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The intra- and extracellular proteome of Aspergillus niger growing on defined medium with xylose or maltose as carbon substrate.

Lu X, Sun J, Nimtz M, Wissing J, Zeng AP, Rinas U - Microb. Cell Fact. (2010)

Bottom Line: For example, glucoamylase is the most efficiently secreted protein of Aspergillus niger, thus the homologous glucoamylase (glaA) promoter as well as the glaA signal sequence are widely used for heterologous protein production.For a more profound understanding of A. niger physiology, a comprehensive analysis of the intra- and extracellular proteome of Aspergillus niger AB1.13 growing on defined medium with xylose or maltose as carbon substrate was carried out using 2-D gel electrophoresis/Maldi-ToF and nano-HPLC MS/MS.The utilization of xylose or maltose was strongly affecting the composition of the secretome but of minor influence on the composition of the intracellular proteome.

View Article: PubMed Central - HTML - PubMed

Affiliation: Helmholtz Center for Infection Research, Inhoffenstr, Braunschweig, Germany.

ABSTRACT

Background: The filamentous fungus Aspergillus niger is well-known as a producer of primary metabolites and extracellular proteins. For example, glucoamylase is the most efficiently secreted protein of Aspergillus niger, thus the homologous glucoamylase (glaA) promoter as well as the glaA signal sequence are widely used for heterologous protein production. Xylose is known to strongly repress glaA expression while maltose is a potent inducer of glaA promoter controlled genes. For a more profound understanding of A. niger physiology, a comprehensive analysis of the intra- and extracellular proteome of Aspergillus niger AB1.13 growing on defined medium with xylose or maltose as carbon substrate was carried out using 2-D gel electrophoresis/Maldi-ToF and nano-HPLC MS/MS.

Results: The intracellular proteome of A. niger growing either on xylose or maltose in well-aerated controlled bioreactor cultures revealed striking similarities. In both cultures the most abundant intracellular protein was the TCA cycle enzyme malate-dehydrogenase. Moreover, the glycolytic enzymes fructose-bis-phosphate aldolase and glyceraldehyde-3-phosphate-dehydrogenase and the flavohemoglobin FhbA were identified as major proteins in both cultures. On the other hand, enzymes involved in the removal of reactive oxygen species, such as superoxide dismutase and peroxiredoxin, were present at elevated levels in the culture growing on maltose but only in minor amounts in the xylose culture. The composition of the extracellular proteome differed considerably depending on the carbon substrate. In the secretome of the xylose-grown culture, a variety of plant cell wall degrading enzymes were identified, mostly under the control of the xylanolytic transcriptional activator XlnR, with xylanase B and ferulic acid esterase as the most abundant ones. The secretome of the maltose-grown culture did not contain xylanolytic enzymes, instead high levels of catalases were found and glucoamylase (multiple spots) was identified as the most abundant extracellular protein. Surprisingly, the intracellular proteome of A. niger growing on xylose in bioreactor cultures differed more from a culture growing in shake flasks using the same medium than from the bioreactor culture growing on maltose. For example, in shake flask cultures with xylose as carbon source the most abundant intracellular proteins were not the glycolytic and the TCA cycle enzymes and the flavohemoglobin, but CipC, a protein of yet unknown function, superoxide dismutase and an NADPH dependent aldehyde reductase. Moreover, vacuolar proteases accumulated to higher and ER-resident chaperones and foldases to lower levels in shake flask compared to the bioreactor cultures.

Conclusions: The utilization of xylose or maltose was strongly affecting the composition of the secretome but of minor influence on the composition of the intracellular proteome. On the other hand, differences in culture conditions (pH control versus no pH control, aeration versus no aeration and stirring versus shaking) have a profound effect on the intracellular proteome. For example, lower levels of ER-resident chaperones and foldases and higher levels of vacuolar proteases render shake flask conditions less favorable for protein production compared to controlled bioreactor cultures.

