VTT Biotechnology, PO Box 1500 (Tietotie 2, Espoo), FIN-02044 VTT, Finland
Correspondence
Tiina Pakula
tiina.pakula{at}vtt.fi
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Present address: University of Helsinki, Biocentrum Helsinki, Cell and Protein Production Unit, PO Box 63, (Haartmaninkatu 8, Biomedicum), 00014 University of Helsinki, Finland.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Regulation of the cellulase genes on various carbon sources has been studied in detail. The cellulase genes are to a large extent induced in a coordinate manner in the presence of cellulose, its hydrolytic products, or certain oligosaccharides, such as sophorose and lactose (Ilmén et al., 1997; Foreman et al., 2003
). For the individual hemicellulase genes also, specific and partially overlapping induction mechanisms have been anticipated to exist based on differential expression of the genes on various carbon sources (Margolles-Clark et al., 1997
). Wide-domain carbon catabolite repression has been shown to control the expression of both cellulase and hemicellulase genes in the presence of glucose (Ilmén et al., 1996
, 1997
; Margolles-Clark et al., 1997
; Strauss et al., 1995
; Takashima et al., 1996
). Many of the transcription factors involved in regulation of the genes, CREI mediating carbon catabolite repression, the repressor ACEI, the activator ACEII, and the CCAAT binding complex Hap2/3/5, have been characterized in molecular detail (reviewed by Kubicek & Penttilä, 1998
; Mach & Zeilinger, 2003
; Schmoll & Kubicek, 2003
).
Although the carbon-source-dependent transcriptional regulation of the cellulase genes in T. reesei is fairly well characterized, information on cellulase gene expression and production of the proteins at different physiological states of the cells is scarce. Furthermore, very little information is available on the cellular responses to protein production. T. reesei has the potential to produce extracellular proteins in very large quantities, which sets a demand for the cells to adjust the capacity of protein synthesis and transport to the level required, and may also provoke stress responses within the cells. To obtain information on protein production and factors affecting the processes at different growth rates of the organism, we have analysed carbon-limited chemostat cultures of the strain RUT-C30 in detail. Specifically, the capacity of the cells to synthesize and secrete proteins has been studied using metabolic labelling of the proteins, a methodology previously set up for analysis of protein synthesis and secretion in defined culture conditions (Pakula et al., 2000). In addition, the expression levels of the major cellulase genes cbh1 and egl1, as well as genes involved in protein folding and transport, have been analysed under these conditions.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Usually steady state in the cultures was achieved after cultivation of four to five residence times. Steady state of the cultures was monitored by measuring biomass dry weight and dissolved oxygen concentration in the culture, carbon dioxide content of the exhaust air, as well as lactose, phosphate and ammonium concentrations in the culture broth.
Analysis of the chemostat cultures.
Dry weight was measured by filtering and drying mycelium samples at 105 °C to a constant weight (24 h). Residual lactose and glucose in the culture filtrate were measured using either HPLC or an enzymic test kit for lactose (Boehringer Mannheim 176 303) and the GOD-Perid method for glucose (Boehringer Mannheim 124 036). The amount of phosphate was measured as described by Basset et al. (1987). An enzymic test kit (Boehringer Mannheim 1112 732) was used for measuring ammonium concentration. Soluble protein was analysed using the Bio-Rad Protein Assay. The carbon content of the biomass was determined using a Carlo Erba C/N analyser, and the value for carbon content determined by Nielsen & Villadsen (1994)
was used for extracellular proteins.
Analysis of intracellular methionine.
Cells resuspended in double-distilled water to 13 mg biomass dry weight ml1 were disrupted by sonication (Pakula et al., 2000) and boiled for 15 min. The extracts were centrifuged at 14 000 g for 10 min. Sulphosalicylic acid was added to the supernatant to a final concentration of 5 % (w/v), and the samples were centrifuged at 14 000 g for 10 min. The amino acids were analysed from the supernatant using HPLC.
