The Effect of Organic Nitrogen Sources on Recombinant Glucoamylase Production by Aspergillus niger in Chemostat Culture

Swift, Richard J.; Karandikar, Atul; Griffen, Alison M.; Punt, Peter J.; Hondel, Cees A. M. J. J. van den; Robson, Geoffrey D.; Trinci, Anthony P. J.; Wiebe, Marilyn G.

doi:10.1006/fgbi.2000.1241

Cited by 28 publications

(19 citation statements)

References 16 publications

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“…In previous studies with A. niger chemostats grown at pH 5.4, the morphologic mutants that appeared were mainly brown conidia bearing mutants, and those were scarcely observed in cultures grown at pH 4.0 (Mainwaring et al, 1999;Swift et al, 1998Swift et al, , 2000Wallis et al, 2001;Withers et al, 1998). In these studies, Swift et al (1998) and Withers et al (1998) reported the appearance of white, nonsporulating mutants, but only as a small proportion of the total population and only at pH 5.4 or 5.5.…”

Section: Isolation Of a Stable Fluffy Mutantmentioning

confidence: 86%

“…These variants had a selective advantage over their parent and were morphologically more stable in chemostat cultures. Similarly, from a glucoamylase-overproducing A. niger strain, mutants with increased selective advantage were isolated, but unfortunately, in most cases their productivity appeared to be reduced (Swift et al, 2000;Withers et al, 1998). However, Withers et al (1998) isolated a mutant with less densely, brown spores, which sustained production for 735 h, and Swift et al (1998) isolated a white aconidial strain with higher glucoamylase production than the parent strain.…”

Section: Introductionmentioning

confidence: 94%

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Isolation of a fluffy mutant of Aspergillus niger from chemostat culture and its potential use as a morphologically stable host for protein production

Vondervoort

Poulsen

Ruijter

et al. 2004

Biotech & Bioengineering

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Chemostat cultivation of Aspergillus niger and other filamentous fungi is often hindered by the spontaneous appearance of morphologic mutants. Using the Variomixing bioreactor and applying different chemostat conditions we tried to optimize morphologic stability in both ammonium- and glucose-limited cultures. In most cultivations mutants with fluffy (aconidial) morphology became dominant. From an ammonium-limited culture, a fluffy mutant was isolated and genetically characterized using the parasexual cycle. The mutant contained a single morphological mutation, causing an increased colony radial growth rate. The fluffy mutant was subjected to transformation and finally conidiospores from a forced heterokaryon were shown to be a proper inoculum for fluffy strain cultivation.

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Section: Isolation Of a Stable Fluffy Mutantmentioning

confidence: 86%

Section: Introductionmentioning

confidence: 94%

Isolation of a fluffy mutant of Aspergillus niger from chemostat culture and its potential use as a morphologically stable host for protein production

Vondervoort

Poulsen

Ruijter

et al. 2004

Biotech & Bioengineering

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“…It also improves barley mash for beer production (36). GA is a key enzyme too in the production of sake and soy sauce.…”

Section: Introductionmentioning

confidence: 99%

Production and characterization of glucoamylase from fungus Aspergillus awamori expressed in yeast Saccharomyces cerevisiae using different carbon sources

Pavezzi¹,

Gomes²,

Silva³

2008

Braz. J. Microbiol.

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Glucoamylase is widely used in the food industry to produce high glucose syrup, and also in fermentation processes for production beer and ethanol. In this work the productivity of the glucoamylase of Aspergillus awamori expressed by the yeast Saccharomyces cerevisiae, produced in submerged fermentation using different starches, was evaluated and characterized physico-chemically. The enzyme presented high specific activity, 13.8 U/mgprotein or 2.9 U/mgbiomass, after 48 h of fermentation using soluble starch as substrate. Glucoamylase presented optimum activity at temperature of 55ºC, and, in the substratum absence, the thermostability was for 1h at 50ºC. The optimum pH of activity was pH 3.5 -4.0 and the pH stability between 5.0 and 7.0. The half life at 65ºC was at 30.2 min, and the thermal energy of denaturation was 234.3 KJ mol -1 . The hydrolysis of different substrate showed the enzyme's preference for the substrate with a larger polymerization degree. The gelatinized corn starch was the substratum most susceptible to the enzymatic action.

