scholarly journals Comparative Investigation into Formycin A and Pyrazofurin A Biosynthesis Reveals Branch Pathways for the Construction of C-Nucleoside Scaffolds

2019 ◽  
Vol 86 (2) ◽  
Author(s):  
Meng Zhang ◽  
Peichao Zhang ◽  
Gudan Xu ◽  
Wenting Zhou ◽  
Yaojie Gao ◽  
...  
Keyword(s):  
Biological Activities ◽  
Genome Mining ◽  
Gene Clusters ◽  
Combinatorial Biosynthesis ◽  
Glycosidic Bond ◽  
Enzymatic Reactions ◽  
Nucleoside Antibiotics ◽  
Enzymatic Mechanism ◽  
Chemical Structures ◽  
The Way

ABSTRACT Formycin A (FOR-A) and pyrazofurin A (PRF-A) are purine-related C-nucleoside antibiotics in which ribose and a pyrazole-derived base are linked by a C-glycosidic bond. However, the logic underlying the biosynthesis of these molecules has remained largely unexplored. Here, we report the discovery of the pathways for FOR-A and PRF-A biosynthesis from diverse actinobacteria and propose that their biosynthesis is likely initiated by a lysine N6-monooxygenase. Moreover, we show that forT and prfT (involved in FOR-A and PRF-A biosynthesis, respectively) mutants are correspondingly capable of accumulating the unexpected pyrazole-related intermediates 4-amino-3,5-dicarboxypyrazole and 3,5-dicarboxy-4-oxo-4,5-dihydropyrazole. We also decipher the enzymatic mechanism of ForT/PrfT for C-glycosidic bond formation in FOR-A/PRF-A biosynthesis. To our knowledge, ForT/PrfT represents an example of β-RFA-P (β-ribofuranosyl-aminobenzene 5ʹ-phosphate) synthase-like enzymes governing C-nucleoside scaffold construction in natural product biosynthesis. These data establish a foundation for combinatorial biosynthesis of related purine nucleoside antibiotics and also open the way for target-directed genome mining of PRF-A/FOR-A-related antibiotics. IMPORTANCE FOR-A and PRF-A are C-nucleoside antibiotics known for their unusual chemical structures and remarkable biological activities. Deciphering the enzymatic mechanism for the construction of a C-nucleoside scaffold during FOR-A/PRF-A biosynthesis will not only expand the biochemical repertoire for novel enzymatic reactions but also permit target-oriented genome mining of FOR-A/PRF-A-related C-nucleoside antibiotics. Moreover, the availability of FOR-A/PRF-A biosynthetic gene clusters will pave the way for the rational generation of designer FOR-A/PRF-A derivatives with enhanced/selective bioactivity via synthetic biology strategies.

scholarly journals Comparative investigation into formycin A and pyrazofurin A biosynthesis reveals branch pathways for the construction of C-nucleoside scaffolds

2019 ◽  
Author(s):  
Meng Zhang ◽  
Peichao Zhang ◽  
Gudan Xu ◽  
Wenting Zhou ◽  
Yaojie Gao ◽  
...  
Keyword(s):  
Biological Activities ◽  
Bond Formation ◽  
Genome Mining ◽  
Comparative Investigation ◽  
Combinatorial Biosynthesis ◽  
Glycosidic Bond ◽  
Enzymatic Reactions ◽  
Natural Product Biosynthesis ◽  
Nucleoside Antibiotics ◽  
Chemical Structures

