discover and develop new antibiotics. We propose both short-term and long-term solutions to overcome the most urgent limitations in the various sectors of research and funding, aiming to bridge the gap between academic, industrial and political stakeholders, and to unite interdisciplinary expertise in order to efficiently fuel the translational pipeline for the benefit of future generations.
255 citations
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01 Apr 2020TL;DR: The multifaceted use of bacteria as biological factories in diverse applications is discussed and recent advances in targeted genetic engineering of bacteria for the production of valuable bioactive compounds are highlighted.
Abstract: Next to plants, bacteria account for most of the biomass on Earth. They are found everywhere, although certain species thrive only in specific ecological niches. These microorganisms biosynthesize a plethora of both primary and secondary metabolites, defined, respectively, as those required for the growth and maintenance of cellular functions and those not required for survival but offering a selective advantage for the producer under certain conditions. As a result, bacterial fermentation has long been used to manufacture valuable natural products of nutritional, agrochemical and pharmaceutical interest. The interactions of secondary metabolites with their biological targets have been optimized by millions of years of evolution and they are, thus, considered to be privileged chemical structures, not only for drug discovery. During the last two decades, functional genomics has allowed for an in-depth understanding of the underlying biosynthetic logic of secondary metabolites. This has, in turn, paved the way for the unprecedented use of bacteria as programmable biochemical workhorses. In this Review, we discuss the multifaceted use of bacteria as biological factories in diverse applications and highlight recent advances in targeted genetic engineering of bacteria for the production of valuable bioactive compounds. Emphasis is on current advances to access nature’s abundance of natural products. This Review highlights bacteria as biological factories in diverse applications, with an emphasis on targeted genetic engineering for the production of bioactive natural products.
83 citations
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TL;DR: In this article, a review of known evolutionary mechanisms underlying the overwhelming chemical diversity of bacterial secondary metabolism, focusing on enzyme promiscuity and the evolution of enzymatic domains that enable metabolic traits is presented.
83 citations
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TL;DR: The structures and small-angle x-ray scattering showNRPSs undergo very large conformational changes and challenge the general assumption that NRPSs have regular higher-order architecture, as well as direct coupling analyses used to confirm the biological relevance and evolutionary conservation of observed interdomain interfaces.
Abstract: Nonribosomal peptide synthetases (NRPSs) are biosynthetic enzymes that synthesize natural product therapeutics using a modular synthetic logic, whereby each module adds one aminoacyl substrate to the nascent peptide. We have determined five x-ray crystal structures of large constructs of the NRPS linear gramicidin synthetase, including a structure of a full core dimodule in conformations organized for the condensation reaction and intermodular peptidyl substrate delivery. The structures reveal differences in the relative positions of adjacent modules, which are not strictly coupled to the catalytic cycle and are consistent with small-angle x-ray scattering data. The structures and covariation analysis of homologs allowed us to create mutants that improve the yield of a peptide from a module-swapped dimodular NRPS.
82 citations