Heterologous protein secretion and fungal morphology in chemostat cultures of a recombinant Aspergillus niger (B1-D)

Abstract

Synthesis of the heterologous marker protein hen egg white lysozyme (HEWL) and a native enzyme involved in substrate (starch) degradation (glucoamylase) was studied in chemostat cultures of a recombinant Aspergillus niger (B1-D) at a range of dilution rates. The fungal morphology was quantitatively assessed by means of computerized image analysis (IA) at all dilution rates. Synthesis of the heterologous protein and the native starch processing enzyme was maximal at low dilution rates, and fell as dilution rate (D) increased due to a combination of specific induction/repression mechanisms influenced by the residual sugar concentration and the decreased mean residence time of fluid elements in the reactor as D increased. Proteolysis in the culture fluid could not, of itself, explain the decrease in enzyme synthesis as D rose. The fungus synthesized significant amounts of ethanol at all dilution rates, but ethanol synthesis tended to increase with D, due to a steady increase in pellet core diameters and an increasing degree of oxygen limitation. In terms of both the micro- and macromorphology of the culture, increasing the dilution rate tended generally to result in longer “organisms” (i.e., increases in the value of the morphological indices) due to the combined effects of increased extensive growth and decreased hydromechanical damage at higher D. It is clear that the duration of exposure of culture elements to hydromechanical damage may well have had a profound morphological influence. Although there was no correlation between the conventional indices of micromorphology and HEWL/glucoamylase levels, there was a correlation between “percentage active length” of hyphae and total soluble protein concentration, and also between “total concentration of tips” (i.e., tip number per “organism”×biomass concentration) and total soluble protein concentration. This latter quantity (total concentration of tips) may be an adequate, and simpler means of assessing protein-synthesizing capability than more involved double-staining techniques.