ABSTRACTFormycin A (FOR-A) and pyrazofurin A (PRF-A) are purine-related C-nucleoside antibiotics, in which ribose and a pyrazole-derived base are linked by a C-glycosidic bond, however, the logic underlying the biosynthesis of these molecules has remained largely unexplored. Here, we report the discovery of the pathways for FOR-A and PRF-A biosynthesis from diverse actinobacteria, and demonstrate that their biosynthesis is initiated by a lysine N6-monooxygenase. Moreover, we show that the forT and prfE (individually related to FOR-A and PRF-A biosynthesis) mutants are correspondingly capable of accumulating the unexpected pyrazole-related intermediates, compound 11 and 9a. We also decipher the enzymatic basis of ForT/PrfE for the C-glycosidic bond formation in FOR-A/PRF-A biosynthesis. To our knowledge, ForT/PrfE represents the first example of β-RFA-P (β-ribofuranosyl-aminobenzene 5’-phosphate) synthase-like enzymes governing C-nucleoside scaffold construction in natural product biosynthesis. These data establish a foundation for combinatorial biosynthesis of related purine nucleoside antibiotics, and also open the way for target-directed genome mining of PRF-A/FOR-A related antibiotics.IMPORTANCEFormycin A (FOR-A) and pyrazofurin A (PRF-A) are well known for their unusual chemical structures and remarkable biological activities. Actually, deciphering FOR-A/PRF-A biosynthesis will not only expand biochemical repertoire for novel enzymatic reactions, but also permit the target-oriented genome mining of FOR-A/PRF-A related C-nucleoside antibiotics.

scholarly journals Coordinated Biosynthesis of the Purine Nucleoside Antibiotics Aristeromycin and Coformycin in Actinomycetes

2018 ◽  
Vol 84 (22) ◽  
Author(s):  
Gudan Xu ◽  
Liyuan Kong ◽  
Rong Gong ◽  
Liudong Xu ◽  
Yaojie Gao ◽  
...  
Keyword(s):  
Biological Activities ◽  
Genome Mining ◽  
Combinatorial Biosynthesis ◽  
Purine Nucleoside ◽  
Enzymatic Reactions ◽  
Single Strain ◽  
Content Type ◽  
Precursor Pool ◽  
Irreversible Reaction ◽  
Chemical Structures

ABSTRACT Purine nucleoside antibiotic pairs, concomitantly produced by a single strain, are an important group of microbial natural products. Here, we report a target-directed genome mining approach to elucidate the biosynthesis of the purine nucleoside antibiotic pair aristeromycin (ARM) and coformycin (COF) in Micromonospora haikouensis DSM 45626 (a new producer for ARM and COF) and Streptomyces citricolor NBRC 13005 (a new COF producer). We also provide biochemical data that MacI and MacT function as unusual phosphorylases, catalyzing an irreversible reaction for the tailoring assembly of neplanocin A (NEP-A) and ARM. Moreover, we demonstrate that MacQ is shown to be an adenosine-specific deaminase, likely relieving the potential “excess adenosine” for producing cells. Finally, we report that MacR, an annotated IMP dehydrogenase, is actually an NADPH-dependent GMP reductase, which potentially plays a salvage role for the efficient supply of the precursor pool. Hence, these findings illustrate a fine-tuned pathway for the biosynthesis of ARM and also open the way for the rational search for purine antibiotic pairs. IMPORTANCE ARM and COF are well known for their prominent biological activities and unusual chemical structures; however, the logic of their biosynthesis has long been poorly understood. Actually, the new insights into the ARM and COF pathway will not only enrich the biochemical repertoire for interesting enzymatic reactions but may also lay a solid foundation for the combinatorial biosynthesis of this group of antibiotics via a target-directed genome mining strategy.

scholarly journals Biosynthesis of 2′-Chloropentostatin and 2′-Amino-2′-Deoxyadenosine Highlights a Single Gene Cluster Responsible for Two Independent Pathways in Actinomadura sp. Strain ATCC 39365

2017 ◽  
Vol 83 (10) ◽  
Author(s):  
Yaojie Gao ◽  
Gudan Xu ◽  
Pan Wu ◽  
Jin Liu ◽  
You-sheng Cai ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Single Gene ◽  
Combinatorial Biosynthesis ◽  
Pathway Engineering ◽  
Enzymatic Reactions ◽  
Strain Atcc ◽  
Nudix Hydrolase ◽  
Nucleoside Antibiotics ◽  
Chemical Structures ◽  
Hydrolysis Of