Analysis of intracellular protein.
Cell extracts were prepared and the protein amount in the extracts was measured as described previously (Pakula et al., 2000). Samples of the cultures were filtered through Millipore HVLP02500 filters to collect the mycelium, and the mycelium was washed with double-distilled water and resuspended in 20 mM N-ethylmaleimide, 10 mM NaN3 (37 mg biomass dry weight ml1). The cells were disrupted by sonication (8x8 s with an MSE 150 W sonicator, 18 µm amplitude, 30 s cooling on ice between the sonication cycles). The cell lysates were first lyophilized, and then resuspended in two-dimensional (2D) lysis buffer [9 M urea, 2 % (v/v) Triton X-100, 286 mM
-mercaptoethanol, 2 % (v/v) Pharmalyte 310; 560 µg biomass dry weight per 25 µl buffer], 3 vols 2D sample buffer [the lysis buffer containing 0·5 % (v/v) Triton X-100] was added, and the samples were incubated at room temperature for 1 h. Insoluble material was removed by centrifugation. Proteins were analysed using the Bio-Rad Protein Assay Kit.
Metabolic labelling of the proteins.
Metabolic labelling of the proteins was carried out essentially as described previously (Pakula et al., 2000). Aliquots (25 ml) of the chemostat culture were gently transferred into shake flasks on a rotary shaker (210 r.p.m., 30 °C), and the labelling was started immediately after the transfer. To avoid changes in concentration of the nutrients, fresh medium was added into the shake flasks. Medium was added at intervals of 15 s, the amount of the added medium corresponding to the amount of medium fed into the chemostat culture during the same time period. [35S]Methionine (Amersham SJ 1015, in vivo cell labelling grade, 1000 Ci mmol1, 10 µCi µl1) was added to the shake flasks, either 5·5 or 12·5 µCi (mg biomass dry weight)1 in the labelling experiment (1 Ci=3·7x1010 Bq), and samples of 1 ml were collected at short intervals. The samples were rapidly filtered through Millipore HVLP02500 filters to separate the culture medium and mycelium. The mycelium on the filters was washed with 10 ml double-distilled water and frozen immediately in liquid nitrogen.
Analysis of the labelled proteins.
The cells were disrupted by sonication and the cell extracts were prepared as previously described (Pakula et al., 2000). Protein samples prepared from the cell extracts and culture medium were subjected to 2D gel electrophoresis (Pakula et al., 2000
) for analysis of specific proteins. Equal amounts of total protein (measured using the Bio-Rad Protein Assay Kit as described above) were loaded in the gels. The amount of protein loaded was confirmed to be in a range giving a linear response between the signal analysed and the sample volume. Typically, 20 µg intracellular protein or 3 µg extracellular protein was loaded. The 2D gels were analysed using a phosphorimager (Molecular Dynamics). The intracellular labelled protein per culture volume and per biomass amount was calculated by taking into account the protein content of the biomass and the biomass amount under each of the culture conditions. For analysis of total labelled protein, the proteins in the cell extracts and in culture supernatant were precipitated using TCA, and the radioactivity in the TCA-insoluble material was measured by scintillation counting (Pakula et al., 2000
).
Parameters describing protein synthesis and secretion.
The parameters, protein synthesis and secretion rate, the mean synthesis time of specific proteins, and the minimum secretion time of the molecules were determined essentially as described by Pakula et al. (2000). The amount of labelled protein per biomass and culture volume in cell extract and in culture supernatant was plotted against time (scintillation counting of TCA-insoluble material was used for quantification of total labelled protein, and specific labelled proteins were quantified in 2D gels using a phosphorimager). The specific rate of protein synthesis was determined as the slope of the linear part of the curve representing intracellular labelled protein at the early time points where protein secretion was not yet detectable. The specific rate of protein secretion was determined as the slope of the linear part of the curve representing extracellular labelled protein. The specific rates of protein synthesis and secretion per biomass and time unit were normalized with the ratio of [35S]methionine taken up by the cells to the total intracellular methionine. The mean synthesis time for a specific protein was determined by extrapolating the linear part of the curve representing the amount of the labelled protein in cell extract to the abscissa. The intercept of the curve and the abscissa corresponds to half the synthesis time of the protein (see also Braakman et al., 1991
; Horwitz et al., 1969
; Loftfield & Eigner, 1958
). The minimum secretion time of the protein was determined as the distance of the intercepts of the intracellular and extracellular protein curves extrapolated to the abscissa.
