Optimizing dimodular nonribosomal peptide synthetases and natural dipeptides in an Escherichia coli heterologous host

2013 ◽  
Vol 91 (4) ◽  
pp. 203-208 ◽  
Author(s):  
Morgan A. Wyatt ◽  
Nathan A. Magarvey
Keyword(s):  
Escherichia Coli ◽  
Biological Activities ◽  
Structural Complexity ◽  
Biosynthetic Gene Cluster ◽  
Nonribosomal Peptides ◽  
Heterologous Host ◽  
T7 Promoter ◽  
Nonribosomal Peptide ◽  
Nonribosomal Peptide Synthetases ◽  
Peptide Synthetases

Nonribosomal peptides are an important class of natural products that have a broad range of biological activities. Their structural complexity often prevents simple chemical synthesis, and production from the natural producer is often low, which deters pharmaceutical development. Expression of biosynthetic machinery in heterologous host organisms like Escherichia coli is one way to access these structures, and subsequent optimization of these systems is critical for future development. We utilized the aureusimine biosynthetic gene cluster as a model system to identify the optimal conditions to produce nonribosomal peptides in the isopropyl β-d-1-thiogalactopyranoside (IPTG)-inducible T7 promoter system of pET28. Single reaction monitoring of nonribosomal products was used to find the optimal concentration of IPTG, postinduction temperature, and the effect of amino acid precursor supplementation. In addition, principle component analysis of these extracts identified 3 previously undiscovered pyrazine products of the aureusimine biosynthetic locus, highlighting the utility of heterologously expressing nonribosomal peptide synthetases to find new products.

scholarly journals In Vivo Production of Artificial Nonribosomal Peptide Products in the Heterologous Host Escherichia coli

2004 ◽  
Vol 70 (6) ◽  
pp. 3282-3291 ◽  
Author(s):  
Stephan Gruenewald ◽  
Henning D. Mootz ◽  
Per Stehmeier ◽  
Torsten Stachelhaus
Keyword(s):  
Escherichia Coli ◽  
Single Amino Acid ◽  
Modular Organization ◽  
Cyclic Dipeptide ◽  
Assembly Lines ◽  
Heterologous Host ◽  
Nonribosomal Peptide ◽  
Nonribosomal Peptide Synthetases ◽  
E Coli ◽  
Peptide Synthetases

ABSTRACT Nonribosomal peptide synthetases represent the enzymatic assembly lines for the biosynthesis of pharmacologically relevant natural peptides, e.g., cyclosporine, vancomycin, and penicillin. Due to their modular organization, in which every module accounts for the incorporation of a single amino acid, artificial assembly lines for the production of novel peptides can be constructed by biocombinatorial approaches. Once transferred into an appropriate host, these hybrid synthetases could facilitate the bioproduction of basically any peptide-based molecule. In the present study, we describe the fermentative production of the cyclic dipeptide d-Phe-Pro-diketopiperazine, as a prototype for the exploitation of the heterologous host Escherichia coli, and the use of artificial nonribosomal peptide synthetases. E. coli provides a tremendous potential for genetic engineering and was manipulated in our study by stable chromosomal integration of the 4′-phosphopantetheine transferase gene sfp to ensure heterologous production of fully active holoenzmyes. d-Phe-Pro-diketopiperazine is formed by the TycA/TycB1 system, whose components represent the first two modules for tyrocidine biosynthesis in Bacillus brevis. Coexpression of the corresponding genes in E. coli gave rise to the production of the expected diketopiperazine product, demonstrating the functional interaction of both modules in the heterologous environment. Furthermore, the cyclic dipeptide is stable and not toxic to E. coli and is secreted into the culture medium without the need for any additional factors. Parameters affecting the productivity were comprehensively investigated, including various genetic setups, as well as variation of medium composition and temperature. By these means, the overall productivity of the artificial system could be enhanced by over 400% to yield about 9 mg of d-Phe-Pro-diketopiperazine/liter. As a general tool, this approach could allow the sustainable bioproduction of peptides, e.g., those used as pharmaceuticals or fine chemicals.

scholarly journals One pathway, two cyclic pentapeptides: heterologous expression of BE-18257 A-C and pentaminomycins A-E from Streptomyces cacaoi CA-170360

2020 ◽  
Author(s):  
Fernando Román-Hurtado ◽  
Marina Sánchez-Hidalgo ◽  
Jesús Martín ◽  
Francisco Javier Ortiz-López ◽  
Daniel Carretero-Molina ◽  
...  
Keyword(s):  
Heterologous Expression ◽  
Gene Cluster ◽  
Binding Protein ◽  
Biosynthetic Gene Cluster ◽  
Protein Family ◽  
Biosynthetic Gene ◽  
Penicillin Binding Protein ◽  
Nonribosomal Peptide ◽  
Nonribosomal Peptide Synthetases ◽  
Peptide Synthetases

1.AbstractThe strain Streptomyces cacaoi CA-170360 produces the cyclic pentapeptides pentaminomycins A-E and BE-18257 A-C, two families of cyclopeptides synthesized by two nonribosomal peptide synthetases encoded in tandem within the same biosynthetic gene cluster. In this work, we have cloned and confirmed the heterologous expression of this biosynthetic gene cluster, demonstrating that each of the nonribosomal peptide synthetases present in the cluster is involved in the biosynthesis of each group of cyclopeptides. In addition, we discuss the involvement of a stand-alone enzyme belonging to the Penicillin Binding Protein family in the release and macrocyclization of the peptides.