ABSTRACT 2′-Chloropentostatin (2′-Cl PTN, 2′-chloro-2′-deoxycoformycin) and 2′-amino-2′-deoxyadenosine (2′-amino dA) are two adenosine-derived nucleoside antibiotics coproduced by Actinomadura sp. strain ATCC 39365. 2′-Cl PTN is a potent adenosine deaminase (ADA) inhibitor featuring an intriguing 1,3-diazepine ring, as well as a chlorination at C-2′ of ribose, and 2′-amino dA is an adenosine analog showing bioactivity against RNA-type virus infection. However, the biosynthetic logic of them has remained poorly understood. Here, we report the identification of a single gene cluster (ada) essential for the biosynthesis of 2′-Cl PTN and 2′-amino dA. Further systematic genetic investigations suggest that 2′-Cl PTN and 2′-amino dA are biosynthesized by independent pathways. Moreover, we provide evidence that a predicted cation/H+ antiporter, AdaE, is involved in the chlorination step during 2′-Cl PTN biosynthesis. Notably, we demonstrate that 2′-amino dA biosynthesis is initiated by a Nudix hydrolase, AdaJ, catalyzing the hydrolysis of ATP. Finally, we reveal that the host ADA (designated ADA1), capable of converting adenosine/2′-amino dA to inosine/2′-amino dI, is not very sensitive to the powerful ADA inhibitor pentostatin. These findings provide a basis for the further rational pathway engineering of 2′-Cl PTN and 2′-amino dA production. IMPORTANCE 2′-Cl PTN/PTN and 2′-amino dA have captivated the great interests of scientists, owing to their unusual chemical structures and remarkable bioactivities. However, the precise logic for their biosynthesis has been elusive for decades. Actually, the identification and elucidation of their biosynthetic pathways not only enrich the biochemical repertoire of novel enzymatic reactions but may also lay solid foundations for the pathway engineering and combinatorial biosynthesis of this family of purine nucleoside antibiotics to generate novel hybrid analogs with improved features.

Expanding the Natural Products Heterologous Expression Repertoire in the Model Cyanobacterium Anabaena sp. Strain PCC 7120: Production of Pendolmycin and Teleocidin B-4

2019 ◽  
Author(s):  
Patrick Videau ◽  
Kaitlyn Wells ◽  
Arun Singh ◽  
Jessie Eiting ◽  
Philip Proteau ◽  
...  
Keyword(s):  
Natural Products ◽  
Genome Mining ◽  
Gene Clusters ◽  
Combinatorial Biosynthesis ◽  
Test Case ◽  
Biosynthetic Gene ◽  
Biosynthetic Gene Clusters ◽  
Cyanobacterium Anabaena ◽  
Anabaena Sp ◽  
Pcc 7120

Cyanobacteria are prolific producers of natural products and genome mining has shown that many orphan biosynthetic gene clusters can be found in sequenced cyanobacterial genomes. New tools and methodologies are required to investigate these biosynthetic gene clusters and here we present the use of <i>Anabaena </i>sp. strain PCC 7120 as a host for combinatorial biosynthesis of natural products using the indolactam natural products (lyngbyatoxin A, pendolmycin, and teleocidin B-4) as a test case. We were able to successfully produce all three compounds using codon optimized genes from Actinobacteria. We also introduce a new plasmid backbone based on the native <i>Anabaena</i>7120 plasmid pCC7120ζ and show that production of teleocidin B-4 can be accomplished using a two-plasmid system, which can be introduced by co-conjugation.

scholarly journals In vivo characterization of the activities of novel cyclodipeptide oxidases: new tools for increasing chemical diversity of bioproduced 2,5-diketopiperazines in Escherichia coli

2020 ◽  
Vol 19 (1) ◽  
Author(s):  
Fabien Le Chevalier ◽  
Isabelle Correia ◽  
Lucrèce Matheron ◽  
Morgan Babin ◽  
Mireille Moutiez ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Biological Activities ◽  
Gene Clusters ◽  
Chemical Diversity ◽  
E Coli ◽  
Chemical Structures ◽  
In Vivo Characterization ◽  
Culture Supernatants