Northern analysis.
Mycelium samples of 50 ml were filtered, washed with equal volume of 0·7 % NaCl, frozen immediately in liquid nitrogen, and stored at 80 °C. Total RNA was isolated using the Trizol Reagent (Gibco-BRL), essentially according to the manufacturer's instructions. Northern blotting and hybridization on nitrocellulose filters were carried out according to standard procedures (Sambrook et al., 1989). Five micrograms of total RNA isolated from each sample was used in the Northern analysis. cDNAs of the genes (cbh1, E00389; egl1, M15665; pdi1, AJ222773; sar1, Y08636; ypt1, AJ277108) were used as probes. The signals were normalized against the signals of act1 encoding actin.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
The highest specific production rate of extracellular proteins was obtained at low specific growth rates (0·0220·033 h1), the maximal value being 4·1 mg g1 h1 at the specific growth rate 0·031 h1 (Fig. 1c). However, at the lowest specific growth rate studied (0·021 h1), the specific protein production rate was reduced close to the level measured at high specific growth rates of 0·0450·066 h1 (1·41·6 mg g1 h1). Similarly, the yield of extracellular protein produced per the amount of carbon source consumed was the highest at the specific growth rate 0·031 h1 (Fig. 1d
). At low specific growth rates (0·0210·031 h1), both the biomass yield and the yield of protein produced increased with increasing specific growth rate, after which much less protein was produced per amount of lactose consumed, whereas the production of biomass related to lactose consumption remained at a high level (Fig. 1d
). Carbon balances accounting for biomass, secreted protein and CO2 produced in the cultures showed a mean closure of 90±7 % total carbon consumed. For the determination of the balances, the carbon content of the mycelial samples was measured, and the elemental composition CH1·72O0·31N0·27S0·004 described by Nielsen & Villadsen (1994)
was used for secreted proteins.
To elucidate how the capacity of protein production was directed to production of intracellular and extracellular proteins, the specific synthesis rates of intracellular and extracellular protein were compared at the different specific growth rates. The amount of intracellular protein in the cell extracts was determined, and the specific synthesis rate of intracellular proteins (grams of intracellular protein per gram of biomass dry weight per hour) was deduced. The specific synthesis rate of intracellular and extracellular protein as well as the specific rate of total protein synthesis (combined synthesis of extracellular and intracellular protein) plotted against the dilution rate in the culture are shown in Fig. 2(a). The specific rate of total protein synthesis increased with increasing specific growth rate until a constant level was reached at the specific growth rate 0·045 h1 (Fig. 2a
). At low specific growth rates, between 0·022 and 0·033 h1, a markedly higher percentage of the total protein synthesis was directed to production of extracellular proteins compared to that of cultures with the high specific growth rates of 0·0450·066 h1 (Fig. 2b
). At most, at the specific growth rate 0·022 h1, 29 % of the synthesized proteins were extracellular, whereas at high specific growth rates (0·0450·066 h1), only 68 % of the synthesized proteins were those transported into the culture medium.
