scholarly journals The role of swnR gene on the biosynthesis pathway of the swainsonine in Metarhizium anisopliae

2019 ◽  
Author(s):  
Lu Sun ◽  
Runjie Song ◽  
Jinglong Wang ◽  
Yiling Liu ◽  
Yu Zhang ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Metarhizium Anisopliae ◽  
Type Strain ◽  
Fermentation Broth ◽  
Biosynthesis Pathway ◽  
Mutant Type ◽  
Knock Out ◽  
Q Exactive ◽  
Precise Role

AbstractSwainsonine (SW) is the principal toxic ingredient of locoweeds, and is produced by fungi including Metarhizium anisopliae, Slafractonia leguminicola, and Alternaria oxytropis. Studies of the SW biosynthesis pathway in these fungi have demonstrated the requirement for a swnK gene and the presence of a variety of other SWN cluster genes, but have not determined a precise role for the swnR gene, which encodes a NADB Rossmann-fold reductase, nor if it is necessary for the biosynthesis of SW. In this study, we used homologous recombination (HR) to knock out the swnR gene of M. anisopliae to determine its effect on the SW biosynthesis pathway. The concentration of SW was measured in the fermentation broth of M. anisopliae at 1 d, 3 d, 5 d and 7 d using a Q Exactive Mass Spectrometer. The gene for swnR was detected by RT-qPCR. To determine the role of the swnR gene in the SW biosynthesis pathway of M. anisopliae, we used PEG-mediated homologous recombination (HR) to transform a wild-type strain (WT) with a Benomyl (ben)-resistant fragment to knock out the swnR gene producing a mutant-type strain (MT). A complemented-type (CT) strain was produced by adding a complementation vector that contains the glufosinate (herbicide) resistance (bar) gene as a marker. The content of SW decreased, but was not eliminated in the fermentation broth of the MT strain, and returned to the original level in the CT strain. These results indicate that the swnR gene plays a crucial role in the SW biosynthesis pathway of M. anisopliae, but suggests that another gene in the fungus may share the function of swnR.

scholarly journals The role of swnR gene on the biosynthesis pathway of the swainsonine in Metarhizium anisopliae

2020 ◽  
Author(s):  
Lu Sun ◽  
Runjie Song ◽  
Jinglong Wang ◽  
Yu Zhang ◽  
Yanli Zhu ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Metarhizium Anisopliae ◽  
Type Strain ◽  
Fermentation Broth ◽  
Biosynthesis Pathway ◽  
Mutant Type ◽  
Knock Out ◽  
Q Exactive ◽  
Precise Role

Abstract Background: Swainsonine (SW) is the principal toxic ingredient of locoweeds, and is produced by fungi including Metarhizium anisopliae, Slafractonia leguminicola, and Alternaria oxytropis (found in locoweeds of Oxytropis). Studies of the SW biosynthesis pathway in these fungi have demonstrated the requirement for a swnK gene and the presence of a variety of other SWN cluster genes, but have not determined a precise role for the swnR gene, which encodes a NADB Rossmann-fold reductase, nor if it is necessary for the biosynthesis of SW. In this study, we used homologous recombination (HR) to knock out the swnR gene of M. anisopliae to determine its effect on the SW biosynthesis pathway.Results: The concentration of SW was measured in the fermentation broth of M. anisopliae at 1 d, 3 d, 5 d and 7 d using a Q Exactive Mass Spectrometer. The gene for swnR was detected by RT-qPCR. To determine the role of the swnR gene in the SW biosynthesis pathway of M. anisopliae, we used PEG-mediated homologous recombination (HR) to transform a wild-type strain (WT) with a Benomyl (ben)-resistant fragment to knock out the swnR gene producing a mutant-type strain (MT). A complemented-type (CT) strain was produced by adding a complementation vector that contains the glufosinate (herbicide) resistance (bar) gene as a marker. The content of SW decreased, but was not eliminated in the fermentation broth of the MT strain, and returned to the original level in the CT strain.Conclusions: These results indicate that the swnR gene plays a crucial role in the SW biosynthesis pathway of M. anisopliae, but suggests that another gene in the fungus may share the function of swnR.

The Critical Role of Polyketide Synthase Gene On The Swainsonine Biosynthesis in The Fungus Metarhizium Anisopliae

2021 ◽  
Author(s):  
Lu Sun ◽  
Enxia Huang ◽  
Yu Zhang ◽  
Ziyu Guo ◽  
Kexin Wu ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Metarhizium Anisopliae ◽  
Type Strain ◽  
Polyketide Synthase ◽  
Wild Type Strain ◽  
Biosynthesis Pathway ◽  
Wild Type ◽  
Knock Out ◽  
Polyketide Synthase Gene

