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.
Highlights
Aspergillus sp. contains ppo genes coding for dioxygenases that belong to the linoleate diol synthase (LDS) protein family
Immunoprecipitation using G. graminis LOX antibody and proteomics analysis A. nidulans WG505 and A. nidulans GG-LOX were grown with 50 mM D-xylose for 48 h and the culture broth and mycelium were separated by funnel filtration The mycelium was disrupted three times by French Press with a pressure of 1,000 psi and the resulting cell free extract was prepared by centrifugation
Immunoprecipitation was performed using 100 μl Dynabeads mRNA identification of the G. graminis lox gene In order to confirm that the G. graminis LOX gene was successfully transcribed in the A. nidulans GG-LOX transformant mRNA was isolated from both the wild type and the A. nidulans GG-LOX
Summary
Aspergillus sp. contains ppo genes coding for dioxygenases that belong to the linoleate diol synthase (LDS) protein family. Contains ppo genes coding for dioxygenases that belong to the linoleate diol synthase (LDS) protein family. Oxylipins are signal molecules that are used by fungi to control sporulation (Brodhun and Feussner 2011). Aspergillus nidulans contains three ppo genes: ppoA, ppoB, and ppoC coding for enzymes that synthesize the oxylipins psiBα, psiBβ, and psiBβ respectively (Brodhun and Feussner 2011). Lipoxygenase (LOX) is a nonheme iron- or managese-containing dioxygenase present in a wide variety of organisms, including fungi (Heshof et al 2013). They catalyze the synthesis of oxylipins, but are absent in A. nidulans and A. niger (Wadman et al 2009).
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