|
The specific rates of CBHI synthesis and secretion (the amount of protein synthesized or produced in the culture medium per biomass and time unit) at different specific growth rates are shown in Fig. 3. The highest specific synthesis and secretion rates of labelled CBHI were both obtained at the specific growth rate 0·031 h1, which is in accordance with the result of total protein production into the culture medium. However, at low specific growth rates (0·0220·045 h1), under conditions where the specific CBHI synthesis rate was high, the ratio between the secretion rate and the synthesis rate was much lower than the ratio at high specific growth rates. At 0·031 h1, the secretion rate was 37 % of the synthesis rate, whereas at 0·066 h1, the secretion rate was 62 % of the synthesis rate. The result indicates that although CBHI was efficiently synthesized at the low specific growth rates, the protein secretion capacity limits protein production under these conditions. The mean time of synthesis of CBHI molecules and the minimum time of secretion of the protein did not differ significantly at the different specific growth rates. The mean synthesis time of CBHI was 3·9±0·3 min and the minimum secretion time of the molecules was 12·6±0·2 min. The values are close to the ones obtained previously for cultures carried out at the specific growth rate 0·07 h1 (Pakula et al., 2000
).
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The dilution rates studied (0·0210·076 h1) covered the range from a very low specific growth rate to a rate exceeding the maximal specific growth rate of the fungus. The maximal specific growth rate estimated from the chemostat data was 0·068 h1, which is close to the value 0·073 h1, determined in batch bioreactor cultures for the strain on the same medium, and to the value previously obtained for T. reesei strain C5 on lactose-containing minimal medium, 0·07 h1 (Chaudhuri & Sahai, 1994). At low specific growth rates (0·0210·033 h1), the fungal biomass increased significantly with increasing growth rate, but at higher specific growth rates (0·0330·066 h1), the biomass amount remained approximately constant. Cultivation at the dilution rate 0·076 h1 resulted in washout of the biomass, as predicted. The specific consumption rate of the carbon source lactose increased until the critical dilution rate was reached. Assuming that the amount of energy source used for maintenance as well as the maximal growth yield would be constant over the range of growth rates studied, the maintenance coefficient (0·027 g g1 h1) and maximal growth yield (0·60 g g1) were determined (Pirt, 1965
). A good linear regression was obtained for the specific lactose consumption rate over the dilution-rate range studied, indicating that the assumptions of the Pirt equation were valid under these conditions.
Protein production into the culture medium was most efficient at low specific growth rates (below 0·033h1). The specific production rate of extracellular proteins increased first with increasing dilution rate, reaching the maximal level, 4·1 mg g1 h1, at the specific growth rate 0·031 h1, after which the production rate decreased significantly. Similar trends for specific protein production rates at different dilution rates have previously been reported for lactase production by Rut-C30 from continuous cultures on lactose medium (Castillo et al., 1984) and for production of cellulase activity by T. reesei strain C5 (Chaudhuri & Sahai, 1994
) and have been modelled for cellulase productivity on medium containing xylose and sorbose by T. reesei strain QM9414 (Schafner & Toledo, 1992
). In accordance with the other data on production of biomass and extracellular protein, at low specific growth rates (0·0220·033 h1), a higher proportion of the total protein synthesis was also directed to production of extracellular proteins compared to the cultures at higher specific growth rates (0·0450·066 h1). At low specific growth rates, up to 29 % of the proteins synthesized were extracellular and at the high growth rates only 68 % were extracellular.
Production of extracellular proteins by filamentous fungi, such as -amylase in Aspergillus oryzae (Carlsen et al., 1996
; Spohr et al., 1998
) and glucoamylase in Aspergillus niger (Pedersen et al., 2000
; Schrickx et al., 1993
; Withers et al., 1998
), has been shown to be growth-associated in many cases. However, examples of growth-rate-disassociated production are also known, such as recombinant protein production by Fusarium venenatum (Wiebe et al., 2000
). As active growth of the cells and the fungal hyphae requires efficient transportation of, for example, cell wall material, protein transport is expected to take place efficiently in the growing hyphae. However, a majority of the extracellular enzymes produced by T. reesei are needed for degradation of polymeric compounds derived from plant material to provide the fungus with a source of carbon and energy. Therefore, production of hydrolytic enzymes would be beneficial for the fungus under low nutrient conditions in which easily metabolized carbon sources, such as glucose, are not available and growth might be slow.
