Expanding the Toolbox for Engineering Nonribosomal Peptide Synthetases PDF Download
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Author: Farzaneh Pourmasoumi
Publisher:
ISBN:
Category :
Languages : de
Pages : 0
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Book Description
Nonribosomal peptide synthetases (NRPSs) are an important source of pharmaceutically valuable natural products, including antibiotics. In depth understanding of enzymatic interactions responsible for the biosynthesis of these compounds by NRPSs is essential for re-engineering of these proteins to create structural diversity. In this work, we establish tools that expand the understanding and engineering of nonribosomal peptide synthetases.
Author: Farzaneh Pourmasoumi
Publisher:
ISBN:
Category :
Languages : de
Pages : 0
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Book Description
Nonribosomal peptide synthetases (NRPSs) are an important source of pharmaceutically valuable natural products, including antibiotics. In depth understanding of enzymatic interactions responsible for the biosynthesis of these compounds by NRPSs is essential for re-engineering of these proteins to create structural diversity. In this work, we establish tools that expand the understanding and engineering of nonribosomal peptide synthetases.
Author: Reto Daniel Zwahlen
Publisher:
ISBN: 9789403406732
Category :
Languages : en
Pages :
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Author: Mathew Allen Rude
Publisher:
ISBN:
Category :
Languages : en
Pages : 296
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Author: Philipp Stephan
Publisher:
ISBN:
Category :
Languages : de
Pages : 0
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Over the last decades, discovery rates of new antibiotics have declined while the occurrence of multi-resistant pathogens has increased, leading towards a post-antibiotic era. Many current antibiotics are natural products or derivatives, known for their excellent bioactivities but complex structures that complicate synthetic production. Many antibiotics, like penicillin and vancomycin, are peptides produced by nonribosomal peptide synthetases (NRPS). These multi-modular enzyme complexes function like assembly lines, with each module adding a specific amino acid to the peptide through the biocatalytic activities of distinct domains. Targeted NRPS engineering to alter structures of the produced peptides has faced challenges, but directed evolution of NRPS domains seems to offer a promising solution. This thesis presents a straightforward method for the directed evolution of adenylation (A) domains using LC-MS/MS detection of dipeptides in cell lysates, demonstrating its power by engineering the GrsB1 A domain from gramicidin S biosynthesis to select for L-piperazic acid (Piz) instead of its native substrate L-Pro without reducing enzyme activity. The modified antibiotic, Piz-gramicidin S, showed improved bioactivity. Despite compatibility issues between NRPS domains, kinetic analyses revealed that A domain substrate preferences ultimately determine final product ratios. Additionally, the thesis proposes a novel DNA-based approach for NRPS engineering, using zinc finger domains that bind specific DNA sequences to simplify module rearrangement by making NRPSs DNA-templated. This method offers a more efficient alternative to traditional cloning techniques by replacing the handling of large NRPS genes with much shorter sequences of the zinc finger domains. Overall, this research advances the engineering of NRPSs for developing new antibiotics with better efficacy against resistant pathogens and highlights the usefulness of directed evolution in this context.
Author: Jennifer Fick Brannock
Publisher:
ISBN:
Category :
Languages : en
Pages : 184
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Author: Pawel Jajesniak
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Category :
Languages : en
Pages :
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Author: Sebastian Leonhard Wenski
Publisher:
ISBN:
Category :
Languages : en
Pages : 0
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Complex peptide natural products exhibit diverse biological functions and a wide range of physico-chemical properties. As a result, many peptides have entered the clinics for various applications. Two main routes for the biosynthesis of complex peptides have evolved in nature: ribosomally synthesized and post-translationally modified peptide (RiPP) biosynthetic pathways and non-ribosomal peptide synthetases (NRPSs). Insights into both bioorthogonal peptide biosynthetic strategies led to the establishment of universal principles for each of the two routes. These universal rules can be leveraged for the targeted identification of novel peptide biosynthetic blueprints in genome sequences and used for the rational engineering of biosynthetic pathways to produce non-natural peptides. In this review, we contrast the key principles of both biosynthetic routes and compare the different biochemical strategies to install the most frequently encountered peptide modifications. In addition, the influence of the fundamentally different biosynthetic principles on past, current and future engineering approaches is illustrated. Despite the different biosynthetic principles of both peptide biosynthetic routes, the arsenal of characterized peptide modifications encountered in RiPP and NRPS systems is largely overlapping. The continuous expansion of the biocatalytic toolbox of peptide modifying enzymes for both routes paves the way towards the production of complex tailor-made peptides and opens up the possibility to produce NRPS-derived peptides using the ribosomal route and vice versa.