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Change of extracellular proteome in response to maltose addition to a culture pregrown on xylose. Change of the extracellular proteome of A. niger AB1.13 in response to maltose addition to a bioreactor culture pregrown on xylose. (A) The composition of the extracellular proteome of cells grown on defined medium with xylose as carbon substrate. (B) Change of the extracellular proteome 24 h after the addition of maltose to a culture pregrown on xylose. Characteristics of proteins indicated by arrows are discussed in more detail. The basic side of the gel is on the right. The inserts on the right show the time-dependent fraction change of two selected proteins from the extracellular proteome after the addition of maltose. A detailed list of all proteins from the extracellular proteome of A. niger growing either on xylose or maltose identified on 2-D gels is found in Additional file 6.
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Figure 4: Change of extracellular proteome in response to maltose addition to a culture pregrown on xylose. Change of the extracellular proteome of A. niger AB1.13 in response to maltose addition to a bioreactor culture pregrown on xylose. (A) The composition of the extracellular proteome of cells grown on defined medium with xylose as carbon substrate. (B) Change of the extracellular proteome 24 h after the addition of maltose to a culture pregrown on xylose. Characteristics of proteins indicated by arrows are discussed in more detail. The basic side of the gel is on the right. The inserts on the right show the time-dependent fraction change of two selected proteins from the extracellular proteome after the addition of maltose. A detailed list of all proteins from the extracellular proteome of A. niger growing either on xylose or maltose identified on 2-D gels is found in Additional file 6.

Mentions: Proteins only present during growth on xylose are D-xylose reductase (XyrA), β-xylosidase (XlnD), arabinofuranosidase B (AbfB) and β-glucosidase (Bgl1 or BglA) (Fig. 3, Additional file 5). XyrA catalyses the NADPH dependent reduction of xylose to xylitol, which represents the first step in D-xylose metabolism. Expression of the xyrA gene is under the control of the xylanolytic transcriptional activator XlnR [25,26]. XlnD, also under control of XlnR, catalyses the hydrolysis of xylo-oligosaccharides [26,27]. It is also present in the medium (Fig. 4), and, thus probably as cell wall associated protein present in the mycelium. AbfB and Bgl1, both not controlled by XlnR [28], are enzymes involved in the hydrolytic release of L-arabinofuranosyl residues from arabinoxylan and arabinan and in glucose moieties from gluco-oligosaccharides, respectively. Both enzymes are also found in the medium, and, thus, are probably as XlnD present as cell wall associated proteins.


The intra- and extracellular proteome of Aspergillus niger growing on defined medium with xylose or maltose as carbon substrate.

Lu X, Sun J, Nimtz M, Wissing J, Zeng AP, Rinas U - Microb. Cell Fact. (2010)

Change of extracellular proteome in response to maltose addition to a culture pregrown on xylose. Change of the extracellular proteome of A. niger AB1.13 in response to maltose addition to a bioreactor culture pregrown on xylose. (A) The composition of the extracellular proteome of cells grown on defined medium with xylose as carbon substrate. (B) Change of the extracellular proteome 24 h after the addition of maltose to a culture pregrown on xylose. Characteristics of proteins indicated by arrows are discussed in more detail. The basic side of the gel is on the right. The inserts on the right show the time-dependent fraction change of two selected proteins from the extracellular proteome after the addition of maltose. A detailed list of all proteins from the extracellular proteome of A. niger growing either on xylose or maltose identified on 2-D gels is found in Additional file 6.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2874515&req=5

Figure 4: Change of extracellular proteome in response to maltose addition to a culture pregrown on xylose. Change of the extracellular proteome of A. niger AB1.13 in response to maltose addition to a bioreactor culture pregrown on xylose. (A) The composition of the extracellular proteome of cells grown on defined medium with xylose as carbon substrate. (B) Change of the extracellular proteome 24 h after the addition of maltose to a culture pregrown on xylose. Characteristics of proteins indicated by arrows are discussed in more detail. The basic side of the gel is on the right. The inserts on the right show the time-dependent fraction change of two selected proteins from the extracellular proteome after the addition of maltose. A detailed list of all proteins from the extracellular proteome of A. niger growing either on xylose or maltose identified on 2-D gels is found in Additional file 6.
Mentions: Proteins only present during growth on xylose are D-xylose reductase (XyrA), β-xylosidase (XlnD), arabinofuranosidase B (AbfB) and β-glucosidase (Bgl1 or BglA) (Fig. 3, Additional file 5). XyrA catalyses the NADPH dependent reduction of xylose to xylitol, which represents the first step in D-xylose metabolism. Expression of the xyrA gene is under the control of the xylanolytic transcriptional activator XlnR [25,26]. XlnD, also under control of XlnR, catalyses the hydrolysis of xylo-oligosaccharides [26,27]. It is also present in the medium (Fig. 4), and, thus probably as cell wall associated protein present in the mycelium. AbfB and Bgl1, both not controlled by XlnR [28], are enzymes involved in the hydrolytic release of L-arabinofuranosyl residues from arabinoxylan and arabinan and in glucose moieties from gluco-oligosaccharides, respectively. Both enzymes are also found in the medium, and, thus, are probably as XlnD present as cell wall associated proteins.