Abstract Background Cyclodipeptide oxidases (CDOs) are enzymes involved in the biosynthesis of 2,5-diketopiperazines, a class of naturally occurring compounds with a large range of pharmaceutical activities. CDOs belong to cyclodipeptide synthase (CDPS)-dependent pathways, in which they play an early role in the chemical diversification of cyclodipeptides by introducing Cα-Cβ dehydrogenations. Although the activities of more than 100 CDPSs have been determined, the activities of only a few CDOs have been characterized. Furthermore, the assessment of the CDO activities on chemically-synthesized cyclodipeptides has shown these enzymes to be relatively promiscuous, making them interesting tools for cyclodipeptide chemical diversification. The purpose of this study is to provide the first completely microbial toolkit for the efficient bioproduction of a variety of dehydrogenated 2,5-diketopiperazines. Results We mined genomes for CDOs encoded in biosynthetic gene clusters of CDPS-dependent pathways and selected several for characterization. We co-expressed each with their associated CDPS in the pathway using Escherichia coli as a chassis and showed that the cyclodipeptides and the dehydrogenated derivatives were produced in the culture supernatants. We determined the biological activities of the six novel CDOs by solving the chemical structures of the biologically produced dehydrogenated cyclodipeptides. Then, we assessed the six novel CDOs plus two previously characterized CDOs in combinatorial engineering experiments in E. coli. We co-expressed each of the eight CDOs with each of 18 CDPSs selected for the diversity of cyclodipeptides they synthesize. We detected more than 50 dehydrogenated cyclodipeptides and determined the best CDPS/CDO combinations to optimize the production of 23. Conclusions Our study establishes the usefulness of CDPS and CDO for the bioproduction of dehydrogenated cyclodipeptides. It constitutes the first step toward the bioproduction of more complex and diverse 2,5-diketopiperazines.

scholarly journals Genome mining of novel rubiginones from Streptomyces sp. CB02414 and characterization of the post-PKS modification steps in rubiginone biosynthesis

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Jingyan Zhang ◽  
Ying Sun ◽  
Yeji Wang ◽  
Xin Chen ◽  
Lu Xue ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Biosynthetic Pathway ◽  
Scale Up ◽  
Genome Mining ◽  
Gene Clusters ◽  
Combinatorial Biosynthesis ◽  
Wild Type ◽  
Aromatic Polyketides ◽  
A Genome

Abstract Background Rubiginones belong to the angucycline family of aromatic polyketides, and they have been shown to potentiate the vincristine (VCR)-induced cytotoxicity against VCR-resistant cancer cell lines. However, the biosynthetic gene clusters (BGCs) and biosynthetic pathways for rubiginones have not been reported yet. Results In this study, based on bioinformatics analysis of the genome of Streptomyces sp. CB02414, we predicted the functions of the two type II polyketide synthases (PKSs) BGCs. The rub gene cluster was predicted to encode metabolites of the angucycline family. Scale-up fermentation of the CB02414 wild-type strain led to the discovery of eight rubiginones, including five new ones (rubiginones J, K, L, M, and N). Rubiginone J was proposed to be the final product of the rub gene cluster, which features extensive oxidation on the A-ring of the angucycline skeleton. Based on the production profiles of the CB02414 wild-type and the mutant strains, we proposed a biosynthetic pathway for the rubiginones in CB02414. Conclusions A genome mining strategy enabled the efficient discovery of new rubiginones from Streptomyces sp. CB02414. Based on the isolated biosynthetic intermediates, a plausible biosynthetic pathway for the rubiginones was proposed. Our research lays the foundation for further studies on the mechanism of the cytochrome P450-catalyzed oxidation of angucyclines and for the generation of novel angucyclines using combinatorial biosynthesis strategies.

scholarly journals Genome mining of a marine-derived Streptomyces sp. PDH23 isolated from sponge in Da Nang sea for secondary metabolite gene clusters

2021 ◽  
Vol 18 (4) ◽  
pp. 709-721
Author(s):  
Le Ngoc Giang ◽  
Le Thi Hong Minh ◽  
Vu Thi Quyen ◽  
Nguyen Mai Anh ◽  
Nguyen Thi Kim Cuc ◽  
...  
Keyword(s):  
Secondary Metabolites ◽  
Antimicrobial Agents ◽  
Biological Activities ◽  
Wide Spectrum ◽  
Genome Mining ◽  
Gc Content ◽  
Gene Clusters ◽  
Gram Positive ◽  
16S Rrna Analysis ◽  
Streptomyces Sp

