Knockdown of phosphoenolpyruvate carboxykinase increases carbon flux to lipid synthesis in Phaeodactylum tricornutum

The tricarboxylic acid (TCA) cycle is a key metabolic pathway that unifies carbohydrate, lipid and protein metabolism. In Phaeodactylum tricornutum, accumulation of triacylglycerols (TAGs) under nitrogen stress is a consequence of re-allocation of carbon mainly from the intermediates of TCA cycle. Oxaloacetate, an intermediate of the TCA cycle, can flux out of the cycle by the decarboxylation enzyme phosphoenolpyruvate carboxykinase (PEPCK), thus decreasing the carbon flow directed to fatty acid biosynthesis. We assayed the expression of genes encoding mitochondrially-localized decarboxylation enzymes, essential for putative C4 metabolism, in P. tricornutum. Targeted knockdown of PEPCK was performed using RNA-interference and the photosynthetic characteristics and TAG contents of knockdown lines were characterized. PEPCK was significantly upregulated at both the mRNA and protein levels during TAG accumulation in P. tricornutum, and both PEPCK transcript abundance and enzyme activity were reduced significantly in two RNAi lines. The mutants exhibited lower photosynthetic activity, while TAG content was markedly increased. Meanwhile, cell density was comparable with that of wild-type. We propose that carbon from decarboxylation of C4 metabolites in the mitochondria plays a minor role in carbon concentration in this diatom. Downregulated decarboxylation succeeded in increasing lipid accumulation by decreasing the loss of carbon skeletons without compromising cell biomass.