scholarly journals Target Accumulation of Selective Anticancer Depsipeptides by Reconstructing Precursor Supply in Neoantimycins Biosynthetic Pathway

2021 ◽  
Author(s):  
Lin Zhou ◽  
Yaoyao Shen ◽  
Nannan Chen ◽  
Wanlu Li ◽  
Hou-wen Lin ◽  
...  
Keyword(s):  
Biosynthetic Pathway ◽  
Polyketide Synthase ◽  
Isolation Procedure ◽  
Anticancer Activities ◽  
Production Improvement ◽  
Peptide Synthetase ◽  
Starter Unit ◽  
Pharmaceutical Molecules ◽  
Significant Production ◽  
Producer Strains

Abstract BackgroundNeoantimycins are a group of 15-membered ring depsipeptides isolated from streptomycetes with a broad-spectrum of anticancer activities. Their biosynthesis is directed by the hybrid multimodular megaenzymes of non-ribosomal peptide synthetase and polyketide synthase. We have previously discovered a new neoantimycin analogue unantimycin B, which was demonstrated with selective anticancer activities and was produced from neoantimycins biosynthetic pathway with a starter unit of 3-hydroxybenzoate, instead of the 3-formamidosalicylate for neoantimycins. However, the low fermentation yield and tough isolation procedure have been hindering in-depth pharmacology investigation of unantimycin B as anticancer agents.ResultsIn the work, we genetically constructed two unantimycin B producer strains with neoantimycins production destroyed by removing natO and natJ-L genes essential for 3-formamidosalicylate biosynthesis and therefore facilitated chromatographic separation of unantimycin B from the complex fermentation extract. Based on the △natO mutant, we improved unantimycin Bproduction by two times, reaching to approximate 12.8 mg/L, by feeding 3-hydroxybenzoate in fermentation. Further, the production was improved by more than six times, reaching to approximate 40.0 mg/L, in the △natO strain introduced with a chorismatase gene highly expressed under a strong promoter for over-producing 3-hydroxybenzoate endogenously.ConclusionThe work gives a case of targeting accumulation and significant production improvement of medicinally interesting natural products via genetically manipulation of precursor biosynthesis in streptomycetes, the talented producers of pharmaceutical molecules.

scholarly journals Targeted accumulation of selective anticancer depsipeptides by reconstructing the precursor supply in the neoantimycin biosynthetic pathway

2021 ◽  
Vol 8 (1) ◽  
Author(s):  
Lin Zhou ◽  
Yaoyao Shen ◽  
Nannan Chen ◽  
Wanlu Li ◽  
Hou-wen Lin ◽  
...  
Keyword(s):  
Biosynthetic Pathway ◽  
Polyketide Synthase ◽  
Genetic Manipulation ◽  
Anticancer Activities ◽  
Production Improvement ◽  
Peptide Synthetase ◽  
Starter Unit ◽  
Pharmaceutical Molecules ◽  
Significant Production ◽  
Producer Strains

Abstract Background Neoantimycins are a group of 15-membered ring depsipeptides isolated from Streptomycetes with a broad-spectrum of anticancer activities. Neoantimycin biosynthesis is directed by the hybrid multimodular megaenzymes of non-ribosomal peptide synthetase and polyketide synthase. We previously discovered a new neoantimycin analogue unantimycin B, which was demonstrated to have selective anticancer activities and was produced from the neoantimycin biosynthetic pathway with a starter unit of 3-hydroxybenzoate, instead of the 3-formamidosalicylate unit that is common for neoantimycins. However, the low fermentation titre and tough isolation procedure have hindered in-depth pharmacological investigation of unantimycin B as an anticancer agent. Results In this work, we genetically constructed two unantimycin B producer strains and inhibited neoantimycins production by removing natO and natJ-L genes essential for 3-formamidosalicylate biosynthesis, therefore facilitating chromatographic separation of unantimycin B from the complex fermentation extract. Based on the ΔnatO mutant, we improved unantimycin B production twofold, reaching approximately 12.8 mg/L, by feeding 3-hydroxybenzoate during fermentation. Furthermore, the production was improved more than sixfold, reaching approximately 40.0 mg/L, in the ΔnatO strain introduced with a chorismatase gene highly expressed under a strong promoter for endogenously over-producing 3-hydroxybenzoate. Conclusion This work provides a case of targeting accumulation and significant production improvement of medicinally interesting natural products via genetic manipulation of precursor biosynthesis in Streptomycetes, the talented producers of pharmaceutical molecules.