Abstract Swainsonine (SW) is the principal toxic ingredient of locoweeds, and is produced by fungi including Metarhizium anisopliae, Slafractonia leguminicola, and Alternaria oxytropis. While the SW biosynthesis pathway of fungi and the catalytic enzyme genes that regulate synthesis are not cleanly. In this study, we used homologous recombination (HR) to knock out and interfere with the polyketide synthase gene (pks) of M. anisopliae to determine its effect on the SW biosynthesis pathway. The concentration of SW was measured in the fermentation broth of M. anisopliae at 1 d, 2 d, 3 d, 4 d, 5 d, 6 d or 7 d using LC-MS. The gene for the pks gene was detected by RT-qPCR. Day 5 of M. anisopliae gave the highest content of SW and the highest expression of the pks gene. To determine the role of the pks gene in the SW biosynthesis pathway of M. anisopliae, we used PEG-mediated homologous recombination (HR) to transform a wild-type strain (WT) with a Benomyl (ben)-resistant fragment to knock out the pks gene producing a mutant-type strain (MT) and used PEG-mediated RNAi to transform a wild-type strain (WT) with a Benomyl (ben)-resistant plasmid to interfere with the pks gene. A complemented-type (CT) strain was produced by adding a complementation vector that contains the geneticin (G418) resistance gene as a marker. The content of SW didn’t detected in MT strain, and returned to the original level in the CT strain, while the content of SW was significantly decreased in RNAi strain. We suggest that mutation and RNAi in the pks gene affect the cell wall formation of M. anisopliae, while the colony diameters, phenotypes, and growth rates did not change significantly, and no obvious changes in other cellular organelles were noted. These results indicate that the pks gene plays a crucial role in the SW biosynthesis of M. anisopliae, which provides an important theoretical basis for illuminating the SW biosynthesis and solving locoism in livestock.

scholarly journals Depletion of Akt1 and Akt2 Impairs the Repair of Radiation-Induced DNA Double Strand Breaks via Homologous Recombination

2019 ◽  
Vol 20 (24) ◽  
pp. 6316 ◽  
Author(s):  
Tahereh Mohammadian Gol ◽  
H. Peter Rodemann ◽  
Klaus Dittmann
Keyword(s):  
Homologous Recombination ◽  
Double Strand Breaks ◽  
Major Protein ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Knock Out ◽  
Strand Breaks ◽  
Protein Levels ◽  
Non Homologous End Joining

Homologous recombination repair (HRR), non-homologous end-joining (NHEJ) and alternative NHEJ are major pathways that are utilized by cells for processing DNA double strand breaks (DNA-DSBs); their function plays an important role in the radiation resistance of tumor cells. Conflicting data exist regarding the role of Akt in homologous recombination (HR), i.e., the regulation of Rad51 as a major protein of this pathway. This study was designed to investigate the specific involvement of Akt isoforms in HRR. HCT116 colon cancer cells with stable AKT-knock-out and siRNA-mediated AKT-knockdown phenotypes were used to investigate the role of Akt1 and Akt2 isoforms in HR. The results clearly demonstrated that HCT116 AKT1-KO and AKT2-KO cells have a significantly reduced Rad51 foci formation 6 h post irradiation versus parental cells. Depletion of Akt1 and Akt2 protein levels as well as inhibition of Akt kinase activity resulted in an increased number of residual-γH2AX in CENP-F positive cells mainly representing the S and G2 phase cells. Furthermore, inhibition of NHEJ and HR using DNA-PK and Rad51 antagonists resulted in stronger radiosensitivity of AKT1 and AKT2 knockout cells versus wild type cells. These data collectively show that both Akt1 and Akt2 are involved in DSBs repair through HRR.

scholarly journals Xanthomonas oryzae pv. oryzae XKK.12 Contains an AroQγ Chorismate Mutase That Is Involved in Rice Virulence

2010 ◽  
Vol 100 (3) ◽  
pp. 262-270 ◽  
Author(s):  
Giuliano Degrassi ◽  
Giulia Devescovi ◽  
Joseph Bigirimana ◽  
Vittorio Venturi
Keyword(s):  
Pathogenic Bacteria ◽  
Shikimate Pathway ◽  
Aromatic Amino Acids ◽  
Xanthomonas Oryzae ◽  
Chorismate Mutase ◽  
In Planta ◽  
Knock Out ◽  
Key Enzyme ◽  
Precise Role

Chorismate mutase (CM) is a key enzyme in the shikimate pathway which is responsible for the synthesis of aromatic amino acids. There are two classes of CMs, AroQ and AroH, and several pathogenic bacteria have been reported to possess a subgroup of CMs designated AroQγ. These CMs are usually exported to the periplasm or outside the cell; in a few cases, they have been reported to be involved in virulence and their precise role is currently unknown. Here, we report that the important rice pathogen Xanthomonas oryzae pv. oryzae XKK.12 produces an AroQγ CM which we have purified and characterized from spent supernatants. This enzyme is synthesized in planta and X. oryzae pv. oryzae knock-out mutants are hypervirulent to rice. The role of this enzyme in X. oryzae pv. oryzae rice virulence is discussed.

