scholarly journals Avant-garde assembly-line biosynthesis expands diversity of cyclic lipodepsipeptide products

2019 ◽  
Author(s):  
Jia Jia Zhang ◽  
Xiaoyu Tang ◽  
Tao Huan ◽  
Avena C. Ross ◽  
Bradley S. Moore
Keyword(s):  
Assembly Line ◽  
Polyketide Synthase ◽  
Structural Features ◽  
Chemical Diversity ◽  
Chain Extension ◽  
Assembly Lines ◽  
Avant Garde ◽  
Peptide Synthetase ◽  
Domain Interactions ◽  
Catalytic Cycles

Modular nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) enzymatic assembly lines are large and dynamic protein machines that generally undergo a linear progression of catalytic cycles via a series of enzymatic domains organized into independent modules. Here we report the heterologous reconstitution and comprehensive characterization of two hybrid NRPS-PKS assembly lines that defy many standard rules of assembly-line biosynthesis to generate a large combinatorial library of cyclic lipodepsipeptide protease inhibitors called thalassospiramides. We generate a series of precise domain-inactivating mutations in thalassospiramide assembly lines and present compelling evidence for an unprecedented biosynthetic model that invokes inter-module substrate activation and tailoring, module skipping, and pass-back chain extension, whereby the ability to pass the growing chain back to a preceding module is flexible and substrate-driven. Expanding bidirectional inter-module domain interactions could represent a viable mechanism for generating chemical diversity without increasing the size of biosynthetic assembly lines and raises new questions regarding our understanding of the structural features of multi-modular megaenzymes.

scholarly journals Thiocysteine lyases as polyketide synthase domains installing hydropersulfide into natural products and a hydropersulfide methyltransferase

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Song Meng ◽  
Andrew D. Steele ◽  
Wei Yan ◽  
Guohui Pan ◽  
Edward Kalkreuter ◽  
...  
Keyword(s):  
Natural Products ◽  
Natural Product ◽  
Assembly Line ◽  
Polyketide Synthase ◽  
Genome Mining ◽  
Assembly Lines ◽  
Biologically Relevant ◽  
Peptide Synthetase ◽  
Adenosyl Methionine

AbstractNature forms S-S bonds by oxidizing two sulfhydryl groups, and no enzyme installing an intact hydropersulfide (-SSH) group into a natural product has been identified to date. The leinamycin (LNM) family of natural products features intact S-S bonds, and previously we reported an SH domain (LnmJ-SH) within the LNM hybrid nonribosomal peptide synthetase (NRPS)-polyketide synthase (PKS) assembly line as a cysteine lyase that plays a role in sulfur incorporation. Here we report the characterization of an S-adenosyl methionine (SAM)-dependent hydropersulfide methyltransferase (GnmP) for guangnanmycin (GNM) biosynthesis, discovery of hydropersulfides as the nascent products of the GNM and LNM hybrid NRPS-PKS assembly lines, and revelation of three SH domains (GnmT-SH, LnmJ-SH, and WsmR-SH) within the GNM, LNM, and weishanmycin (WSM) hybrid NRPS-PKS assembly lines as thiocysteine lyases. Based on these findings, we propose a biosynthetic model for the LNM family of natural products, featuring thiocysteine lyases as PKS domains that directly install a -SSH group into the GNM, LNM, or WSM polyketide scaffold. Genome mining reveals that SH domains are widespread in Nature, extending beyond the LNM family of natural products. The SH domains could also be leveraged as biocatalysts to install an -SSH group into other biologically relevant scaffolds.

Protein–protein interactions in polyketide synthase–nonribosomal peptide synthetase hybrid assembly lines

2018 ◽  
Vol 35 (11) ◽  
pp. 1185-1209 ◽  
Author(s):  
Akimasa Miyanaga ◽  
Fumitaka Kudo ◽  
Tadashi Eguchi
Keyword(s):  
Protein Interactions ◽  
Polyketide Synthase ◽  
Nonribosomal Peptide Synthetase ◽  
Hybrid Assembly ◽  
Protein Protein Interactions ◽  
Assembly Lines ◽  
Nonribosomal Peptide ◽  
Peptide Synthetase

The protein–protein interactions in polyketide synthase–nonribosomal peptide synthetase hybrids are summarized and discussed.

scholarly journals Diversity of Bacterial Biosynthetic Genes in Maritime Antarctica

