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

Synthesis of the heterologous marker protein hen egg white lysozyme (HEWL) and a native enzyme involved in substrate (starch) degradation (glucoamylas e) 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 en zyme was maximal at low dilution rates, and fell as dilution rate (D) incre ased due to a combination of specific induction/repression mechanisms influ enced by the residual sugar concentration and the decreased mean residence time of fluid elements in the reactor as D increased. Proteolysis in the cu lture fluid could not, of itself explain the decrease in enzyme synthesis a s D rose. The fungus synthesized significant amounts of ethanol at all dilu tion rates, but ethanol synthesis tended to increase with D, due to a stead y increase in pellet core diameters and an increasing degree of oxygen limi tation. In terms of both the micro- and macromorphology of the culture, inc reasing the dilution rate tended generally to result in longer "organisms" (i.e., increases in the value of the morphological indices) doe to the comb ined effects of increased extensive growth and decreased hydromechanical da mage at higher D. It is clear that the duration of exposure of culture elem ents to hydromechanical damage may well have had a profound morphological i nfluence. Although there was no correlation between the conventional indice s of micromorphology and HEWL/glucoamylase levels, there was a correlation between "percentage active length" of hyphae and total soluble protein conc entration, and also between "total concentration of tips" (i.e., tip number per "organism " X biomass concentration) and total soluble protein concent ration. This latter quantity (total concentration of tips) may be an adequa te, and simpler means of assessing protein-synthesizing capability than mor e involved double-staining techniques. (C) 1999 Elsevier Science Inc. All r ights reserved