Production of GFP and glucoamylase by recombinant Aspergillus niger: effects of fermentation conditions on fungal morphology and protein secretion.

The effect of filamentous fungal morphology on heterologous protein secretion was investigated using the recombinant Aspergillus niger strain AB4.1[pgpdAGLAGFP], which contained the gene coded for the GLA-GFP (glucoamylase-green fluorescence protein) fusion protein. Three culturing systems were studied to develop different morphological forms of the fungus. Free-cell cultures in conventional stirred-tank bioreactors grew in pellet form with various sizes depending on culturing conditions. Cells immobilized on cotton cloth grew in mycelial form in a rotating fibrous bed (RFB) and a static fibrous bed (SFB) bioreactors. The expression of the fusion protein was growth-associated and dependent on the fungal morphology. Immobilized cells produced 10-fold more GFP and glucoamylase than well-oxygenated free-cell pellets. In free-cell cultures, excretion of the fusion protein occurred mainly from cell autolysis, when oxygen or nutrient were depleted, whereas protein secretion took place from the beginning of the fermentation in immobilized-cell cultures. Also, protein secretion was found to be strongly dependent on morphology. Small pellets of a 1-mm size secreted 82% of GFP produced, whereas 43% of GFP remained intracellular in larger pellets of 5 mm. Complete secretion of GFP was obtained with cells immobilized on the fibrous matrix. The improvement in heterologous protein synthesis and secretion can be attributed to the filamentous mycelial morphology since protein secretion occurred predominantly at the tips of growing hyphae. Secretion of proteases occurred mainly in the stationary phase or when cell autolysis were induced by nutrient depletion and was not dependent on morphology, although immobilizing the cells also reduced protease activity. The RFB bioreactor gave the best fermentation performance because of its ability to control the cell morphology that was amenable to efficient oxygen transfer and protein secretion.