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

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 (qp) 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 (qp, 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.

Uptake and Reduction of Hexavalent Chromium by Aspergillus Niger and Aspergillus Parasiticus

“…For the growth of the fungi in suspension culture, a stock solution containing 0 to 20 mg/l Cr(VI) was prepared by dissolving analytical grade potassium dichromate (K 2 Cr 2 O 7 ) in 1 litre of sterile modified Vogel's mineral salts medium [20][21][22][23]. The solution was adjusted to a final pH of 4.5 ± 0.5 and 100 ml was used as culture medium for the growth of fungi.…”

“…1 filter paper, washing the biomass with distilled water and drying to constant weight at 70°C [20]. The biomass concentration was calculated as follows: …”

Enhanced heterologous protein production in Aspergillus niger through pH control of extracellular protease activity

“…First, total GLA was measured using PNPG (4-nitrophenyl-␣-dglucopyranoside) as substrate [22]. Freshly made 0.1% (w/v) PNPG in 0.1 M Na acetate buffer (pH 4.3) was equilibrated to 30 • C. The reaction mixture, which was incubated at 30 • C for 20 min, contained 250 l culture filtrate (or GLA standard) and 500 l PNPG solution.…”

“…Sev-eral strategies have been developed to increase these yields. Those employed include the introduction of a large number of gene copies [22], the use of strong promoters and efficient fungal secretion signals [1] and the development of protease-deficient host strains [16]. One of the most successful strategies involves the construction of gene fusions of the target gene to genomic sequences encompassing the complete or partial coding region of a highly expressed fungal gene such as Aspergillus niger glucoamylase (GLA) [21].…”

Quantitative proteomics reveals the mechanism and consequence of gliotoxin-mediated dysregulation of the methionine cycle in Aspergillus niger

“…niger is a filamentous fungus widely used as a host for the production of native and heterologous proteins, and multiple enzymes with biotechnological uses have been identified in, and purified from, A. niger [14,15]. Indeed, the availability of the genome sequence of this highly useful biological cell factory has been available since 2007 which has facilitated its further development as a tractable system for recombinant protein expression [16].…”