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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

SDS-PAGE and western blot analysis ofG. graminisLOX expressionA. nidulans. Samples T1-T3 are transformants of A. nidulans carrying the G. graminis lox gene, while M1-M3 are transformants of A. nidulans carrying the mutated G. graminis lox gene. As positive controls 10 ng, 100 ng, and 1000 ng of the G. graminis LOX were applied. The wild type (WT) sample is used as a negative control. a) SDS-PAGE of the proteins in the culture broth of A. nidulans; b) SDS-PAGE of intracellular proteins of A. nidulans; c) Western blot of the proteins in the culture broth reacting to the antibodies of the G. graminis LOX; d) Western blot of intracellular proteins reacting to the antibodies of G. graminis LOX.
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Figure 3: SDS-PAGE and western blot analysis ofG. graminisLOX expressionA. nidulans. Samples T1-T3 are transformants of A. nidulans carrying the G. graminis lox gene, while M1-M3 are transformants of A. nidulans carrying the mutated G. graminis lox gene. As positive controls 10 ng, 100 ng, and 1000 ng of the G. graminis LOX were applied. The wild type (WT) sample is used as a negative control. a) SDS-PAGE of the proteins in the culture broth of A. nidulans; b) SDS-PAGE of intracellular proteins of A. nidulans; c) Western blot of the proteins in the culture broth reacting to the antibodies of the G. graminis LOX; d) Western blot of intracellular proteins reacting to the antibodies of G. graminis LOX.

Mentions: A western blot analysis was performed on three different transformants carrying the G. graminis lox gene (T1-T3) and three different G. graminis lox H306Q-H310E mutant transformants (M1-M3) for which the results are shown in Figure 3. Western blot analysis revealed A. nidulans has two different protein band fingerprints seen in samples WT, T2, T3, M2 and T1, M1, M3 (Figure 3a). This coincides with the single and double band found in the western blot analysis of the intracellular proteins of A. nidulans (Figure 3d). The presence of the bands found in the wild type samples showed non-selective binding of polyclonal antibodies. Production of the G. graminis LOX was neither detected in the supernatants nor in the cell free extract. Also the mutant version of the G. graminis LOX was not be detected in the M1-M3 samples. Since the expression of the G. graminis LOX was not be detected at protein level by SDS-PAGE nor by western blot, a proteomic analysis was performed of the proteins isolated by immunoprecipitation using the polyclonal antibodies against G. graminis LOX.


Heterologous expression of Gaeumannomyces graminis lipoxygenase in Aspergillus nidulans.

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

SDS-PAGE and western blot analysis ofG. graminisLOX expressionA. nidulans. Samples T1-T3 are transformants of A. nidulans carrying the G. graminis lox gene, while M1-M3 are transformants of A. nidulans carrying the mutated G. graminis lox gene. As positive controls 10 ng, 100 ng, and 1000 ng of the G. graminis LOX were applied. The wild type (WT) sample is used as a negative control. a) SDS-PAGE of the proteins in the culture broth of A. nidulans; b) SDS-PAGE of intracellular proteins of A. nidulans; c) Western blot of the proteins in the culture broth reacting to the antibodies of the G. graminis LOX; d) Western blot of intracellular proteins reacting to the antibodies of G. graminis LOX.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: SDS-PAGE and western blot analysis ofG. graminisLOX expressionA. nidulans. Samples T1-T3 are transformants of A. nidulans carrying the G. graminis lox gene, while M1-M3 are transformants of A. nidulans carrying the mutated G. graminis lox gene. As positive controls 10 ng, 100 ng, and 1000 ng of the G. graminis LOX were applied. The wild type (WT) sample is used as a negative control. a) SDS-PAGE of the proteins in the culture broth of A. nidulans; b) SDS-PAGE of intracellular proteins of A. nidulans; c) Western blot of the proteins in the culture broth reacting to the antibodies of the G. graminis LOX; d) Western blot of intracellular proteins reacting to the antibodies of G. graminis LOX.
Mentions: A western blot analysis was performed on three different transformants carrying the G. graminis lox gene (T1-T3) and three different G. graminis lox H306Q-H310E mutant transformants (M1-M3) for which the results are shown in Figure 3. Western blot analysis revealed A. nidulans has two different protein band fingerprints seen in samples WT, T2, T3, M2 and T1, M1, M3 (Figure 3a). This coincides with the single and double band found in the western blot analysis of the intracellular proteins of A. nidulans (Figure 3d). The presence of the bands found in the wild type samples showed non-selective binding of polyclonal antibodies. Production of the G. graminis LOX was neither detected in the supernatants nor in the cell free extract. Also the mutant version of the G. graminis LOX was not be detected in the M1-M3 samples. Since the expression of the G. graminis LOX was not be detected at protein level by SDS-PAGE nor by western blot, a proteomic analysis was performed of the proteins isolated by immunoprecipitation using the polyclonal antibodies against G. graminis LOX.

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