From cyanobacteria to cell organelle – Engineering and studying minimal endosymbiotic metabolism

In eukaryotes, photosynthesis evolved through endosymbiotic incorporation of a photosynthetic cyanobacterium, which became the ancestor of plastids. The transformation entailed gene transfer from the endosymbiont to the host nucleus, evolution of protein targeting systems, as well as synchronization of host and endosymbiont cell cycles. The ménage-à-trois (MAT) hypothesis proposes that the linkage of carbon and energy metabolism, established by the integration of transporters gained by horizontal gene transfer from Chlamydia-like endoparasites, was the primary driver of the endosymbiosis (Karkar et al., 2015).

In order to investigate whether the linkage of host and endosymbiont carbon and energy metabolism could be sufficient for the establishment of endosymbiosis, we aim to enforce a synthetic endosymbiont-like metabolism in the model cyanobacterium Synechocystis sp. PCC 6803. By deletion of the glgC gene, the precursor for the energy storage compound glycogen will no longer be produced. Implementation of the Chlamydia-like transporter UhpC will alleviate carbon overflow reactions (Gründel et al., 2012) via export of excess carbon out of the cell during periods of light. Implementation of the transporter NTT will allow uptake of external ATP for energy during periods of darkness. Markerless deletion will be used to sequentially erode the genome, while diurnal growth, transcriptomes and metabolomes will be characterized. Our microfluidic platform (Witting et al., 2024) will be used to mimic the host cytoplasm during early stages of endosymbiosis, enabling analysis of cyanobacterial physiology in response to metabolomic rerouting and special constraints at single cell resolution. Additionally, the introduction of metabolite sensors will enable real-time readouts of metabolic changes within the cell.

The successful knockout of glgC has been achieved and three independent Synechocystis ∆glgC knockout lines have been characterized under diurnal growth conditions. The transcriptomes and metabolomes of the mutant lines reveal stressed phenotypes and increased energy charge as well as metabolic re-routing. Strains expressing Chlamydia-like transporters are tested for their efficiency to complement the metabolic overflow in light and energy shortage in darkness.