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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

Distribution ofppoandloxgenes amongst four differentAspergillusspecies. The black box indicates the presence of the ppo gene. The blue box indicates the presence of a lox gene coding for an intracellular LOX, and the yellow box represents a lox gene coding for an extracellular LOX. A. nidulans and A. niger have three different ppo genes. A. flavus has four different ppo genes and a lox gene. A. fumigatus has three different ppo genes and two lox genes.
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Figure 1: Distribution ofppoandloxgenes amongst four differentAspergillusspecies. The black box indicates the presence of the ppo gene. The blue box indicates the presence of a lox gene coding for an intracellular LOX, and the yellow box represents a lox gene coding for an extracellular LOX. A. nidulans and A. niger have three different ppo genes. A. flavus has four different ppo genes and a lox gene. A. fumigatus has three different ppo genes and two lox genes.

Mentions: Aspergillus sp. contains ppo genes coding for dioxygenases that belong to the linoleate diol synthase (LDS) protein family. These dioxygenases produce oxylipins called precocious sexual inducer (psi) factors, which is a collective term for C18:1, C18:2 and C18:3 derived oxylipins (Gao et al. [2007]). Oxylipins are signal molecules that are used by fungi to control sporulation (Brodhun and Feussner [2011]). These oxylipins are categorized in three groups depending on the position of hydroxyl groups on the polyunsaturated fatty acid (PUFA): psiB (8′-hydroxy-PUFA), psiC (5′,8′-dihydroxy-PUFA), and psiA, which has a δ-lactone ring at the 5′ position of the psiC oxylipin (Tsitsigiannis et al. [2004]). Depending on the PUFA it is further categorized as α (18:2, linoleic acid), β (18:1, oleic acid), and γ (18:3, linolenic acid) (Tsitsigiannis and Keller [2007]). 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]). Aspergillus niger also contains three ppo genes where it lacks ppoB but instead contains ppoD (Wadman et al. [2009]) In A. nidulans PpoA and PpoC have antagonistic roles and PpoB upregulates ppoA and represses ppoC (Tsitsigiannis et al. [2005]; Brodhun and Feussner [2011]). The deletion of the ppoA and ppoB genes increase the ratio of asexual to sexual sporulation, while deletion of the ppoC gene decreases the ratio of asexual to sexual sporulation. However, it is speculated that these sporulation phenotypes cannot be explained by the psiB oxylipin levels alone (Tsitsigiannis et al. [2005]). Therefore it is suggested that other oxylipins, produced by either Ppo’s or other enzymes, are involved in these phenotypic differences. Lipoxygenase (LOX) is a non-heme 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]). However, the human pathogenic Aspergillus flavus and Aspergillus fumigatus contain both ppo genes and lox genes (Brown et al. [2008]; Affeldt et al. [2012]). The distribution of ppo and lox genes is schematically given in Figure 1. Studies revealed that LOX in A. flavus is involved in quorum-sensing and phenotypic differences are seen when the gene is disrupted (Brown et al. [2008]; Affeldt et al. [2012]). The Gaeumannomyces graminis LOX is a secreted enzyme that is capable of producing 11S-HPODE and 13R-HPODE oxylipins and is hypothesized to be involved in invading plant tissue (Oliw [2002]). Plant oxylipins 9S-HPODE and 13S-HPODE induce sporogenic effects in A. nidulans similar to the ones produced by the psi factor (Calvo et al. [1999]). Both oxylipins caused decreased mycelial growth where 13S-HPODE in a 10–100 μM concentration also reduces mycotoxin production of aflatoxin and sterigmatocystin (Burow et al. [1997]). In this study we introduced the G. graminis LOX in the A. nidulans WG505 production host. The goal of the study was to verify whether A. nidulans is a suitable host for the heterologous expression of 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)

Distribution ofppoandloxgenes amongst four differentAspergillusspecies. The black box indicates the presence of the ppo gene. The blue box indicates the presence of a lox gene coding for an intracellular LOX, and the yellow box represents a lox gene coding for an extracellular LOX. A. nidulans and A. niger have three different ppo genes. A. flavus has four different ppo genes and a lox gene. A. fumigatus has three different ppo genes and two lox genes.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Distribution ofppoandloxgenes amongst four differentAspergillusspecies. The black box indicates the presence of the ppo gene. The blue box indicates the presence of a lox gene coding for an intracellular LOX, and the yellow box represents a lox gene coding for an extracellular LOX. A. nidulans and A. niger have three different ppo genes. A. flavus has four different ppo genes and a lox gene. A. fumigatus has three different ppo genes and two lox genes.
Mentions: Aspergillus sp. contains ppo genes coding for dioxygenases that belong to the linoleate diol synthase (LDS) protein family. These dioxygenases produce oxylipins called precocious sexual inducer (psi) factors, which is a collective term for C18:1, C18:2 and C18:3 derived oxylipins (Gao et al. [2007]). Oxylipins are signal molecules that are used by fungi to control sporulation (Brodhun and Feussner [2011]). These oxylipins are categorized in three groups depending on the position of hydroxyl groups on the polyunsaturated fatty acid (PUFA): psiB (8′-hydroxy-PUFA), psiC (5′,8′-dihydroxy-PUFA), and psiA, which has a δ-lactone ring at the 5′ position of the psiC oxylipin (Tsitsigiannis et al. [2004]). Depending on the PUFA it is further categorized as α (18:2, linoleic acid), β (18:1, oleic acid), and γ (18:3, linolenic acid) (Tsitsigiannis and Keller [2007]). 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]). Aspergillus niger also contains three ppo genes where it lacks ppoB but instead contains ppoD (Wadman et al. [2009]) In A. nidulans PpoA and PpoC have antagonistic roles and PpoB upregulates ppoA and represses ppoC (Tsitsigiannis et al. [2005]; Brodhun and Feussner [2011]). The deletion of the ppoA and ppoB genes increase the ratio of asexual to sexual sporulation, while deletion of the ppoC gene decreases the ratio of asexual to sexual sporulation. However, it is speculated that these sporulation phenotypes cannot be explained by the psiB oxylipin levels alone (Tsitsigiannis et al. [2005]). Therefore it is suggested that other oxylipins, produced by either Ppo’s or other enzymes, are involved in these phenotypic differences. Lipoxygenase (LOX) is a non-heme 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]). However, the human pathogenic Aspergillus flavus and Aspergillus fumigatus contain both ppo genes and lox genes (Brown et al. [2008]; Affeldt et al. [2012]). The distribution of ppo and lox genes is schematically given in Figure 1. Studies revealed that LOX in A. flavus is involved in quorum-sensing and phenotypic differences are seen when the gene is disrupted (Brown et al. [2008]; Affeldt et al. [2012]). The Gaeumannomyces graminis LOX is a secreted enzyme that is capable of producing 11S-HPODE and 13R-HPODE oxylipins and is hypothesized to be involved in invading plant tissue (Oliw [2002]). Plant oxylipins 9S-HPODE and 13S-HPODE induce sporogenic effects in A. nidulans similar to the ones produced by the psi factor (Calvo et al. [1999]). Both oxylipins caused decreased mycelial growth where 13S-HPODE in a 10–100 μM concentration also reduces mycotoxin production of aflatoxin and sterigmatocystin (Burow et al. [1997]). In this study we introduced the G. graminis LOX in the A. nidulans WG505 production host. The goal of the study was to verify whether A. nidulans is a suitable host for the heterologous expression of 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