Taking into account the data from the wide range of growth rates studied, the results in our study as well as those obtained by other groups (Castillo et al., 1984; Chaudhuri & Sahai, 1994
; Schafner & Toledo, 1992
) indicate that production of the hydrolytic enzymes that constitute the major part of the extracellular proteins produced by T. reesei under these conditions is not directly growth-rate associated. In terms of specific protein production rate as well as the yield of extracellular protein per amount of carbon source consumed, the production of extracellular proteins was maximal at the specific growth rate 0·031 h1, but at higher growth rates a significant reduction in production was observed. However, in the range of the low dilution rates 0·0210·031 h1, both protein and biomass production are positively correlated with the specific growth rate. The reduction in biomass and protein production at very low specific growth rates is due to the maintenance requirement of the cells. At low specific growth rates, the specific consumption rate of the carbon and energy source lactose was very low. At the dilution rate 0·021 h1, 0·068 g lactose g1 h1 was consumed, of which the estimated maintenance coefficient 0·027 g g1 h1 forms 40 %. The high substrate consumption for maintenance under these conditions would limit production of both biomass and extracellular protein.
As cellulase gene expression by T. reesei is known to be regulated by the carbon source, one explanation for the differences in the protein production levels at different growth rates could be differences in the concentrations of the residual carbon sources. However, the concentration of the repressing carbon source glucose in the cultures in this study was very low, and thus unlikely to cause repression of cellulase production. In addition, the strain Rut-C30 is defective in the cre1-mediated carbon catabolite mechanism (Ilmén et al., 1996) and therefore cellulase production by the strain is partially derepressed even in a glucose medium. Further, the residual levels of lactose were low in the cultures and no positive correlation between cellulase expression and the residual lactose concentration in the cultures was observed. Thus the residual lactose in the cultures was unlikely to act as an inducer for cellulase production. Interestingly, under high protein production conditions at low specific growth rates, the specific consumption rate of lactose was low. Previous studies have shown that cellulase gene expression is induced in batch cultures after the carbon source is exhausted (Ilmén et al., 1997
). It is possible that the low consumption rate of the carbon source might trigger a similar type of induction as does carbon and energy source starvation. In natural habitats of the fungus, this type of mechanism might be important for activation of synthesis of hydrolytic enzymes to enable sequestering of easily metabolizable carbon sources from complex plant polymers.
To address the question of transcriptional regulation of cellulase genes at different specific growth rates, we carried out a Northern analysis of the genes cbh1 and egl1. The transcript levels of both genes were slightly increased at low specific growth rates, thus being in accordance with the results on production of total extracellular proteins. In addition, metabolic labelling of the cultures was used for analysis of the synthesis and secretion of CBHI specifically. The results showed that both the specific synthesis and secretion rate of labelled CBHI were the highest at the low specific growth rate 0·031 h1. Thus, the differences in transcript levels at the different growth rates could, at least in part, explain the differences in the amounts of the corresponding proteins synthesized per time unit. However, further inspection of the full range of dilution rates studied indicated that the transcript levels alone do not fully explain the differences in protein production. Especially at high specific growth rates, the specific protein production rate (Fig. 1) and the specific synthesis rate of CBHI (Fig. 3
) were lower than that which could have been anticipated based on the transcript levels of the major cellulase genes cbh1 and egl1.
