Regulating phosphoenolpyruvate carboxylase activity by copper-induced expression method and exploring its role of carbon flux distribution in Synechocystis PCC 6803

Abstract

The petE gene transcription can be regulated by copper titer in some cyanobacteria. In order to regulate the activity of phosphoenolpyruvate carboxylase (PEPC) in Synechocystis PCC 6803, “Ω-PpetE” fragment, containing T4 transcription terminating sequence, anti-spectinomycin gene and petE promoter sequence was inserted between the native promoter and the ORF of ppc (gene encoding PEPC). After several rounds of selection, a stable and completely segregated mutant was attained. Cultured in different copper titers, the mutant presented narrow enzyme activity variance in the late exponential phase. A basal enzyme activity was observed in the culture without copper. When copper titer was increased to more than 280 nM which was the normal level of BG11 broth, the activity was stable. When PEPC activity was downregulated, the content of intracellular total carbonhydrate increased significantly and the content of intracellular total protein decreased. In central metabolic network, regulation characteristics of PEPC in Synechocystis PCC 6803 showed considerable robustness. Within the studied range of regulation, it can be regarded a pivotal node in central metabolic network which controls carbon flux distribution and decides the ratio of major biomass constituents.