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Heterologous expression of Gaeumannomyces graminis lipoxygenase in Aspergillus nidulans.

Heshof R, van Schayck JP, Tamayo-Ramos JA, de Graaff LH - AMB Express (2014)

Bottom Line: The presence of the recombinant LOX induced phenotypic changes in A. nidulans transformants.We observed that the presence of LOX induced the specific production of aminopeptidase Y that possibly degrades the G. graminis lipoxygenase intercellularly.Also the presence of the protein thioredoxin reductase suggests that the G. graminis lipoxygenase is actively repressed in A. nidulans.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Systems and Synthetic Biology, Microbial Systems and Synthetic Biology, Dreijenplein 10, Wageningen 6703 HB, Netherlands.

ABSTRACT
Aspergillus sp. contain ppo genes coding for Ppo enzymes that produce oxylipins from polyunsaturated fatty acids. These oxylipins function as signal molecules in sporulation and influence the asexual to sexual ratio of Aspergillus sp. Fungi like Aspergillus nidulans and Aspergillus niger contain just ppo genes where the human pathogenic Aspergillus flavus and Aspergillus fumigatus contain ppo genes as well as lipoxygenases. Lipoxygenases catalyze the synthesis of oxylipins and are hypothesized to be involved in quorum-sensing abilities and invading plant tissue. In this study we used A. nidulans WG505 as an expression host to heterologously express Gaeumannomyces graminis lipoxygenase. The presence of the recombinant LOX induced phenotypic changes in A. nidulans transformants. Also, a proteomic analysis of an A. nidulans LOX producing strain indicated that the heterologous protein was degraded before its glycosylation in the secretory pathway. We observed that the presence of LOX induced the specific production of aminopeptidase Y that possibly degrades the G. graminis lipoxygenase intercellularly. Also the presence of the protein thioredoxin reductase suggests that the G. graminis lipoxygenase is actively repressed in A. nidulans.

No MeSH data available.


Related in: MedlinePlus

Immunoprecipitation ofG. graminisLOX in cell free extract (CFE) and culture broth (CB) ofA. nidulanswild type (WT) and the transformant (T) using polyclonal antibodies againstG. graminisLOX. In sample Tcfe a protein band is found at ~67 kDa, which corresponds to the size G. graminis LOX using proteomics (1), a protein band of ~54 kDa represents aminopeptidase Y (2), and a protein band ~41 kDa is thioredoxin reductase (3).
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Figure 4: Immunoprecipitation ofG. graminisLOX in cell free extract (CFE) and culture broth (CB) ofA. nidulanswild type (WT) and the transformant (T) using polyclonal antibodies againstG. graminisLOX. In sample Tcfe a protein band is found at ~67 kDa, which corresponds to the size G. graminis LOX using proteomics (1), a protein band of ~54 kDa represents aminopeptidase Y (2), and a protein band ~41 kDa is thioredoxin reductase (3).

Mentions: Immunoprecipitation was done on both cell free extract and culture broth for the identification of G. graminis LOX. Three different proteins were identified in the A. nidulans WG505 wild type and the A. nidulans carrying the G. graminis LOX as shown in Figure 4. Analysis of the A. nidulans transformant revealed intracellular production of G. graminis LOX (1) but the LOX was not found in the culture broth. Also, aminopeptidase Y (2) was identified in the A. nidulans transformant. A third difference is the transformant showed production of thioredoxin reductase (3). This protein functions as a defense agent against oxidative damage and could well be a reflection of the production and activity of G. graminis LOX (Missall and Lodge [2005]). Finally a difference is found in the production of the PpoC enzyme. In the A. nidulans WG505 wild type this protein is present in the cell free extract and the culture broth. However, only small traces of PpoC were found in the transformant.


Heterologous expression of Gaeumannomyces graminis lipoxygenase in Aspergillus nidulans.

Heshof R, van Schayck JP, Tamayo-Ramos JA, de Graaff LH - AMB Express (2014)

Immunoprecipitation ofG. graminisLOX in cell free extract (CFE) and culture broth (CB) ofA. nidulanswild type (WT) and the transformant (T) using polyclonal antibodies againstG. graminisLOX. In sample Tcfe a protein band is found at ~67 kDa, which corresponds to the size G. graminis LOX using proteomics (1), a protein band of ~54 kDa represents aminopeptidase Y (2), and a protein band ~41 kDa is thioredoxin reductase (3).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4230170&req=5

Figure 4: Immunoprecipitation ofG. graminisLOX in cell free extract (CFE) and culture broth (CB) ofA. nidulanswild type (WT) and the transformant (T) using polyclonal antibodies againstG. graminisLOX. In sample Tcfe a protein band is found at ~67 kDa, which corresponds to the size G. graminis LOX using proteomics (1), a protein band of ~54 kDa represents aminopeptidase Y (2), and a protein band ~41 kDa is thioredoxin reductase (3).
Mentions: Immunoprecipitation was done on both cell free extract and culture broth for the identification of G. graminis LOX. Three different proteins were identified in the A. nidulans WG505 wild type and the A. nidulans carrying the G. graminis LOX as shown in Figure 4. Analysis of the A. nidulans transformant revealed intracellular production of G. graminis LOX (1) but the LOX was not found in the culture broth. Also, aminopeptidase Y (2) was identified in the A. nidulans transformant. A third difference is the transformant showed production of thioredoxin reductase (3). This protein functions as a defense agent against oxidative damage and could well be a reflection of the production and activity of G. graminis LOX (Missall and Lodge [2005]). Finally a difference is found in the production of the PpoC enzyme. In the A. nidulans WG505 wild type this protein is present in the cell free extract and the culture broth. However, only small traces of PpoC were found in the transformant.

Bottom Line: The presence of the recombinant LOX induced phenotypic changes in A. nidulans transformants.We observed that the presence of LOX induced the specific production of aminopeptidase Y that possibly degrades the G. graminis lipoxygenase intercellularly.Also the presence of the protein thioredoxin reductase suggests that the G. graminis lipoxygenase is actively repressed in A. nidulans.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Systems and Synthetic Biology, Microbial Systems and Synthetic Biology, Dreijenplein 10, Wageningen 6703 HB, Netherlands.

ABSTRACT
Aspergillus sp. contain ppo genes coding for Ppo enzymes that produce oxylipins from polyunsaturated fatty acids. These oxylipins function as signal molecules in sporulation and influence the asexual to sexual ratio of Aspergillus sp. Fungi like Aspergillus nidulans and Aspergillus niger contain just ppo genes where the human pathogenic Aspergillus flavus and Aspergillus fumigatus contain ppo genes as well as lipoxygenases. Lipoxygenases catalyze the synthesis of oxylipins and are hypothesized to be involved in quorum-sensing abilities and invading plant tissue. In this study we used A. nidulans WG505 as an expression host to heterologously express Gaeumannomyces graminis lipoxygenase. The presence of the recombinant LOX induced phenotypic changes in A. nidulans transformants. Also, a proteomic analysis of an A. nidulans LOX producing strain indicated that the heterologous protein was degraded before its glycosylation in the secretory pathway. We observed that the presence of LOX induced the specific production of aminopeptidase Y that possibly degrades the G. graminis lipoxygenase intercellularly. Also the presence of the protein thioredoxin reductase suggests that the G. graminis lipoxygenase is actively repressed in A. nidulans.

No MeSH data available.


Related in: MedlinePlus