Abstract
Solid-state fermentation (SSF) with Aspergillus oryzae results in high levels of secreted protein. However, control mechanisms of gene expression in SSF have been only poorly studied. In this study we show that both glucoamylase (glaB) and protease (alpA, nptB) genes are highly expressed during surface cultivation on wheat-based solid medium, and even higher during cultivation on wheat kernels. In wheat-based liquid medium, low levels of gene expression are observed. Typical SSF cultivation conditions, such as low water activity and the formation of aerial hyphae, did not contribute to the high-level gene expression on wheat-based solid medium. Analysis of wheat-based solid and liquid cultivations showed differences in carbon and nitrogen utilisation and external pH. The results presented show that the difference in regulation of transcription of the alpA and nptB genes in wheat-based liquid and solid medium could be pH dependent, involving a pH-dependent transcription regulator. The results obtained suggest that the difference in regulation of transcription of the glaB gene in wheat-based liquid and solid medium is caused by a difference in carbohydrate degradation and consumption under the different culture conditions.
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References
Adler-Nissen J (1979) Determination of the degree of hydrolysis of food protein hydrolysates by trinitrobenzenesulfonic acid. J Agric Food Chem 27:1256–1262
Burrin JM, Price CP (1985) Measurement of blood glucose. Ann Clin Biochem 22:327–342
Cheevahanarak S, Renno DV, Saunders G, Holt G (1991) Cloning and selective overexpression of an alkaline protease-encoding gene from Aspergillus oryzae. Gene 108:151–155
Dowzer CEA, Kelly JM (1991) Analysis of creA, a regulator of carbon catabolite repression in Aspergillus nidulans. Mol Cell Biol 11:5701–5709
Dynesen J, Nielsen J (2003) Branching is coordinated with mitosis in growing hyphae of Aspergillus nidulans. Fungal Genet Biol 40:15–24
Gomi K, Arikawa K, Kamiya K, Kitamoto K, Kumagai C (1993) Cloning and nucleotide sequence of the acid protease-encoding gene (pepA) from Aspergillus oryzae. Biosci Biotechnol Biochem 57:1095–1100
Hata Y, Ishida H, Ichikawa E, Kawato A, Suginami K, Imayasu S (1998) Nucleotide sequence of an alternative glucoamylase-encoding gene (glaB) expressed in solid state culture of Aspergillus oryzae. Gene 207:127–134
Hesse SJA, Ruijter GJG, Dijkema C, Visser J (2002) Intracellular pH homeostasis in the filamentous fungus Aspergillus niger. Eur J Biochem 269:3485–3494
Hoogschagen M, Zhu Y, van As H, Tramper J, Rinzema A (2002) Influence of wheat type and pretreatment on fungal growth in solid state fermentation. Biotechnol Lett 23:1183–1187
Ishida H, Hata Y, Ichikawa E, Kawato A, Suginami K, Imayasu S (1998) Regulation of the glucoamylase-encoding gene (glaB), expressed in solid state culture (koji) of Aspergillus oryzae. J Ferment Bioeng 86:301–307
Ishida H, Hata Y, Kawato A, Abe Y, Suginami K, Imayasu S (2000) Identification of functional elements that regulate the glucoamylase-encoding gene (glaB) expressed in solid state culture of Aspergillus oryzae. Curr Genet 37:373–379
Iwashita K (2002) Recent studies of protein secretion by filamentous fungi. J Biosci Bioeng 94:530–535
Kitano H, Kataoka K, Furukawa K, Hara S (2002) Specific expression and temperature-dependent expression of the acid protease encoding gene (pepA) in Aspergillus oryzae in solid state culture (rice-koji). J Biosci Bioeng 93:563–567
Kolar M, Punt PJ, van den Hondel CAMJJ, Schwab H (1988) Transformation of Penicillium chrysogenum using dominant selection markers and expression of an Escherichia coli lacZ fusion gene. Gene 62:127–134
Machida M, Chang YC, Manabe M, Yasukawa M, Kunihiro S, Jigami Y (1996) Molecular cloning of a cDNA encoding enolase from the filamentous fungus, Aspergillus oryzae. Curr Genet 30:81–106
Malathi S, Chakraborthy R (1991) Production of alkaline protease by a new Aspergillus flavus isolate under solid-substrate fermentation conditions for use as a depilation agent. Appl Environ Microbiol 57:712–716
Mary F, Hua X, John TL (1994) Fine structure analysis of the Drosophila and Saccharomyces heat shock factor-heat shock element interactions. Nucleic Acids Res 22:167–173
Nakajima K, Kunihiro S, Sano M, Zhang Y, Eto S, Chang YC, Suzuki T, Jigami Y, Machida M (2000) Comprehensive cloning and expression analysis of glycolytic genes from the filamentous fungus, Aspergillus oryzae. Curr Genet 37:322–327
Pandey A, Selvakumar P, Soccol CR, Nigam P (1999) Solid state fermentation for the production of industrial enzymes. Curr Sci 77:149–162
Panelva MA, Arst HN (2002) Regulation of gene expression by ambient pH in filamentous fungi and yeast. Microbiol Mol Biol Rev 66:426–446
Prosser JI (1994) Kinetics of filamentous growth and branching. In: Gow NAR, Gadd GM (eds) The growing fungus. Chapman & Hall, London
Rahardjo YS, Weber FJ, le Compte EP, Tramper J, Rinzema A (2001) Contribution of aerial hyphae of Aspergillus oryzae to respiration in a model solid state fermentation. Biotechnol Bioeng 78:539–544
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning—a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
Sanders D, Slayman CL (1982) Control of intracellular pH: predominant role of oxidative metabolism, not proton transport, in the eukaryotic microorganism Neurospora. J Gen Physiol 80:377–402
Su NW, Lee MH (2001) Screening and characterization of koji molds producing saline-tolerant protease. J Ind Microbiol Biotechnol 26:230–234
Tatsumi H, Murakami S, Tsuji RF, Ishida Y, Murakami K, Masaki A, Kawabe H, Arimura H, Nakano E, Motai H (1991) Cloning and expression in yeast of a cDNA clone encoding Aspergillus oryzae neutral protease II, a unique metalloprotease. Mol Gen Genet 228:97–103
Te Biesebeke R, Ruijter JG, Rahardjo SP, Hoogschagen MJ, Heerikhuisen M, Levin A, van Driel KGA, Schutyser MAI, Dijksterhuis J, Zhu Y, Weber FJ, de Vos WM, van den Hondel CAMJJ, Rinzema A, Punt PJ (2002) Aspergillus oryzae in solid state and submerged fermentations. FEMS Yeast Res 2:245–248
Trinci APJ (1974) A study of the kinetics of hyphal extension and branch initiation of fungal mycelia. J Gen Microbiol 81:225–236
Tunga R, Banerjee R, Bhattacharyya BC (2001) Optimization of some additives to improve protease production under SSF. Indian J Exp Biol 39:1144–1148
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This work was supported by the Wageningen Centre for Food Sciences (WCFS), an alliance of research organizations and food industry partners in the Netherlands to carry out strategic fundamental research
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te Biesebeke, R., van Biezen, N., de Vos, W.M. et al. Different control mechanisms regulate glucoamylase and protease gene transcription in Aspergillus oryzae in solid-state and submerged fermentation. Appl Microbiol Biotechnol 67, 75–82 (2005). https://doi.org/10.1007/s00253-004-1807-z
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DOI: https://doi.org/10.1007/s00253-004-1807-z