The intra- and extracellular proteome of Aspergillus niger growing on defined medium with xylose or maltose as carbon substrate.
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.
Lu
Xin
X
Helmholtz Center for Infection Research, Inhoffenstr, Braunschweig, Germany.
Sun
Jibin
J
Nimtz
Manfred
M
Wissing
Josef
J
Zeng
An-Ping
AP
Rinas
Ursula
U
D000388:Air; D001234:Aspergillus niger / Q000737:chemistry* / Q000378:metabolism; D019149:Bioreactors; D002244:Carbon / Q000378:metabolism; D005656:Fungal Proteins / Q000032:analysis / Q000187:drug effects; D006863:Hydrogen-Ion Concentration; D008320:Maltose / Q000378:metabolism* / Q000494:pharmacology; D020543:Proteome / Q000032:analysis* / Q000187:drug effects; D040901:Proteomics / Q000379:methods; D014994:Xylose / Q000378:metabolism* / Q000494:pharmacology
D005656:Fungal Proteins; D020543:Proteome; D008320:Maltose; D002244:Carbon; D014994:Xylose
Chem Biol Interact. 2001 Jan 30;130-132(1-3):685-98
11306086
BMC Microbiol. 2009 Dec 10;9:255
20003296
Anal Biochem. 1976 May 7;72:248-54
942051
Eur J Biochem. 1997 Apr 1;245(1):164-73
9128738
Fungal Genet Biol. 2009 Mar;46 Suppl 1:S141-52
18824119
J Biol Chem. 1996 Oct 11;271(41):25131-8
8810268
J Proteome Res. 2006 Apr;5(4):954-62
16602703
Microbiology (Reading). 2009 Dec;155(Pt 12):3893-3902
19696104
Gene. 1993 Mar 15;125(1):57-64
8449413
Biochemistry. 1987 Nov 17;26(23):7363-71
3427078
J Bacteriol. 1997 May;179(10):3284-92
9150225
Gene. 1994 Aug 5;145(2):171-8
8056328
J Mol Biol. 1997 Oct 17;273(1):114-21
9367751
J Biol Chem. 1998 Apr 17;273(16):9527-33
9545281
Mol Cell Biol. 1984 Nov;4(11):2306-15
6440004
Arch Mikrobiol. 1969;66(4):396-405
5384636
Appl Environ Microbiol. 1998 Oct;64(10):3615-9
9758775
Curr Genet. 2007 Feb;51(2):89-98
17119968
Antonie Van Leeuwenhoek. 1987;53(5):349-52
3120641
J Bacteriol. 1993 Jun;175(12):3703-9
8509325
Biochemistry (Mosc). 2004 Nov;69(11):1252-67
15627379
Mol Genet Genomics. 2008 Jun;279(6):545-61
18320228
Curr Genet. 1988 Nov;14(5):437-43
3224384
Appl Microbiol Biotechnol. 2003 Mar;61(1):10-20
12658510
Res Microbiol. 2006 Apr;157(3):275-81
16171982
Mol Gen Genet. 1987 Jan;206(1):71-5
3472035
Appl Biochem Biotechnol. 2001 Jan;90(1):57-66
11257807
Mol Gen Genet. 1992 Nov;235(2-3):349-58
1465107
Mol Cell Proteomics. 2004 Dec;3(12):1181-93
15475568
Mol Microbiol. 2000 Apr;36(1):193-200
10760176
Electrophoresis. 2004 May;25(9):1327-33
15174055
Nat Biotechnol. 2007 Feb;25(2):221-31
17259976
Z Allg Mikrobiol. 1983;23(1):17-25
6346702
Mol Gen Genet. 1992 Aug;234(2):332-6
1508158
Zentralbl Mikrobiol. 1985;140(6):475-84
4072456
Microbiology (Reading). 1998 Nov;144 ( Pt 11):3079-3086
9846753
Biochim Biophys Acta. 1995 Apr 4;1261(2):319-20
7711083
Fungal Genet Biol. 2009 Mar;46 Suppl 1:S153-S160
19618504
J Gen Microbiol. 1993 Nov;139(11):2801-10
8277260
Curr Genet. 1990 Dec;18(6):537-45
2076554
Fungal Biol. 2010 Feb-Mar;114(2-3):135-43
20960969
Yeast. 1994 Aug;10(8):1075-82
7992507
Fungal Genet Biol. 2000 Dec;31(3):205-17
11273682
Mol Cell Proteomics. 2010 Jun;9(6):1182-98
20154335
Biochim Biophys Acta. 1994 Sep 13;1219(1):148-50
8086452
Genome Biol. 2007;8(9):R182
17784953
Appl Biochem Biotechnol. 1999 Jul;81(1):1-11
10533711
Biotechnol Bioeng. 2001 Sep;76(2):164-74
11505386
Appl Microbiol Biotechnol. 1995 Nov;43(6):978-84
8590664
J Biotechnol. 2008 Jun 30;135(3):266-71
18501461
Antonie Van Leeuwenhoek. 1992 Feb;61(2):153-60
1580617
Fungal Genet Biol. 2008 Dec;45(12):1591-9
18930158
BMC Genomics. 2009 Jan 23;10:44
19166577
Biotechnol Prog. 1999 Jan;15(1):43-50
9933512
Mol Genet Genomics. 2002 Aug;267(6):695-702
12207217
Gene. 1990 Oct 15;94(2):147-54
2258049
Biochem Biophys Res Commun. 2009 Mar 27;381(1):7-11
19351585
Proteome Sci. 2003 Dec 3;1:6
14653859
J Gen Microbiol. 1974 Apr;81(2):425-34
4365536
Appl Environ Microbiol. 1996 Jul;62(7):2303-10
8779568
Gene. 1994 Aug 5;145(2):179-87
8056329