Interestingly, the metabolic labelling studies also showed that the ratio between the specific secretion rate and the synthesis rate of labelled CBHI was much lower at the low specific growth rates than at the high specific growth rates. The result indicates that at the low growth rates the capacity of the cells to transport the proteins would become limiting as the amount of secreted protein synthesized increases. Accumulation of unfolded proteins in the endoplasmic reticulum is known to activate the UPR, in both lower and higher eukaryotes. In filamentous fungi, UPR induction has been reported to take place under conditions in which protein transport or folding is impaired by treatment of the cultures with different chemical agents or under conditions where a heterologous protein is produced (Mulder et al., 2004; Ngiam et al., 1997
; Pakula et al., 2003
; Punt et al., 1998
; Saloheimo et al., 1999
, 2003
; van Gemeren et al., 1997
). In addition, we have recently shown that the UPR pathway is activated along with cellulase gene induction when the cultures are shifted from a glucose repressed state to an induced state (Collén et al., 2004
). In the present study, the transcript levels of the UPR transcription factor gene hac1 and the UPR target genes pdi1 and bip1 were more abundant at low specific growth rates than at high growth rates. The result indicates that the UPR pathway was activated in response to increased production of secreted proteins, such as CBHI, and also in response to the postulated limitation in the transport process under those conditions. For comparison, no such increase at low specific growth rates was observed in the transcript levels of ypt1 and sar1, which encode proteins involved in other functions in protein transport. It has been previously shown that ypt1 and sar1 transcript levels are not induced by endoplasmic reticulum stress caused by treatment of the cultures with DTT, an agent known to induce UPR via inhibition of protein folding and transport (Saloheimo et al., 2004
).
In conclusion, our study offers new insight into the protein production characteristics of the industrially important host organism T. reesei. Production of extracellular proteins was favoured at low specific growth rates, which is beneficial for fed-batch processes where efficient protein production without extensive biomass formation is required. However, our study indicated that the capacity of the cells to transport the newly synthesized proteins is a limiting factor in the process under these conditions, and, for optimal protein production, strategies to improve the transport step should be designed. In addition, information on cellulase gene regulation at different growth rates and on consumption of the carbon and energy source was obtained to form a basis for further studies, especially in the field of starvation signalling for cellulase expression.
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Biely, P. & Tenkanen, M. (1998). Enzymology of hemicellulose degradation. In Trichoderma and Gliocladium, pp. 2547. Edited by G. E. Harman & C. P. Kubicek. London. Bristol: Taylor & Francis Ltd.
Braakman, I., Hoover-Litty, H., Wagner, K. R. & Helenius, A. (1991). Folding of influenza hemagglutinin in the endoplasmic reticulum. J Cell Biol 114, 401411.[Abstract]
Carlsen, M., Nielsen, J. & Villadsen, J. (1996). Growth and -amylase production by Aspergillus oryzae during continuous cultivations. J Biotechnol 45, 8193.[CrossRef]
Castillo, F. J., Blanch, H. W. & Wilke, C. R. (1984). Lactase production in continuous culture by Trichoderma reesei Rut-C30. Biotechnol Lett 6, 593596.[CrossRef]
Chaudhuri, B. K. & Sahai, V. (1994). Comparison of growth and maintenance parameters for cellulase biosynthesis by Trichoderma reesei-C5 with some published data. Enzyme Microb Technol 16, 10791083.[CrossRef]
Collén, A., Saloheimo, M., Bailey, M., Penttilä, M. & Pakula, T. M. (2004). Protein production and induction of the unfolded protein response in Trichoderma reesei strain Rut-C30 and its transformant expressing endoglucanase I with a hydrophobic tag. Biotechnol Bioeng, (in press).
Foreman, P. K., Brown, D., Dankmeyer, L. & 14 other authors (2003). Transcriptional regulation of biomass-degrading enzymes in the filamentous fungus Trichoderma reesei. J Biol Chem 278, 3198831997.