2020 ◽  
Vol 8 (2) ◽  
pp. 279 ◽  
Author(s):  
Adriana Rego ◽  
António G. G. Sousa ◽  
João P. Santos ◽  
Francisco Pascoal ◽  
João Canário ◽  
...  
Keyword(s):  
Secondary Metabolites ◽  
Polyketide Synthase ◽  
Amplicon Sequencing ◽  
Soil Samples ◽  
Chemical Diversity ◽  
Maritime Antarctica ◽  
Biosynthetic Genes ◽  
Peptide Synthetase ◽  
Drug Leads ◽  
Culture Dependent

Bacterial natural products (NPs) are still a major source of new drug leads. Polyketides (PKs) and non-ribosomal peptides (NRP) are two pharmaceutically important families of NPs and recent studies have revealed Antarctica to harbor endemic polyketide synthase (PKS) and non-ribosomal peptide synthetase (NRPS) genes, likely to be involved in the production of novel metabolites. Despite this, the diversity of secondary metabolites genes in Antarctica is still poorly explored. In this study, a computational bioprospection approach was employed to study the diversity and identity of PKS and NRPS genes to one of the most biodiverse areas in maritime Antarctica—Maxwell Bay. Amplicon sequencing of soil samples targeting ketosynthase (KS) and adenylation (AD) domains of PKS and NRPS genes, respectively, revealed abundant and unexplored chemical diversity in this peninsula. About 20% of AD domain sequences were only distantly related to characterized biosynthetic genes. Several PKS and NRPS genes were found to be closely associated to recently described metabolites including those from uncultured and candidate phyla. The combination of new approaches in computational biology and new culture-dependent and -independent strategies is thus critical for the recovery of the potential novel chemistry encoded in Antarctica microorganisms.

Towards the structure of an unusual nonribosomal peptide synthetase

2014 ◽  
Vol 70 (a1) ◽  
pp. C433-C433
Author(s):  
Diego Alonzo ◽  
Janice Reimer ◽  
Nathan Magarvey ◽  
T. Martin Schmeing
Keyword(s):  
Assembly Line ◽  
Biochemical Characterization ◽  
Food Poisoning ◽  
Structural Features ◽  
Nonribosomal Peptide Synthetase ◽  
Hydroxy Acids ◽  
Nonribosomal Peptide ◽  
X Ray Crystallography ◽  
Family Protein ◽  
Peptide Synthetase

Cereulide synthetase (Ces) is a macromolecular machine that produces a toxin involved in severe and lethal food poisoning. Ces belongs to the nonribosomal peptide synthetase (NRPS) family protein, and as such, it employs an assembly line mechanism to generate its cereulide product. The assembly line for cereulide differs from the canonical NRPS pathway because it adds hydroxy acids instead of amino acids at half of the positions in the molecule. To achieve this, the megaenzyme selects alpha keto acids through specialized adenylation domains and then reduces them to hydroxy acids in embedded ketoreductase domains, before adding them to the growing depsipeptide. Several details on the structural features of this unusual NRPS remain unknown. Here we report a full biochemical characterization of this highly complex megaenzyme. We obtained the kinetic constants of each of its four adenylation domains and probed the side chain selectivity for its alpha keto acid – recognizing adenylation domains. Also, we report that the activating protein MbtH is not required for full activity of the assembly line. Finally, we show that each of the modules can be inhibited through mechanism-based inhibitors, which can restrain the conformational flexibility paving the way for structural studies by X-ray crystallography.

scholarly journals Engineering of chimeric polyketide synthases using SYNZIP docking domains

2018 ◽  
Author(s):  
Maja Klaus ◽  
Alicia D. D’Souza ◽  
Aleksandra Nivina ◽  
Chaitan Khosla ◽  
Martin Grininger
Keyword(s):  
Turnover Rate ◽  
Assembly Line ◽  
Polyketide Synthases ◽  
Chain Elongation ◽  
Different Types ◽  
Domain Interactions ◽  
Covalent Docking ◽  
Polyketide Chain ◽  
Extender Unit ◽  
Catalytic Cycles

