Pommerenke, B. | Search bioRxiv

bioRxiv2024

Bottom-up reconstruction of minimal pyrenoids provides insights into the evolution and mechanisms of carbon concentration by EPYC1 proteins

Membraneless organelles play essential roles in cellular processes. In various photosynthetic organisms, they confer carbon concentrating mechanisms (CCMs). One example is the pyrenoid in Chlamydomonas reinhardtii, a liquid-phase separated organelle that localizes and improves carbon fixation via the intrinsically-disordered protein (IDP) EPYC1. Modern-day pyrenoids are complex structures, which makes it impossible to study the function of EPYC1, especially whether EPYC1 alone confers carbon concentration, and how EPYC1 could have initiated the evolution of pyrenoids. Here, we developed a bottom-up approach to study the function of EPYC1 and its sequence-function space across evolution. We demonstrate that modern-day EPYC1-sequences, but not other IDPs, induce liquid-phase separation of Rubisco into minimal pyrenoids with functional CCMs. Using ancestral sequence reconstruction, we trace the evolution of pyrenoids and demonstrate that selection acted on carboxylation rate, and in selected cases on specificity, providing unexpected perspectives on the design and function of natural and synthetic pyrenoids.

biochemistry↗

bioRxiv2024

Multiple levels of transcriptional regulation control glycolate metabolism in Paracoccus denitrificans

The hydroxyacid glycolate is a highly abundant carbon source in the environment. Glycolate is produced by unicellular photosynthetic organisms and excreted at petagram scales to the environment, where it serves as growth substrate for heterotrophic bacteria. In microbial metabolism, glycolate is first oxidized to glyoxylate by the enzyme glycolate oxidase. The recently described {beta}-hydroxyaspartate cycle (BHAC) subsequently mediates the carbon-neutral assimilation of glyoxylate into central metabolism in ubiquitous Alpha- and Gammaproteobacteria. While the reaction sequence of the BHAC was elucidated in Paracoccus denitrificans, little is known about the regulation of glycolate and glyoxylate assimilation in this relevant alphaproteobacterial model organism. Here, we show that regulation of glycolate metabolism in P. denitrificans is surprisingly complex, involving two regulators, the IclR-type transcription factor BhcR that acts as an activator for the BHAC gene cluster, as well as the GntR-type transcriptional regulator GlcR, a previously unidentified repressor that controls the production of glycolate oxidase. Furthermore, an additional layer of regulation is exerted at the global level, which involves the transcriptional regulator CceR that controls the switch between glycolysis and gluconeogenesis in P. denitrificans. Together, these regulators control glycolate metabolism in P. denitrificans, allowing the organism to assimilate glycolate together with other carbon substrates in a simultaneous fashion, rather than sequentially. Our results show that the metabolic network of Alphaproteobacteria shows a high degree of flexibility to react to the availability of multiple substrates in the environment.

microbiology↗