Highly active, citrate inhibition resistant form of Aspergillus niger 6-phosphofructo-1-kinase encoded by a modified pfkA gene.

In Aspergillus niger cells spontaneous posttranslational modification of 6-phosphofructo-1-kinase (PFK1) occurs. In a two step process the native enzyme (85kDa) is first cleaved to an inactive fragment (49kDa) that regains its activity after phosphorylation of the protein. The shorter PFK1 fragment exhibits changed kinetics, such as resistance to citrate inhibition. In order to avoid spontaneous complex posttranslational modification, modified gene was prepared encoding an active shorter PFK1 fragment. Since no appropriate microbial strains with disrupted native pfkA genes were available, Aspergillus niger strain with reduced likelihood for spontaneous posttranslational modification of PFK1 has been chosen for in vivo tests. First, the appropriate length of a truncated gene was defined after a number of enzymes encoded by genes of different lengths had been tested. After adding sodium azide to the medium, phosphorylation was induced in the transformed hyphae to activate the shorter fragments which were subsequently screened for changed PFK1 kinetics. In the second step the responsible threonine residue was replaced with glutamic acid to elude the need for phosphorylation. An active shorter PFK1 fragment, resistant to citrate inhibition and activated to a higher level by fructose-2,6-bisphosphate with respect to the native enzyme was encoded directly from the modified gene.