Horwitz, M. S., Scharff, M. D. & Maizel, J. V. (1969). Synthesis and assembly of adenovirus 2. I. Polypeptide synthesis, assembly of capsomeres, and morphogenesis of the virion. Virology 39, 682694.[Medline]
Ilmén, M., Thrane, C. & Penttilä, M. (1996). The glucose repressor gene cre1 of Trichoderma: isolation and expression of a full-length and a truncated mutant form. Mol Gen Genet 251, 451460.[CrossRef][Medline]
Ilmén, M., Saloheimo, A., Onnela, M.-L. & Penttilä, M. E. (1997). Regulation of cellulase gene expression in the filamentous fungus Trichoderma reesei. Appl Environ Microbiol 63, 12981306.[Abstract]
Keränen, S. & Penttilä, M. (1995). Production of recombinant proteins in the filamentous fungus Trichoderma reesei. Curr Opin Biotechnol 6, 534537.[CrossRef][Medline]
Kubicek, C. & Penttilä, M. (1998). Regulation of production of plant polysaccharide degrading enzymes by Trichoderma. In Trichoderma and Gliocladium, pp. 4972. Edited by G. E. Harman & C. P. Kubicek. London: Taylor & Francis Ltd.
Loftfield, R. B. & Eigner, E. A. (1958). The time required for the synthesis of a ferritin molecule in rat liver. J Biol Chem 231, 925943.
Mach, R. L. & Zeilinger, S. (2003). Regulation of gene expression in industrial fungi: Trichoderma. Appl Microbiol Biotechnol 60, 515522.[Medline]
Margolles-Clark, E., Ilmén, M. & Penttilä, M. (1997). Expression patterns of ten hemicellulase genes of the filamentous fungus Trichoderma reesei on various carbon sources. J Biotechnol 57, 167179.[CrossRef]
Montenecourt, B. S. & Eveleigh, D. E. (1979). Selective screening methods for the isolation of high yielding cellulase mutants of Trichoderma reesei. Adv Chem Ser 181, 289301.
Mulder, H. J., Saloheimo, M., Penttila, M. & Madrid, S. M. (2004). The transcription factor HACA mediates the unfolded protein response in Aspergillus niger, and up-regulates its own transcription. Mol Genet Genomics 271, 130140.[CrossRef][Medline]
Ngiam, C., Jeenes, D. J. & Archer, D. B. (1997). Isolation and characterisation of a gene encoding protein disulphide isomerase, pdiA, from Aspergillus niger. Curr Genet 31, 133138.[CrossRef][Medline]
Nielsen, J. & Villadsen, J. (1994). Bioreaction Engineering Principles, pp. 480. New York: Plenum.
Pakula, T. M., Uusitalo, J., Saloheimo, M., Salonen, K., Aarts, R. J. & Penttilä, M. (2000). Monitoring the kinetics of glycoprotein synthesis and secretion in the filamentous fungus Trichoderma reesei: cellobiohydrolase I (CBHI) as a model protein. Microbiology 146, 223232.[Medline]
Pakula, T. M., Laxell, M., Huuskonen, A., Uusitalo, J., Saloheimo, M. & Penttilä, M. (2003). The effects of drugs inhibiting protein secretion in the filamentous fungus Trichoderma reesei. Evidence for down-regulation of genes that encode secreted proteins in the stressed cells. J Biol Chem 278, 4501145020.
Pedersen, H., Beyer, M. & Nielsen, J. (2000). Glucoamylase production in batch, chemostat, and fed-batch cultivations by an industrial strain of Aspergillus niger. Appl Microbiol Biotechnol 53, 272277.[CrossRef][Medline]
Penttilä, M. (1998). Heterologous protein production in Trichoderma. In Trichoderma and Gliocladium, pp. 365382. Edited by G. E. Harman & C. P. Kubicek. London: Taylor & Francis Ltd.
Penttilä, M., Limón, C. & Nevalainen, H. (2004). Molecular biology of Trichoderma and biotechnological applications. In Mycology, Vol. 20, Handbook of Fungal Biotechnology, pp. 413427. Edited by D. K. Arora. 2nd edn. New York, Basel: Marcel Dekker.