Author:
Publisher: Academic Press
ISBN: 0323952666
Category : Science
Languages : en
Pages : 394
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Book Description
Chemical Microbiology, Part B, Volume 665, the latest release in the Methods of Enzymology series, highlights new advances in the field, including comprehensive chapters on the Application of Antibiotic-derived Fluorescent Probes to Bacterial Studies, Metabolomic approaches for enzyme function and pathway discovery in bacteria, Adding a diazo-transfer reagent to culture to generate secondary metabolite probes for click chemistry, Customized Peptidoglycan Surfaces to Investigate Innate Immune Recognition via Surface Plasmon Resonance, Development and application of highly sensitive labeling reagents for amino acids, Bacterial Cell Wall Modification with a Glycolipid Substrate, and much more. Provides the authority and expertise of leading contributors from an international board of authors Presents the latest release in the Methods in Enzymology series Updated release includes the latest information on chemical microbiology
Author: Yanyan Li
Publisher: Springer
ISBN: 1493910108
Category : Medical
Languages : en
Pages : 113
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Book Description
Lasso peptides form a growing family of fascinating ribosomally-synthesized and post-translationally modified peptides produced by bacteria. They contain 15 to 24 residues and share a unique interlocked topology that involves an N-terminal 7 to 9-residue macrolactam ring where the C-terminal tail is threaded and irreversibly trapped. The ring results from the condensation of the N-terminal amino group with a side-chain carboxylate of a glutamate at position 8 or 9, or an aspartate at position 7, 8 or 9. The trapping of the tail involves bulky amino acids located in the tail below and above the ring and/or disulfide bridges connecting the ring and the tail. Lasso peptides are subdivided into three subtypes depending on the absence (class II) or presence of one (class III) or two (class I) disulfide bridges. The lasso topology results in highly compact structures that give to lasso peptides an extraordinary stability towards both protease degradation and denaturing conditions. Lasso peptides are generally receptor antagonists, enzyme inhibitors and/or antibacterial or antiviral (anti-HIV) agents. The lasso scaffold and the associated biological activities shown by lasso peptides on different key targets make them promising molecules with high therapeutic potential. Their application in drug design has been exemplified by the development of an integrin antagonist based on a lasso peptide scaffold. The biosynthesis machinery of lasso peptides is therefore of high biotechnological interest, especially since such highly compact and stable structures have to date revealed inaccessible by peptide synthesis. Lasso peptides are produced from a linear precursor LasA, which undergoes a maturation process involving several steps, in particular cleavage of the leader peptide and cyclization. The post-translational modifications are ensured by a dedicated enzymatic machinery, which is composed of an ATP-dependent cysteine protease (LasB) and a lactam synthetase (LasC) that form an enzymatic complex called lasso synthetase. Microcin J25, produced by Escherichia coli AY25, is the archetype of lasso peptides and the most extensively studied. To date only around forty lasso peptides have been isolated, but genome mining approaches have revealed that they are widely distributed among Proteobacteria and Actinobacteria, particularly in Streptomyces, making available a rich resource of novel lasso peptides and enzyme machineries towards lasso topologies.
Author: Kristian Müller
Publisher: Springer Science & Business Media
ISBN: 1597451878
Category : Science
Languages : en
Pages : 318
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Book Description
Protein engineering is a fascinating mixture of molecular biology, protein structure analysis, computation, and biochemistry, with the goal of developing useful or valuable proteins. Protein Engineering Protocols will consider the two general, but not mutually exclusive, strategies for protein engineering. The first is known as rational design, in which the scientist uses detailed knowledge of the structure and function of the protein to make desired changes. The s- ond strategy is known as directed evolution. In this case, random mutagenesis is applied to a protein, and selection or screening is used to pick out variants that have the desired qualities. By several rounds of mutation and selection, this method mimics natural evolution. An additional technique known as DNA shuffling mixes and matches pieces of successful variants to produce better results. This process mimics recombination that occurs naturally during sexual reproduction. The first section of Protein Engineering Protocols describes rational p- tein design strategies, including computational methods, the use of non-natural amino acids to expand the biological alphabet, as well as impressive examples for the generation of proteins with novel characteristics. Although procedures for the introduction of mutations have become routine, predicting and und- standing the effects of these mutations can be very challenging and requires profound knowledge of the system as well as protein structures in general.