AbstractEngineering of assembly line polyketide synthases (PKSs) to produce novel bioactive compounds has been a goal for over twenty years. The apparent modularity of PKSs has inspired many engineering attempts in which entire modules or single domains were exchanged. In recent years, it has become evident that certain domain-domain interactions are evolutionarily optimized, and if disrupted, cause a decrease of the overall turnover rate of the chimeric PKS. In this study, we compared different types of chimeric PKSs in order to define the least invasive interface and to expand the toolbox for PKS engineering. We generated bimodular chimeric PKSs in which entire modules were exchanged, while either retaining a covalent linker between heterologous modules or introducing a non-covalent docking domain- or SYNZIP domain-mediated interface. These chimeric systems exhibited non-native domain-domain interactions during intermodular polyketide chain translocation. They were compared to otherwise equivalent bimodular PKSs in which a non-covalent interface was introduced between the condensing and processing parts of a module, resulting in non-native domain interactions during the extender unit acylation and polyketide chain elongation steps of their catalytic cycles. We show that the natural PKS docking domains can be efficiently substituted with SYNZIP domains and that the newly introduced non-covalent interface between the condensing and processing parts of a module can be harnessed for PKS engineering. Additionally, we established SYNZIP domains as a new tool for engineering PKSs by efficiently bridging non-native interfaces without perturbing PKS activity.

scholarly journals Tetrodotoxins (TTXs) and Vibrio alginolyticus in Mussels from Central Adriatic Sea (Italy): Are They Closely Related?

Marine Drugs ◽  
2021 ◽  
Vol 19 (6) ◽  
pp. 304
Author(s):  
Simone Bacchiocchi ◽  
Debora Campacci ◽  
Melania Siracusa ◽  
Alessandra Dubbini ◽  
Francesca Leoni ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Mytilus Galloprovincialis ◽  
Polyketide Synthase ◽  
Adriatic Sea ◽  
Tandem Mass ◽  
Hydrophilic Interaction ◽  
Marche Region ◽  
Peptide Synthetase ◽  
Scientific Opinion ◽  
Vibrio Spp

Tetrodotoxins (TTXs), potent neurotoxins, have become an increasing concern in Europe in recent decades, especially because of their presence in mollusks. The European Food Safety Authority published a Scientific Opinion setting a recommended threshold for TTX in mollusks of 44 µg equivalent kg−1 and calling all member states to contribute to an effort to gather data in order to produce a more exhaustive risk assessment. The objective of this work was to assess TTX levels in wild and farmed mussels (Mytilus galloprovincialis) harvested in 2018–2019 along the coastal area of the Marche region in the Central Adriatic Sea (Italy). The presence of Vibrio spp. carrying the non-ribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) genes, which are suspected to be involved in TTX biosynthesis, was also investigated. Out of 158 mussel samples analyzed by hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry (HILIC-MS/MS), 11 (7%) contained the toxins at detectable levels (8–26 µg kg−1) and 3 (2%) contained levels above the EFSA safety threshold (61–76 µg kg−1). Contaminated mussels were all harvested from natural beds in spring or summer. Of the 2019 samples, 70% of them contained V. alginolyticus strains with the NRPS and/or PKS genes. None of the strains containing NRPS and/or PKS genes showed detectable levels of TTXs. TTXs in mussels are not yet a threat in the Marche region nor in Europe, but further investigations are surely needed.

Properties of a “Split-and-Stuttering” Module of an Assembly Line Polyketide Synthase

2021 ◽  
Author(s):  
Katarina M. Guzman ◽  
Kai P. Yuet ◽  
Stephen R. Lynch ◽  
Corey W. Liu ◽  
Chaitan Khosla
Keyword(s):  
Assembly Line ◽  
Polyketide Synthase

scholarly journals Draft Genome Sequence of Saccharomonospora sp. Strain LRS4.154, a Moderately Halophilic Actinobacterium with the Biotechnologically Relevant Polyketide Synthase and Nonribosomal Peptide Synthetase Systems

2017 ◽  
Vol 5 (21) ◽  
Author(s):  
Scarlett Alonso-Carmona ◽  
Blanca Vera-Gargallo ◽  
Rafael R. de la Haba ◽  
Antonio Ventosa ◽  
Horacio Sandoval-Trujillo ◽  
...  
Keyword(s):  
Genome Sequence ◽  
Polyketide Synthase ◽  
Draft Genome ◽  
Nonribosomal Peptide Synthetase ◽  
Open Reading Frames ◽  
Draft Genome Sequence ◽  
Nonribosomal Peptide ◽  
Moderately Halophilic ◽  
Peptide Synthetase ◽  
Reading Frames

ABSTRACT The draft genome sequence of Saccharomonospora sp. strain LRS4.154, a moderately halophilic actinobacterium, has been determined. The genome has 4,860,108 bp, a G+C content of 71.0%, and 4,525 open reading frames (ORFs). The clusters of PKS and NRPS genes, responsible for the biosynthesis of a large number of biomolecules, were identified in the genome.

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