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“…The type of fermentation medium also affects the secretory capacity of a given expression host. Most importantly, the expression of extracellular proteases, such as those encoded by pepA, pepB, and pepF, which significantly affect secreted protein yield, are regulated by the types of carbon and nitrogen sources and by the pH of the medium (39,41). Kurzatkowski et al (22) have presented evidence that glucose also has a negative effect on the development of an efficient secretory apparatus.…”

mentioning

confidence: 99%

Metabolically Independent and Accurately AdjustableAspergillussp. Expression System

Pachlinger

Mitterbauer

Adam

et al. 2005

Appl Environ Microbiol

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Filamentous fungi are well-established expression hosts often used to produce extracellular proteins of use in the food and pharmaceutical industries. The expression systems presently used in Aspergillus species rely on either strong constitutive promoters, e.g., that for glyceraldehyde-3-phosphate dehydrogenase, or inducible systems derived from metabolic pathways, e.g., glaA (glucoamylase) or alc (alcohol dehydrogenase). We describe for Aspergillus nidulans and Aspergillus niger a novel expression system that utilizes the transcriptional activation of the human estrogen receptor by estrogenic substances. The system functions independently from metabolic signals and therefore can be used with low-cost, complex media. A combination of positive and negative regulatory elements in the promoter drives the expression of a reporter gene, yielding a linear dose response to the inducer. The off status is completely tight, yet the system responds within minutes to induction and reaches a level of expression of up to 15% of total cell protein after 8 h. Both Aspergillus species are very sensitive to estrogenic substances, and low-cost inducers function in the picomolar concentration range, at which estrogenic substances also can be found in the environment. Given this high sensitivity to estrogens, Aspergillus cells carrying estrogen-responsive units could be used to detect xenoestrogens in food or in the environment.In their natural environment, fungi use extracellular enzymes to gain access to complex, often water-insoluble carbon and nitrogen sources. Extracellular protein production by filamentous fungi usually is very efficient for homologous proteins, for which levels of grams per liter can be obtained. Aspergillus and Trichoderma strains have been reported to secrete up to 30 g of homologous proteins per liter in fermentation processes (e.g., glucoamylases) (11). The production of recombinant proteins of mammalian origin can be up to 4 orders of magnitude lower even when the same expression signals are used.The reason for this difference is not completely understood (2). Along with mRNA stability, codon usage, and translational efficiency, overloading the secretory machinery with a protein that has a "foreign" structure appears to be a critical bottleneck (16,34). High concentrations of such heterologous proteins can result in incorrect folding-leading to translational feedback inhibition by phosphorylation of the ␣ subunit of eukaryotic initiation factor 2 (23) and subsequent intracellular degradation of the misfolded protein. Thus, strong but highly regulatable expression systems are needed to control the flow of proteins through the secretory pathway.The use of strong promoters derived from housekeeping genes, such as those of the various fungal glyceraldehyde-3-phosphate dehydrogenase genes, has the disadvantage of continuous, growth-related expression levels (31). This problem may reduce yields of proteins susceptible to degradation or lead to cell death if the overexpressed foreign proteins are toxic to the ...

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The Effect of Organic Nitrogen Sources on Recombinant Glucoamylase Production by Aspergillus niger in Chemostat Culture

Cited by 28 publications

References 16 publications

Isolation of a fluffy mutant of Aspergillus niger from chemostat culture and its potential use as a morphologically stable host for protein production

Isolation of a fluffy mutant of Aspergillus niger from chemostat culture and its potential use as a morphologically stable host for protein production

Production and characterization of glucoamylase from fungus Aspergillus awamori expressed in yeast Saccharomyces cerevisiae using different carbon sources

Metabolically Independent and Accurately AdjustableAspergillussp. Expression System

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