The streptomyces is one of the best characterized ubiquitous filamentous bacteria from the actinobacteriaclass. They are known to produce thousands of specialized metabolite biosynthesis gene clusters (SMBG). Their SMBG clusters have multiple activities ranging from antimicrobial, antitumor, antiviral and probiotic. Streptomyces strain and their isolates with interesting biological activities against gram-positive and gram-negative indicator strains was recently characterised. Currently, they are employed in more than half of all antibiotics used in human and veterinary medicine. With the increase in drug resistance bacteria, it is important to mine for new natural chemicals.In this study, screening via disk-diffusion agar method revealed that Streptomyces sp. PDH23 isolated from the Rhabdastrellaglobostellata marine sponge sample from Da Nang, Vietnam produce antimicrobial agents with a wide spectrum of activities. This species can produce highly active enzymes, which breakdown celluloses, amyloses and proteins. On top of that they are shown to restrict the grow of the gram positive Bacillus cereus ATCC14579 (BC), Staphylococcus aureus ATCC25923 (SA), the gram-negativeVibrio parahaemolyticus ATCC17802 (VP) and the Candida albicans ATCC10231 fungus (CA). They are antimethicillin-resistant S. aureus(MRSA) ATCC33591 andmethicillin-resistantS. epidermidis (MRSE) ATCC35984. The taxonomy of PDH23 was characterized using 16S rRNA analysis. Whole genome sequencing of PDH23 showed 8594820 base pairs with GC content of 72.03%. Mining of secondary metabolites reveals gene clusters responsible for the biosynthesis of known and/or novel secondary metabolites, including different types of terpene, NRPS-like , PKS, PKS-like, hglE-KS, betalactone, melanin, t1pks, t2pks, t3pks, nrps, indole, siderophore, bacteriocin, ectoine, butyrolactone, phenazine.

scholarly journals Genome mining and UHPLC-MS/MS illuminate the specificity of secondary metabolite synthetic gene clusters in Bacillus subtilis NCD-2

2020 ◽  
Author(s):  
Zhenhe Su ◽  
Xiuye Chen ◽  
Xiaomeng Liu ◽  
Qinggang Guo ◽  
Shezeng Li ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Secondary Metabolites ◽  
Secondary Metabolite ◽  
Biological Activities ◽  
Genome Mining ◽  
Gene Clusters ◽  
Synthetic Gene ◽  
Integrative Approach ◽  
Amino Acid Sequence Similarity ◽  
Genetic Characteristics

Abstract Background Bacillus subtilisstrain NCD-2 is anexcellent biocontrol agent against plant soil-borne diseases and shows broad-spectrum antifungal activities. This study aimed to explore all the secondary metabolite synthetic gene clusters and related bioactive compounds in NCD-2. An integrative approach, which coupled genome mining with structural identification technologies using ultra-high-performance liquid chromatography coupled to quadrupole time-of-flight tandem mass spectrometry (UHPLC-MS/MS), was conducted to interpret the chemical origins of the significant biological activities in NCD-2. Results Genome mining revealed that NCD-2 contained nine gene clustershaving predicted functionsinvolving secondary metabolites with bioactive abilities. They encoded six known products-fengycin, surfactin, bacillaene, subtilosin, bacillibactin, and bacilysin-as well as three unknown products.Interestingly, the synthetic gene clusters for surfactin and fengycin showed 83% and 92% amino acid sequence similarity levels with the corresponding productsin Bacillus velezensisstrain FZB42. A further comparison of gene clusters encoding fengycin and surfactinrevealed that strain NCD-2 had lost thefenC and fenDgenes in the fengycinbiosynthetic operon, and that the surfactin synthetic enzyme-related gene srfAB was divided into two parts.Abioinformatics analysis showed that fenEAmay function as fenCD in synthesizing fengycinand that the structure of thisfengycin synthetic gene clusteris likely unique to NCD-2.Five kinds of fengycin,with 26 homologs, and surfactin,with 4 homologs,were detectedfrom strain NCD-2, which indicated the non-typical and unique nature of this fengycin biosynthetic gene cluster.To the best of our knowledge, this is the first report of a non-typical gene cluster related to fengycin synthesis. Conclusions The data provide the genetic characteristics of secondary metabolite synthetic gene clusters for fengycinand surfactin in B. subtilis NCD-2, including the unique synthetic mechanism for fengycin, and suggest that bioactive secondary metabolites explain the biological activities of NCD-2.

