GlaA promoter controlled production of a mutant green fluorescent protein (S65T) by recombinant aspergillus niger during growth on defined medium in batch and fed-batch cultures

The first successful expression of the Aequorea victoria green fluorescent protein (GFP) gene in Aspergillus niger is described. When the wild-type GFP gene was expressed in A. niger, neither the fluorescence nor the full translation product of the wtGFP gene was detectable. However, the expression of a mutant form of the green fluorescent protein (S65TGFP) gene resulted in the formation of a functional fluorescent polypeptide. The synthesis of S65TGFP was used to study glaA promoter controlled heterologous gene expression by recombinant A. niger in batch and fed-batch cultures using a defined growth medium. Cells were grown on xylose as noninducing carbon source, and the production of S65TGFP was accomplished by the addition of maltose. The recombinant protein accumulated up to 10 or 25 mg of S65TGFP g-1 cell dry weight using either a maltose pulse for induction or continuous addition of the inducing carbon source, respectively. Irrespective of the induction protocol, the recombinant protein started to accumulate 2 h after addition of the inducing carbon source and reached its maximum specific concentration 10 h after induction. Bright green fluorescing fungal pellets were first detectable by fluorescence microscopy 4-5 h after the onset of maltose addition.