Cassier-Chauvat, C. | Search bioRxiv

bioRxiv2023

Phosphoribulokinase abundance is not limiting the Calvin-Benson-Bassham cycle in Chlamydomonas reinhardtii

Improving photosynthetic efficiency in plants and microalgae is of utmost importance to support the growing world population and to enable the bioproduction of energy and chemicals. Limitations in photosynthetic light conversion efficiency can be directly attributed to kinetic bottlenecks within the Calvin-Benson-Bassham cycle (CBBC) responsible for carbon fixation. A better understanding of these bottlenecks in vivo is crucial to overcome these limiting factors through bio-engineering. The present study is focused on the analysis of phosphoribulokinase (PRK) in the unicellular green alga Chlamydomonas reinhardtii. We have characterized a PRK knock-out mutant strain and showed that in the absence of PRK, Chlamydomonas cannot grow photoautotrophically while functional complementation with a synthetic construct allowed restoration of photoautotrophy. Nevertheless, using standard genetic elements, the expression of PRK was limited to 40% of the reference level in complemented strains and could not restore normal growth in photoautotrophic conditions suggesting that the CBBC is limited. We were subsequently able to overcome this initial limitation by improving the design of the transcriptional unit expressing PRK using diverse combinations of DNA parts including PRK endogenous promoter and introns. This enabled us to obtain strains with PRK levels comparable to the reference strain and even overexpressing strains. A collection of strains with PRK levels between 16% and 250% of WT PRK levels was generated and characterized. Immunoblot and growth assays revealed that a PRK content of {approx}86% is sufficient to fully restore photoautotrophic growth. This result suggests that PRK is present in moderate excess in Chlamydomonas. Consistently, the overexpression of PRK did not increase photosynthetic growth indicating that that the endogenous level of PRK in Chlamydomonas is not limiting the Calvin-Benson-Bassham cycle under optimal conditions.

synthetic biology↗

bioRxiv2021

A new gene family diagnostic for intracellular biomineralization of amorphous Ca-carbonates by cyanobacteria

Cyanobacteria have massively contributed to carbonate deposit formation over the geological history. They are traditionally thought to biomineralize CaCO3 extracellularly as an indirect byproduct of photosynthesis. However, the recent discovery of freshwater cyanobacteria forming intracellular amorphous calcium carbonates (iACC) challenges this view. Despite the geochemical interest of such a biomineralization process, its molecular mechanisms and evolutionary history remain elusive. Here, using comparative genomics, we identify a new gene (ccyA) and protein (calcyanin) family specifically associated with cyanobacterial iACC biomineralization. Calcyanin is composed of a conserved C-terminal domain, which likely adopts an original fold, and a variable N-terminal domain whose structure allows differentiating 4 major types among the 35 known calcyanin homologues. Calcyanin lacks detectable full-length homologs with known function. Yet, genetic and comparative genomic analyses suggest a possible involvement in Ca homeostasis, making this gene family a particularly interesting target for future functional studies. Whatever its function, this new gene family appears as a gene diagnostic of intracellular calcification in cyanobacteria. By searching for ccyA in publicly available genomes, we identified 13 additional cyanobacterial strains forming iACC. This significantly extends our knowledge about the phylogenetic and environmental distribution of cyanobacterial iACC biomineralization, especially with the detection of multicellular genera as well as a marine species. Phylogenetic analyses indicate that iACC biomineralization is ancient, with independent losses in various lineages and some HGT cases that resulted in the broad but patchy distribution of calcyanin across modern cyanobacteria. Overall, iACC biomineralization emerges as a new case of genetically controlled biomineralization in bacteria. Significance statementFew freshwater species of Cyanobacteria have been known to mineralize amorphous CaCO3 (ACC) intracellularly. Despite the geochemical interest of this biomineralization, its evolutionary history and molecular mechanism remain poorly known. Here, we report the discovery of a new gene family that has no homolog with known function, which proves to be a good diagnostic marker of this process. It allowed to find cyanobacteria in several phyla and environments such as seawater, where ACC biomineralization had not been reported before. Moreover, this gene is ancient and was independently lost in various lineages with some later horizontal transfers, resulting in a broad and patchy phylogenetic distribution in modern cyanobacteria.

evolutionary biology↗