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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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Growth profile of A. niger. Growth profile of A. niger AB1.13 in controlled bioreactor (filled symbols) or shake flask cultures (open symbols) using either xylose (squares) or maltose (circles) as carbon substrate. Bioreactor and shake flask cultivations were carried out in duplicate and triplicate, respectively. The arrow points to the sampling point for proteome analysis.
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Figure 1: Growth profile of A. niger. Growth profile of A. niger AB1.13 in controlled bioreactor (filled symbols) or shake flask cultures (open symbols) using either xylose (squares) or maltose (circles) as carbon substrate. Bioreactor and shake flask cultivations were carried out in duplicate and triplicate, respectively. The arrow points to the sampling point for proteome analysis.

Mentions: Xylose and maltose are both sugar carbon substrates which can be used by A. niger as sole carbon substrate leading to comparable growth kinetics and final biomass concentrations in controlled bioreactor cultures (Fig. 1), although both sugars are catabolized by different metabolic routes (Fig. 2). Uptake of xylose occurs in the unmodified form followed by intracellular reduction to xylitol through xylose reductase [15]. Xylitol is then converted to L-xylulose and after phosphorylation further degraded in the pentose phosphate pathway [15]. Maltose cleavage into glucose moieties occurs extracellular by glucoamylase as A. niger has no functional uptake system for maltose in contrast to many yeast and other filamentous fungi [16,17]. Glucose uptake occurs either directly with subsequent catabolic degradation of glucose via the glycolytic and the pentose phosphate pathway [15] or extracellular oxidation of glucose to gluconic acid by glucose oxidase with concomitant generation of hydrogen peroxide can occur as an alternative route of glucose catabolism. Gluconic acid is then further metabolized via the pentose phosphate pathway [18,19].


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)

Growth profile of A. niger. Growth profile of A. niger AB1.13 in controlled bioreactor (filled symbols) or shake flask cultures (open symbols) using either xylose (squares) or maltose (circles) as carbon substrate. Bioreactor and shake flask cultivations were carried out in duplicate and triplicate, respectively. The arrow points to the sampling point for proteome analysis.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Growth profile of A. niger. Growth profile of A. niger AB1.13 in controlled bioreactor (filled symbols) or shake flask cultures (open symbols) using either xylose (squares) or maltose (circles) as carbon substrate. Bioreactor and shake flask cultivations were carried out in duplicate and triplicate, respectively. The arrow points to the sampling point for proteome analysis.
Mentions: Xylose and maltose are both sugar carbon substrates which can be used by A. niger as sole carbon substrate leading to comparable growth kinetics and final biomass concentrations in controlled bioreactor cultures (Fig. 1), although both sugars are catabolized by different metabolic routes (Fig. 2). Uptake of xylose occurs in the unmodified form followed by intracellular reduction to xylitol through xylose reductase [15]. Xylitol is then converted to L-xylulose and after phosphorylation further degraded in the pentose phosphate pathway [15]. Maltose cleavage into glucose moieties occurs extracellular by glucoamylase as A. niger has no functional uptake system for maltose in contrast to many yeast and other filamentous fungi [16,17]. Glucose uptake occurs either directly with subsequent catabolic degradation of glucose via the glycolytic and the pentose phosphate pathway [15] or extracellular oxidation of glucose to gluconic acid by glucose oxidase with concomitant generation of hydrogen peroxide can occur as an alternative route of glucose catabolism. Gluconic acid is then further metabolized via the pentose phosphate pathway [18,19].

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