Monosaccharide Biosynthesis Pathways Database

Glycobiology ◽  
2021 ◽  
Author(s):  
Jaya Srivastava ◽  
P Sunthar ◽  
Petety V Balaji
Keyword(s):  
Expression Profiling ◽  
Experimental Studies ◽  
Structural Complexity ◽  
Evolutionary Relationships ◽  
Enzyme Assays ◽  
Biosynthesis Pathway ◽  
Knock Out ◽  
Set Up ◽  
Function Relationship ◽  
Natural Glycans

Abstract A distinctive feature of glycans vis-à-vis proteins and nucleic acids is its structural complexity which arises from the huge repertoire of monosaccharides, isomeric linkages and branching. A very large number of monosaccharides have so far been discovered in natural glycans. Experimentally, pathways for the biosynthesis have been characterized completely for 55 monosaccharides and partially for a few more. However, there is no single platform which provides information about monosaccharide biosynthesis pathways and associated enzymes We have gathered 572 experimentally characterized enzymes of 66 biosynthesis pathways from literature and set up a first of its kind database called the Monosaccharide Biosynthesis Pathways Database http://www.bio.iitb.ac.in/mbpd/). Annotations such as the reaction catalysed, substrate specificity, biosynthesis pathway and PubMed IDs are provided for all the enzymes in the database. Sequence homologs of the experimentally characterized enzymes found in nearly 13,000 completely sequenced genomes from Bacteria and Archaea have also been included in the database. This platform will help in the deduction of evolutionary relationships among enzymes such as aminotransferases, nucleotidyltransferases, acetyltransferases and SDR family enzymes. It can also facilitate experimental studies such as direct enzyme assays to validate putative annotations, establish structure–function relationship, expression profiling to determine the function, determine the phenotypic consequences of gene knock-out/knock-in and complementation studies.

scholarly journals Epigenomic Analysis of RAD51 ChIP-seq Data Reveals cis-regulatory Elements Associated with Autophagy in Cancer Cell Lines

Cancers ◽  
2021 ◽  
Vol 13 (11) ◽  
pp. 2547
Author(s):  
Keunsoo Kang ◽  
Yoonjung Choi ◽  
Hyeonjin Moon ◽  
Chaelin You ◽  
Minjin Seo ◽  
...  
Keyword(s):  
Gene Regulation ◽  
Homologous Recombination ◽  
Cell Lines ◽  
Regulatory Elements ◽  
Binding Motif ◽  
Genome Wide ◽  
E Box ◽  
Autophagy Pathway ◽  
Mcf 7

RAD51 is a recombinase that plays a pivotal role in homologous recombination. Although the role of RAD51 in homologous recombination has been extensively studied, it is unclear whether RAD51 can be involved in gene regulation as a co-factor. In this study, we found evidence that RAD51 may contribute to the regulation of genes involved in the autophagy pathway with E-box proteins such as USF1, USF2, and/or MITF in GM12878, HepG2, K562, and MCF-7 cell lines. The canonical USF binding motif (CACGTG) was significantly identified at RAD51-bound cis-regulatory elements in all four cell lines. In addition, genome-wide USF1, USF2, and/or MITF-binding regions significantly coincided with the RAD51-associated cis-regulatory elements in the same cell line. Interestingly, the promoters of genes associated with the autophagy pathway, such as ATG3 and ATG5, were significantly occupied by RAD51 and regulated by RAD51 in HepG2 and MCF-7 cell lines. Taken together, these results unveiled a novel role of RAD51 and provided evidence that RAD51-associated cis-regulatory elements could possibly be involved in regulating autophagy-related genes with E-box binding proteins.

scholarly journals Loss of microglial SIRPα promotes synaptic pruning in preclinical models of neurodegeneration

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Xin Ding ◽  
Jin Wang ◽  
Miaoxin Huang ◽  
Zhangpeng Chen ◽  
Jing Liu ◽  
...  
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Knock Out ◽  
Classical Complement Pathway ◽  
Synaptic Remodeling ◽  
The Central Nervous System ◽  
System Activation ◽  
Knock Out Mice ◽  
Synaptic Pruning

AbstractMicroglia play a key role in regulating synaptic remodeling in the central nervous system. Activation of classical complement pathway promotes microglia-mediated synaptic pruning during development and disease. CD47 protects synapses from excessive pruning during development, implicating microglial SIRPα, a CD47 receptor, in synaptic remodeling. However, the role of microglial SIRPα in synaptic pruning in disease remains unclear. Here, using conditional knock-out mice, we show that microglia-specific deletion of SIRPα results in decreased synaptic density. In human tissue, we observe that microglial SIRPα expression declines alongside the progression of Alzheimer’s disease. To investigate the role of SIRPα in neurodegeneration, we modulate the expression of microglial SIRPα in mouse models of Alzheimer’s disease. Loss of microglial SIRPα results in increased synaptic loss mediated by microglia engulfment and enhanced cognitive impairment. Together, these results suggest that microglial SIRPα regulates synaptic pruning in neurodegeneration.

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