scholarly journals Recent development of antiSMASH and other computational approaches to mine secondary metabolite biosynthetic gene clusters

2017 ◽  
Vol 20 (4) ◽  
pp. 1103-1113 ◽  
Author(s):  
Kai Blin ◽  
Hyun Uk Kim ◽  
Marnix H Medema ◽  
Tilmann Weber
Keyword(s):  
Natural Products ◽  
Small Molecules ◽  
Sequence Similarity ◽  
Genome Mining ◽  
Gene Clusters ◽  
Biosynthetic Gene ◽  
Biosynthetic Gene Clusters ◽  
Rule Based ◽  
Chemical Structures ◽  
Annotation Quality

Abstract Many drugs are derived from small molecules produced by microorganisms and plants, so-called natural products. Natural products have diverse chemical structures, but the biosynthetic pathways producing those compounds are often organized as biosynthetic gene clusters (BGCs) and follow a highly conserved biosynthetic logic. This allows for the identification of core biosynthetic enzymes using genome mining strategies that are based on the sequence similarity of the involved enzymes/genes. However, mining for a variety of BGCs quickly approaches a complexity level where manual analyses are no longer possible and require the use of automated genome mining pipelines, such as the antiSMASH software. In this review, we discuss the principles underlying the predictions of antiSMASH and other tools and provide practical advice for their application. Furthermore, we discuss important caveats such as rule-based BGC detection, sequence and annotation quality and cluster boundary prediction, which all have to be considered while planning for, performing and analyzing the results of genome mining studies.

scholarly journals Genomic charting of ribosomally synthesized natural product chemical space facilitates targeted mining

2016 ◽  
Vol 113 (42) ◽  
pp. E6343-E6351 ◽  
Author(s):  
Michael A. Skinnider ◽  
Chad W. Johnston ◽  
Robyn E. Edgar ◽  
Chris A. Dejong ◽  
Nishanth J. Merwin ◽  
...  
Keyword(s):  
Natural Products ◽  
Genome Sequencing ◽  
Sequence Data ◽  
Chemical Space ◽  
Biological Activities ◽  
Global Analysis ◽  
Gene Clusters ◽  
Systematic Investigation ◽  
Chemical Structures ◽  
Modified Peptides

Microbial natural products are an evolved resource of bioactive small molecules, which form the foundation of many modern therapeutic regimes. Ribosomally synthesized and posttranslationally modified peptides (RiPPs) represent a class of natural products which have attracted extensive interest for their diverse chemical structures and potent biological activities. Genome sequencing has revealed that the vast majority of genetically encoded natural products remain unknown. Many bioinformatic resources have therefore been developed to predict the chemical structures of natural products, particularly nonribosomal peptides and polyketides, from sequence data. However, the diversity and complexity of RiPPs have challenged systematic investigation of RiPP diversity, and consequently the vast majority of genetically encoded RiPPs remain chemical “dark matter.” Here, we introduce an algorithm to catalog RiPP biosynthetic gene clusters and chart genetically encoded RiPP chemical space. A global analysis of 65,421 prokaryotic genomes revealed 30,261 RiPP clusters, encoding 2,231 unique products. We further leverage the structure predictions generated by our algorithm to facilitate the genome-guided discovery of a molecule from a rare family of RiPPs. Our results provide the systematic investigation of RiPP genetic and chemical space, revealing the widespread distribution of RiPP biosynthesis throughout the prokaryotic tree of life, and provide a platform for the targeted discovery of RiPPs based on genome sequencing.

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