Bottom Line: For example, glucoamylase is the most efficiently secreted protein of Aspergillus niger, thus the homologous glucoamylase (glaA) promoter as well as the glaA signal sequence are widely used for heterologous protein production.For a more profound understanding of A. niger physiology, a comprehensive analysis of the intra- and extracellular proteome of Aspergillus niger AB1.13 growing on defined medium with xylose or maltose as carbon substrate was carried out using 2-D gel electrophoresis/Maldi-ToF and nano-HPLC MS/MS.The utilization of xylose or maltose was strongly affecting the composition of the secretome but of minor influence on the composition of the intracellular proteome.

View Article: PubMed Central - HTML - PubMed

Affiliation: Helmholtz Center for Infection Research, Inhoffenstr, Braunschweig, Germany.

ABSTRACT

Background: The filamentous fungus Aspergillus niger is well-known as a producer of primary metabolites and extracellular proteins. For example, glucoamylase is the most efficiently secreted protein of Aspergillus niger, thus the homologous glucoamylase (glaA) promoter as well as the glaA signal sequence are widely used for heterologous protein production. Xylose is known to strongly repress glaA expression while maltose is a potent inducer of glaA promoter controlled genes. For a more profound understanding of A. niger physiology, a comprehensive analysis of the intra- and extracellular proteome of Aspergillus niger AB1.13 growing on defined medium with xylose or maltose as carbon substrate was carried out using 2-D gel electrophoresis/Maldi-ToF and nano-HPLC MS/MS.

Results: The intracellular proteome of A. niger growing either on xylose or maltose in well-aerated controlled bioreactor cultures revealed striking similarities. In both cultures the most abundant intracellular protein was the TCA cycle enzyme malate-dehydrogenase. Moreover, the glycolytic enzymes fructose-bis-phosphate aldolase and glyceraldehyde-3-phosphate-dehydrogenase and the flavohemoglobin FhbA were identified as major proteins in both cultures. On the other hand, enzymes involved in the removal of reactive oxygen species, such as superoxide dismutase and peroxiredoxin, were present at elevated levels in the culture growing on maltose but only in minor amounts in the xylose culture. The composition of the extracellular proteome differed considerably depending on the carbon substrate. In the secretome of the xylose-grown culture, a variety of plant cell wall degrading enzymes were identified, mostly under the control of the xylanolytic transcriptional activator XlnR, with xylanase B and ferulic acid esterase as the most abundant ones. The secretome of the maltose-grown culture did not contain xylanolytic enzymes, instead high levels of catalases were found and glucoamylase (multiple spots) was identified as the most abundant extracellular protein. Surprisingly, the intracellular proteome of A. niger growing on xylose in bioreactor cultures differed more from a culture growing in shake flasks using the same medium than from the bioreactor culture growing on maltose. For example, in shake flask cultures with xylose as carbon source the most abundant intracellular proteins were not the glycolytic and the TCA cycle enzymes and the flavohemoglobin, but CipC, a protein of yet unknown function, superoxide dismutase and an NADPH dependent aldehyde reductase. Moreover, vacuolar proteases accumulated to higher and ER-resident chaperones and foldases to lower levels in shake flask compared to the bioreactor cultures.

Conclusions: The utilization of xylose or maltose was strongly affecting the composition of the secretome but of minor influence on the composition of the intracellular proteome. On the other hand, differences in culture conditions (pH control versus no pH control, aeration versus no aeration and stirring versus shaking) have a profound effect on the intracellular proteome. For example, lower levels of ER-resident chaperones and foldases and higher levels of vacuolar proteases render shake flask conditions less favorable for protein production compared to controlled bioreactor cultures.

Show MeSH
Related in: MedlinePlus