scholarly journals Structure elucidation of the syringafactin lipopeptides provides insight in the evolution of nonribosomal peptide synthetases

2019 ◽  
Vol 10 (48) ◽  
pp. 10979-10990 ◽  
Author(s):  
Sebastian Götze ◽  
Johannes Arp ◽  
Gerald Lackner ◽  
Shuaibing Zhang ◽  
Hajo Kries ◽  
...  
Keyword(s):  
Structure Elucidation ◽  
Nonribosomal Peptides ◽  
Biosynthetic Genes ◽  
Nonribosomal Peptide ◽  
Nonribosomal Peptide Synthetases ◽  
Peptide Synthetases

A snapshot of evolution in flagrante shows that recombination within and between biosynthetic genes leads to diversification of nonribosomal peptides.

Structure, biosynthesis, and biological activities of cyclooligomer depsipeptides from fungi

2021 ◽  
Vol 66 (1) ◽  
pp. 124-133
Author(s):  
Van Nguyen Thi Thuy ◽  
Viet Nguyen Dinh ◽  
Lam Duong Minh
Keyword(s):  
Hydroxy Acid ◽  
Biological Activities ◽  
Ring Structure ◽  
Nonribosomal Peptide ◽  
Nonribosomal Peptide Synthetases ◽  
Peptide Synthetases ◽  
Natural Substances ◽  
Symmetric Structure ◽  
Potential Applications ◽  
Antiviral Activities

Cyclooligomer depsipeptides (CODs) are peptolides, in which their symmetric structure ring structure has two or more ester bonds formed and alternately arranged between amino and α-hydroxy acid. CODs belong to four main groups of cyclotetrapeptides, cyclohexadepsipeptides, cyclooctadepsipeptides and diketomorpholines. These compounds have been mainly isolated from Acremonium, Aspergillus, Beauveria, Cordyceps, Fusarium, Isaria, Nigrospora, Peacilomyces, and Verticillium. The biosynthesis of CODs takes place outside the ribosome by giant multi-domain enzymes called nonribosomal peptide synthetases (NRPSs). Two different models about the formation of these depsipeptides were proposed. Fungal CODs are known to exhibit various biological activities, especially insecticidal, antitumoral, antimicrobial and antiviral activities. Therefore, CODs are considered to be natural substances with extremely potential applications in medicine and agriculture. This review highlights the structures, classification, biosynthesis, and biological activities of the fungal CODs.

scholarly journals Functional Analysis of All Nonribosomal Peptide Synthetases in Cochliobolus heterostrophus Reveals a Factor, NPS6, Involved in Virulence and Resistance to Oxidative Stress

2005 ◽  
Vol 4 (3) ◽  
pp. 545-555 ◽  
Author(s):  
Bee-Na Lee ◽  
Scott Kroken ◽  
David Y. T. Chou ◽  
Barbara Robbertse ◽  
O. C. Yoder ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Polyketide Synthase ◽  
Biological Activities ◽  
Cochliobolus Heterostrophus ◽  
Pcr Analysis ◽  
Human Pathogens ◽  
Fungal Virulence ◽  
Nonribosomal Peptide ◽  
Nonribosomal Peptide Synthetases ◽  
Peptide Synthetases

ABSTRACT Nonribosomal peptides, made by nonribosomal peptide synthetases, have diverse biological activities, including roles as fungal virulence effectors. Inspection of the genome of Cochliobolus heterostrophus, a fungal pathogen of maize and a member of a genus noted for secondary metabolite production, revealed eight multimodular nonribosomal peptide synthase (NPS) genes and three monomodular NPS-like genes, one of which encodes a nonribosomal peptide synthetase/polyketide synthase hybrid enzyme presumed to be involved in synthesis of a peptide/polyketide molecule. Deletion of each NPS gene and phenotypic analyses showed that the product of only one of these genes, NPS6, is required for normal virulence on maize. NPS6 is also required for resistance to hydrogen peroxide, suggesting it may protect the fungus from oxidative stress. This and all other nps mutants had normal growth, mating ability, and appressoria. Real-time PCR analysis showed that expression of all NPS genes is low (relative to that of actin), that all (except possibly NPS2) are expressed during vegetative growth, and that expression is induced by nitrogen starvation. Only NPS6 is unfailingly conserved among euascomycete fungi, including plant and human pathogens and saprobes, suggesting the possibility that NPS6 activity provides oxidative stress protection during both saprobic and parasitic growth.

scholarly journals Nonprocessive [2 + 2]e- off-loading reductase domains from mycobacterial nonribosomal peptide synthetases

2012 ◽  
Vol 109 (15) ◽  
pp. 5681-5686 ◽  
Author(s):  
A. Chhabra ◽  
A. S. Haque ◽  
R. K. Pal ◽  
A. Goyal ◽  
R. Rai ◽  
...  
Keyword(s):  
Nonribosomal Peptide ◽  
Nonribosomal Peptide Synthetases ◽  
Peptide Synthetases
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