Molecular genetics of 6-phosphofructokinase in Pichia pastoris

Abstract

Previously, studies on glucose-induced microautophagy in the methylotrophic yeast Pichia pastoris provided evidence that the glucose-induced selective autophagy-1-protein is the alpha-subunit of 6-phosphofructokinase (Pfk), a key enzyme in the glycolytic pathway. In our work, we could clearly demonstrate that two types of subunits of Pfk exist in P. pastoris. Investigating the yeast cell-free extract by Western blot analysis, two distinct signals of Pfk were obtained. In addition, we isolated a DNA sequence containing the complete ORF of PpPFK2 encoding the beta-subunit of Pfk from P. pastoris with a deduced molecular mass of 103.7 kDa. On the basis of these results, a hetero-oligomeric structure of Pfk in P. pastoris became obvious. Because the molecular and kinetic properties of a homo-oligomeric yeast Pfk appear to be more similar to those of mammalian Pfk, as described in the literature, our results are of interest for the growing number of studies on P. pastoris as a heterologous production system. Furthermore, the 3'- and 5'-non-coding regions of PpPFK2 were isolated and several putative binding sites for regulatory factors could be identified in the promoter region.