De novo design and engineering of non-ribosomal peptide synthetases

Bozhüyük, Kenan A. J.; Fleischhacker, Florian; Linck, Annabell; Wesche, Frank; Tietze, Andreas; Niesert, Claus-Peter; Bode, Helge B.

doi:10.1038/nchem.2890

Cited by 182 publications

(270 citation statements)

References 38 publications

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“…While we do not dispute that structural novelty is important, we do observe that natural structural diversity present in bacteria that make compound libraries may also be important for structure–function studies. To that effect, the generation of new derivatives of known SM from these bacteria, through in vitro combinatorial biosynthesis, is ongoing with a view to identifying compounds with higher bioactivities (Bozhüyük et al ., ). What our analysis suggests is that there is a strong possibility that many of these derivatives may also exist ‘naturally’ in the environment as evidenced by the extensive molecular networks containing ‘known’ compounds.…”

Section: Discussionmentioning

confidence: 97%

Cyclo(tetrahydroxybutyrate) production is sufficient to distinguish between Xenorhabdus and Photorhabdus isolates in Thailand

Tobias

Parra‐Rojas

Shi

et al. 2019

Environmental Microbiology

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Summary Bacteria of the genera Photorhabdus and Xenorhabdus produce a plethora of natural products to support their similar symbiotic life cycles. For many of these compounds, the specific bioactivities are unknown. One common challenge in natural product research when trying to prioritize research efforts is the rediscovery of identical (or highly similar) compounds from different strains. Linking genome sequence to metabolite production can help in overcoming this problem. However, sequences are typically not available for entire collections of organisms. Here, we perform a comprehensive metabolic screening using HPLC‐MS data associated with a 114‐strain collection (58 Photorhabdus and 56 Xenorhabdus) across Thailand and explore the metabolic variation among the strains, matched with several abiotic factors. We utilize machine learning in order to rank the importance of individual metabolites in determining all given metadata. With this approach, we were able to prioritize metabolites in the context of natural product investigations, leading to the identification of previously unknown compounds. The top three highest ranking features were associated with Xenorhabdus and attributed to the same chemical entity, cyclo(tetrahydroxybutyrate). This work also addresses the need for prioritization in high‐throughput metabolomic studies and demonstrates the viability of such an approach in future research.

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Section: Discussionmentioning

confidence: 97%

Cyclo(tetrahydroxybutyrate) production is sufficient to distinguish between Xenorhabdus and Photorhabdus isolates in Thailand

Tobias

Parra‐Rojas

Shi

et al. 2019

Environmental Microbiology

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“…In contrast to alteringt he substrate specificity of as ingle Adomain,B ozhüyüka nd co-workers established ad en ovo design platform for NRPSs based on the use of newly defined exchange units (XUs). [42] They identified an ew interdomain site located in the linker region between the A-and C-domains from Photorhabdus, Xenorhabdus,a nd Bacillus. By using XU assembly,t he modified NRPS systemsw ere shown to be capable of producingd erivatives of knownN RPs.…”

Section: Exploitation Of Nrps Machinerymentioning

confidence: 99%

Chemical Strategies for Visualizing and Analyzing Endogenous Nonribosomal Peptide Synthetase (NRPS) Megasynthetases

Ishikawa

Tanabe

2019

ChemBioChem

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Nonribosomal peptide (NRP) natural products are among the most promising resources for drug discovery and development, owing to their wide range of biological activities and therapeutic applications. These peptide metabolites are biosynthesized by large multienzyme machinery known as NRP synthetases (NRPSs). The structural complexity of a number of NRPs poses an enormous challenge in their synthesis. A major issue in this field is reprogramming NRPS machineries to allow the biosynthetic production of artificial peptides. NRPS adenylation (A) domains are responsible for the incorporation of a wide variety of amino acids and can be considered as reprogramming sites; therefore, advanced methods to accelerate the functional prediction and assessment of A‐domains are required. This Concept article demonstrates that activity‐based protein profiling of NRPSs offers a simple, rapid, and robust analytical platform for A‐domains and provides insights into enzyme–substrate candidates and active‐site microenvironments. It also describes the background associated with the development and application of a method to analyze endogenous NRPS machinery in its natural environment.

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“…Furthermore, we observed a more than 100-fold increase in levels of thalassospiramide E4 (25), suggesting that the inability to pass growing chains back via C2 forces the assembly line to pass intermediates forward, resulting in enhanced production of the "premature" termination product E4 (25) (Fig. 3b).…”

mentioning

confidence: 94%

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

Zhang

Tang

Huan

et al. 2019

Preprint

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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. Nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) enzymes are molecular-scale assembly lines that construct complex polymeric products, many of which are useful to humans as life-saving drugs. The first characterized assembly lines exhibited an elegant co-linear biosynthetic logic, whereby the linear arrangement of functional units, called modules, along an NRPS/PKS polypeptide directly correlates to the chemical structure of the product 1 . The polyketide antibiotic erythromycin 2 and the nonribosomal peptide antibiotic daptomycin 3 are two such examples. The core components of an assembly line elongation module include adenylation (A) or acyltransferase (AT) domains for substrate selection, thiolation (T) or carrier protein domains for covalent substrate tethering, and condensation (C) or ketosynthase (KS) domains for chain extension. Optional tailoring domains such as methyltransferase (MT), ketoreductase (KR), or dehydratase (DH)domains, if present, chemically modify building blocks or chain-extension intermediates. This one-to-one correlation between product residues and assembly line modules with the requisite catalytic domains is one feature that makes NRPS/PKS enzymes among the largest proteins found within the tree of life.

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De novo design and engineering of non-ribosomal peptide synthetases

Cited by 182 publications

References 38 publications

Cyclo(tetrahydroxybutyrate) production is sufficient to distinguish between Xenorhabdus and Photorhabdus isolates in Thailand

Cyclo(tetrahydroxybutyrate) production is sufficient to distinguish between Xenorhabdus and Photorhabdus isolates in Thailand

Chemical Strategies for Visualizing and Analyzing Endogenous Nonribosomal Peptide Synthetase (NRPS) Megasynthetases

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

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