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.
Enzyme assays of the roots of barley (Hordeum vulgare L.) fed NH 4 (+) show high glutamate-dehydrogenase (GDH; EC 1.4.1.3) activity compared with glutamine-synthetase (GS; EC 6.3.1.2) activity, indicating that GDH may be involved in ammonium assimilation in the root. When (15)NH 4 (+) is fed to barley roots, a high accumulation of (15)N takes place in free amino compounds, particularly glutamine and glutamate. When the GS inhibitor, methionine sulfoximine (MSO), is added to the (15)NH 4 (+) feeding medium the free amino compounds remain unlabelled while (15)NH 4 (+) accumulates rapidly in the roots. Root enzyme assays demonstrate that GS is completely inhibited by MSO treatment, while the activity of GDH remains unaffected. The feeding of (15)N-amido glutamine to the roots in the presence of MSO and the subsequent (15)N enrichment of the free amino compounds of the root show that MSO does not interfere substantially with nitrogen assimilation after the formation of glutamine. These results indicate that in the barley root, ammonium absorbed from the soil is assimilated entirely via the GS-glutamate synthase (GOGAT) pathway, and that GDH plays little, if any, part in this process.
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