OVEREXPRESSION OF PHOSPHOFRUCTOKINASE AND PYRUVATE-KINASE IN CITRIC ACID-PRODUCING ASPERGILLUS-NIGER

Phosphofructokinase and pyruvate kinase were overexpressed in the fila mentous fungus Aspergillus niger. Moderate overexpression of these gly colytic enzymes in A. niger N400 (3-5-fold the wild-type level), eithe r individually or simultaneously, did not increase citric acid product ion by the fungus significantly. Thus, phosphofructokinase and pyruvat e kinase do not seem to contribute in a major way to flux control of t he metabolism involved in the conversion of glucose to citric acid. Ov erexpression of phosphofructokinase and pyruvate kinase did not influe nce the activities of other enzymes in the pathway, nor did it change intermediary metabolite levels. However, in strains overexpressing pho sphofructokinase, the level of fructose 2,6-bisphosphate, a positive a llosteric effector of phosphofructokinase, was reduced almost 2-fold c ompared to the wild-type strain. Measurements with purified phosphofru ctokinase, using substrate, product and effector concentrations found intracellularly, showed that such a reduction in the fructose-2,6-bisp hosphate level could decrease the specific activity of phosphofructoki nase in the cell significantly. Thus, the fungus seems to adapt to ove rexpression of phosphofructokinase by decreasing the specific activity of the enzyme through a reduction in the level of fructose 2,6-bispho sphate.