Expression rekombinanter pflanzlicher Phosphoenolpyruvat-Carboxylasen zur Beeinflussung des Kohlenstoffmetabolismus von Solanum tuberosum

Phosphoenolpyruvate carboxylase (PEPC) is a ubiquitous cytosolic enzyme in plants. Besides several housekeeping functions in plant metabolism, PEPC plays a cardinal role in the initial fixation of CO2 in C4 and CAM plants. The goal of this work was to regenerate transgenic C3-plants (Solanum tuberosum) with enhanced PEPC activity by overexpessing a altered endogenous PEPC. Therefore the ppc1 gene from Solanum tuberosum was modified, expressed in E. coli and analysed in vitro. By site-directed mutagenesis of the N-terminal phosphorylation motive of the recombinant potato PEPC the substrate affinity was increased and the sensitivity to malate, an allosteric inhibitor, was decreased. The catalytic properties were further increased by the exchange of an internal part for the homologous stretch from the C4-PEPC from Flaveria trinervia. By overexpressing of the mutated proteins constitutivly in potato, PEPC activities up to 5-fold compared to the wild-type were measured in vitro. The growth and tuber yield of the high expressing transgenic plants was strongly diminished. Metabolite analysis showed that carbon flow in the transgenic plants was redirected from sugars and starch to organic acids and amino acids. The effects on growth and metabolism indicated that the modified PEPC was highly active in planta