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Optimization and stability of glucoamylase production by recombinant strains of Aspergillus niger in chemostat culture

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Author: Withers, J.M. · Swift, R.J. · Wiebe, M.G. · Robson, G.D. · Punt, P.J. · Hondel, C.A.M.J.J. van den
Type:article
Date:1998
Institution: Centraal Instituut voor Voedingsonderzoek TNO
Source:Biotechnology and Bioengineering, 4, 59, 407-418
Identifier: 86191
doi: DOI:10.1002/(SICI)1097-0290(19980820)59:4<407::AID-BIT3>3.0.CO;2-K
Keywords: Nutrition · Aspergillus niger · Chemostat culture · Glucoamylase (GAM) · Protein secretion · Recombinant protein · Strain stability · Ammonium compounds · Batch cell culture · Chemostats · Genetic engineering · Glucose · Magnesium · Morphology · Phosphates · Physiology · Potassium · Sulfur compounds · Chemostat culture · Glucoamylase · Shake flask culture · Enzymes · glucan 1,4 alpha glucosidase · article · aspergillus niger · chemostat · enzyme stability · enzyme synthesis · fungus culture · fungus growth · genetic recombination · nonhuman · protein secretion · Aspergillus niger · Blotting, Western · Cell Culture Techniques · Cell Division · Culture Media · Fermentation · Glucan 1,4-alpha-Glucosidase · Glucose · Hydrogen-Ion Concentration · Polysaccharides · Recombinant Proteins · Temperature · Time Factors

Abstract

When grown on a medium containing 5 g maltodextrin L-1, Aspergillus niger transformant N402[pAB6-10]B1, which has an additional 20 copies of the glucoamylase (glaA) gene, produced 320 ± 8 mg (mean ± S.E.) glucoamylase (GAM) L-1 in batch culture and 373 ± 9 mg GAM L-1 in maltodextrin- limited chemostat culture at a dilution rate of 0.13 h-1. These values correspond to specific production rates (q(p)) of 5.6 and 16.0 mg GAM [g biomass]-1 h-1, respectively. In maltodextrin-limited chemostat cultures grown at dilution rates from 0.06 to 0.14 h-1, GAM was produced by B1 in a growth-correlated manner, demonstrating that a continuous flow culture system operated at a high dilution rate is an efficient way of producing this enzyme. In chemostat cultures grown at high dilution rates, GAM production in chemostat cultures was repressed when the limiting nutrient was fructose or xylose, but derepressed when the limiting nutrient was glucose (q(p), 12.0), potassium (6.2), ammonium (4.1), phosphate (2.0), magnesium (1.5) or sulphate (0.9). For chemostat cultures grown at a dilution rate of 0.13 h-1, the addition of 5 g mycopeptone L-1 to a glucose-mineral salts medium resulted in a 64% increase in GAM concentration (from 303 ± 12 to 496 ± 10 mg GAM L-1) and a 37% increase in specific production rate (from 12.0 ± 0.4 to 16.4 ± 1.6 mg GAM [g biomass]-1 h-1). However, although recombinant protein production was stable for at least 948 h (191 generations) when A. niger B1 was grown in chemostat culture on glucose-mineral salts medium, it was stable for less than 136 h (27 generations) on medium containing mycopeptone. The predominant morphological mutants occurring after prolonged chemostat culture were shown to have selective advantage in the chemostat over the parental strain. Compared to their parental strains, two morphological mutants had similar GAM production levels, while a third had a reduced production level. Growth tests and molecular analysis revealed that the number of glaA gene copies in this latter strain (B1-M) was reduced, which could explain its reduced GAM production. Shake-flask cultures carried out with the various morphological mutants revealed that in batch culture all three strains produced considerably less GAM than their parent strains and even less than N402. We show that physiological changes in these morphological mutants contribute to this decreased level of GAM production. The predominant morphological mutants occurring after prolonged chemostat culture were shown to have selective advantage in the chemostat over the parental strain. Compared to their parental strains, two morphological mutants had similar glucoamylase (GAM) production levels, while a third had a reduced production level. Growth tests and molecular analysis revealed that the number of GAM gene copies in the later strain was reduced, which could explain its reduced GAM production. Shake-flask cultures of morphological mutants revealed that in batch culture all three strains produced considerably less GAM than their parent strains. The physiological changes in these morphological mutants which contribute to this decreased level of GAM production were shown