scholarly journals Biosynthesis of depsipeptides with a 3-hydroxybenzoate moiety and selective anticancer activities involves a chorismatase

2020 ◽  
Vol 295 (16) ◽  
pp. 5509-5518
Author(s):  
Yaoyao Shen ◽  
Fan Sun ◽  
Liu Zhang ◽  
Yijia Cheng ◽  
Hongrui Zhu ◽  
...  
Keyword(s):  
Biochemical Analysis ◽  
Polyketide Synthase ◽  
Site Directed Mutagenesis ◽  
Human Lung Cancer ◽  
Type I ◽  
Anticancer Activities ◽  
Anticancer Compounds ◽  
Significant Toxicity ◽  
Peptide Synthetase ◽  
Starter Unit

Neoantimycins are anticancer compounds of 15-membered ring antimycin-type depsipeptides. They are biosynthesized by a hybrid multimodular protein complex of nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS), typically from the starting precursor 3-formamidosalicylate. Examining fermentation extracts of Streptomyces conglobatus, here we discovered four new neoantimycin analogs, unantimycins B–E, in which 3-formamidosalicylates are replaced by an unusual 3-hydroxybenzoate (3-HBA) moiety. Unantimycins B–E exhibited levels of anticancer activities similar to those of the chemotherapeutic drug cisplatin in human lung cancer, colorectal cancer, and melanoma cells. Notably, they mostly displayed no significant toxicity toward noncancerous cells, unlike the serious toxicities generally reported for antimycin-type natural products. Using site-directed mutagenesis and heterologous expression, we found that unantimycin productions are correlated with the activity of a chorismatase homolog, the nat-hyg5 gene, from a type I PKS gene cluster. Biochemical analysis confirmed that the catalytic activity of Nat-hyg5 generates 3-HBA from chorismate. Finally, we achieved selective production of unantimycins B and C by engineering a chassis host. On the basis of these findings, we propose that unantimycin biosynthesis is directed by the neoantimycin-producing NRPS–PKS complex and initiated with the starter unit of 3-HBA. The elucidation of the biosynthetic unantimycin pathway reported here paves the way to improve the yield of these compounds for evaluation in oncotherapeutic applications.

scholarly journals Mapping the biosynthetic pathway of a hybrid polyketide-nonribosomal peptide in a metazoan

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Likui Feng ◽  
Matthew T. Gordon ◽  
Ying Liu ◽  
Kari B. Basso ◽  
Rebecca A. Butcher
Keyword(s):  
Protein Interactions ◽  
Biosynthetic Pathway ◽  
Polyketide Synthase ◽  
Carrier Protein ◽  
Nonribosomal Peptides ◽  
Direct Transfer ◽  
Protein Protein Interactions ◽  
Nonribosomal Peptide ◽  
Peptide Synthetase ◽  
Complex Protein

AbstractPolyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) hybrid systems typically use complex protein-protein interactions to facilitate direct transfer of intermediates between these multimodular megaenzymes. In the canal-associated neurons (CANs) of Caenorhabditis elegans, PKS-1 and NRPS-1 produce the nemamides, the only known hybrid polyketide-nonribosomal peptides biosynthesized by animals, through a poorly understood mechanism. Here, we use genome editing and mass spectrometry to map the roles of individual PKS-1 and NRPS-1 enzymatic domains in nemamide biosynthesis. Furthermore, we show that nemamide biosynthesis requires at least five additional enzymes expressed in the CANs that are encoded by genes distributed across the worm genome. We identify the roles of these enzymes and discover a mechanism for trafficking intermediates between a PKS and an NRPS. Specifically, the enzyme PKAL-1 activates an advanced polyketide intermediate as an adenylate and directly loads it onto a carrier protein in NRPS-1. This trafficking mechanism provides a means by which a PKS-NRPS system can expand its biosynthetic potential and is likely important for the regulation of nemamide biosynthesis.

scholarly journals Biosynthesis of Mycotoxin Fusaric Acid and Application of a PLP-Dependent Enzyme for Chemoenzymatic Synthesis of Substituted L-Pipecolic Acids

2020 ◽  
Author(s):  
Yang Hai ◽  
Mengbin Chen ◽  
Arthur Huang ◽  
Yi Tang
Keyword(s):  
Biosynthetic Pathway ◽  
Polyketide Synthase ◽  
Picolinic Acid ◽  
Fusaric Acid ◽  
Biosynthetic Gene Cluster ◽  
Chemoenzymatic Synthesis ◽  
Flavin Mononucleotide ◽  
Biosynthetic Gene ◽  
Bond Forming ◽  
Peptide Synthetase