Pirt, S. J. (1965). The maintenance energy of bacteria in growing cultures. Proc R Soc B 163, 224231.[Medline]
Punt, P. J., van Gemeren, I. A., Drint-Kuijvenhoven, J., Hessing, J. G., van Muijlwijk-Harteveld, G. M., Beijersbergen, A., Verrips, C. T. & van den Hondel, C. A. (1998). Analysis of the role of the gene bipA, encoding the major endoplasmic reticulum chaperone protein in the secretion of homologous and heterologous proteins in black Aspergilli. Appl Microbiol Biotechnol 50, 447454.[CrossRef][Medline]
Saloheimo, M., Lund, M. & Penttilä, M. E. (1999). The protein disulphide isomerase gene of the fungus Trichoderma reesei is induced by endoplasmic reticulum stress and regulated by the carbon source. Mol Gen Genet 262, 3545.[CrossRef][Medline]
Saloheimo, M., Valkonen, M. & Penttilä, M. (2003). Activation mechanisms of the HACI-mediated unfolded protein response in filamentous fungi. Mol Microbiol 47, 11491161.[CrossRef][Medline]
Saloheimo, M., Wang, H., Valkonen, M., Vasara, T., Huuskonen, A., Riikonen, M., Pakula, T., Ward, M. & Penttilä, M. (2004). Characterization of secretory genes ypt1/yptA and nsf1/nsfA from two filamentous fungi: induction of secretory pathway genes of Trichoderma reesei under secretion stress conditions. Appl Environ Microbiol 70, 459467.
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Schafner, D. W. & Toledo, R. T. (1992). Cellulase production in continuous culture by Trichoderma reesei on xylose-based media. Biotechnol Bioeng 39, 865869.[CrossRef]
Schmoll, M. & Kubicek, C. P. (2003). Regulation of Trichoderma cellulase formation: lessons in molecular biology from an industrial fungus. A review. Acta Microbiol Immunol Hung 50, 125145.[CrossRef][Medline]
Schrickx, J. M., Krave, A. S., Verdoes, J. C., van den Hondel, C. A., Stouthamer, A. H. & van Verseveld, H. W. (1993). Growth and product formation in chemostat and recycling cultures by Aspergillus niger N402 and a glucoamylase overproducing transformant, provided with multiple copies of the glaA gene. J Gen Microbiol 139, 28012810.[Medline]
Spohr, A., Carlsen, M., Nielsen, J. & Villadsen, J. (1998). -amylase production in recombinant Aspergillus oryzae during fed-batch and continuous cultivations. J Ferment Bioeng 86, 4956.[CrossRef]
Strauss, J., Mach, R. L., Zeilinger, S., Hartler, G., Stoffler, G., Wolschek, M. & Kubicek, C. P. (1995). Cre1, the carbon catabolite repressor protein from Trichoderma reesei. FEBS Lett 376, 103107.[CrossRef][Medline]
Takashima, S., Nakamura, A., Iikura, H., Masaki, H. & Uozumi, T. (1996). Cloning of a gene encoding a putative carbon catabolite repressor from Trichoderma reesei. Biosci Biotechnol Biochem 60, 173176.[Medline]
van Gemeren, I. A., Punt, P. J., Drint-Kuyvenhoven, A., Broekhuijsen, M. P., van't Hoog, A., Beijersbergen, A., Verrips, C. T. & van den Hondel, C. A. (1997). The ER chaperone encoding bipA gene of black Aspergilli is induced by heat shock and unfolded proteins. Gene 198, 4352.[CrossRef][Medline]
Wiebe, M. G., Robson, G. D., Shuster, J. & Trinci, A. P. (2000). Growth-rate-independent production of recombinant glucoamylase by Fusarium venenatum JeRS 325. Biotechnol Bioeng 68, 245251.[CrossRef][Medline]
Withers, J. M., Swift, R. J., Wiebe, M. G., Robson, G. D., Punt, P. J., van den Hondel, C. A. M. J. J. & Trinci, A. P. J. (1998). Optimisation and stability of glucoamylase production by recombinant strains of Aspergillus niger in chemostat culture. Biotechnol Bioeng 59, 407418.[Medline]
Received 2 July 2004;
revised 23 September 2004;
accepted 24 September 2004.
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
J MED MICROBIOL | ALL SGM JOURNALS |