<div><p>Fusaric acid (FA) is a well-known mycotoxin that plays an important role in plant pathology. The biosynthetic gene cluster for FA has been identified but the biosynthetic pathway remains unclarified. Here, we elucidated the biosynthesis of FA, which features a two-enzyme catalytic cascade, a pyridoxal 5’-phosphate (PLP)-dependent enzyme (Fub7) and a flavin mononucleotide (FMN)-dependent oxidase (Fub9) in synthesizing the picolinic acid scaffold. FA biosynthesis also involves an off-line collaboration between a highly reducing polyketide synthase (HRPKS, Fub1) and a nonribosomal peptide synthetase (NRPS)-like carboxylic acid reductase (Fub8) in making an aliphatic alpha,beta-unsaturated aldehyde. By harnessing the stereoselective C-C bond forming activity of Fub7, we established a chemoenzymatic route for stereoconvergent synthesis of a series of 5-alkyl, 5,5-dialkyl, and 5,5,6-trialkyl-L-pipecolic acids of high diastereomeric ratio. </p></div>

scholarly journals Biosynthesis of Mycotoxin Fusaric Acid and Application of a PLP-Dependent Enzyme for Chemoenzymatic Synthesis of Substituted L-Pipecolic Acids

2020 ◽  
Author(s):  
Yang Hai ◽  
Mengbin Chen ◽  
Arthur Huang ◽  
Yi Tang
Keyword(s):  
Biosynthetic Pathway ◽  
Polyketide Synthase ◽  
Picolinic Acid ◽  
Fusaric Acid ◽  
Biosynthetic Gene Cluster ◽  
Chemoenzymatic Synthesis ◽  
Flavin Mononucleotide ◽  
Biosynthetic Gene ◽  
Bond Forming ◽  
Peptide Synthetase

<div><p>Fusaric acid (FA) is a well-known mycotoxin that plays an important role in plant pathology. The biosynthetic gene cluster for FA has been identified but the biosynthetic pathway remains unclarified. Here, we elucidated the biosynthesis of FA, which features a two-enzyme catalytic cascade, a pyridoxal 5’-phosphate (PLP)-dependent enzyme (Fub7) and a flavin mononucleotide (FMN)-dependent oxidase (Fub9) in synthesizing the picolinic acid scaffold. FA biosynthesis also involves an off-line collaboration between a highly reducing polyketide synthase (HRPKS, Fub1) and a nonribosomal peptide synthetase (NRPS)-like carboxylic acid reductase (Fub8) in making an aliphatic alpha,beta-unsaturated aldehyde. By harnessing the stereoselective C-C bond forming activity of Fub7, we established a chemoenzymatic route for stereoconvergent synthesis of a series of 5-alkyl, 5,5-dialkyl, and 5,5,6-trialkyl-L-pipecolic acids of high diastereomeric ratio. </p></div>

scholarly journals Mapping the biosynthetic pathway of a hybrid polyketide-nonribosomal peptide in a metazoan

2021 ◽  
Author(s):  
Likui Feng ◽  
Matthew T. Gordon ◽  
Ying Liu ◽  
Kari B. Basso ◽  
Rebecca A. Butcher
Keyword(s):  
Protein Interactions ◽  
Biosynthetic Pathway ◽  
Polyketide Synthase ◽  
Carrier Protein ◽  
Direct Transfer ◽  
Protein Protein Interactions ◽  
Nematode Caenorhabditis Elegans ◽  
Nonribosomal Peptide ◽  
Peptide Synthetase ◽  
Complex Protein

Hybrid polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) systems typically use complex protein-protein interactions to facilitate direct transfer of intermediates between megasynthases. In the nematode Caenorhabditis elegans, PKS-1 and NRPS-1 produce the nemamides, the only known hybrid polyketide-nonribosomal peptides in animals, through a poorly understood mechanism. Here, we use genome editing and mass spectrometry to map the roles of individual PKS-1 and NRPS-1 enzymatic domains in nemamide biosynthesis. Furthermore, we show that nemamide biosynthesis requires at least five additional stand-alone enzymes that are encoded by genes distributed across the worm genome. We identify the roles of these enzymes in the biosynthetic pathway and discover a novel mechanism of trafficking intermediates between a PKS and an NRPS. Specifically, we show that the enzyme PKAL-1 activates an advanced polyketide intermediate as an adenylate and directly loads it onto a carrier protein in NRPS-1. This trafficking provides a means by which a PKS-NRPS system can expand its biosynthetic potential and is likely important for the regulation of nemamide biosynthesis.

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.

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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