De novo transcriptome and lipidome analysis of Desmodesmus abundans under model flue gas reveals adaptive changes after ten years of acclimation to high CO2.

Microalgae's ability to mitigate flue gas is an attractive technology that can valorize gas components through biomass conversion. However, tolerance and growth must be ideal; therefore, acclimation strategies are suggested. Here, we compared the transcriptome and lipidome of Desmodesmus abundans strains acclimated to high CO2 (HCA) and low CO2 (LCA) under continuous supply of model flue gas (MFG) and incomplete culture medium (BG11-N-S). Initial growth and nitrogen consumption from MFG were superior in strain HCA, reaching maximum productivity a day before strain LCA. However, similar productivities were attained at the end of the run, probably because maximum photobioreactor capacity was reached. RNA-seq analysis during exponential growth resulted in 16,435 up-regulated and 4,219 down-regulated contigs in strain HCA compared to LCA. Most differentially expressed genes (DEGs) were related to nucleotides, amino acids, C fixation, central carbon metabolism, and proton pumps. In all pathways, a higher number of up-regulated contigs with a greater magnitude of change were observed in strain HCA. Also, cellular component GO terms of chloroplast and photosystems, N transporters, and secondary metabolic pathways of interest, such as starch and triacylglycerols (TG), exhibited this pattern. RT-qPCR confirmed N transporters expression. Lipidome analysis showed increased glycerophospholipids in strain HCA, while LCA exhibited glycerolipids. Cell structure and biomass composition also revealed strains differences. HCA possessed a thicker cell wall and presented a higher content of pigments, while LCA accumulated starch and lipids, validating transcriptome and lipidome data. Overall, results showed significant differences between strains, where characteristic features of adaptation and tolerance to high CO2 might be related to the capacity to maintain a higher flux of internal C, regulate intracellular acidification, active N transporters, and synthesis of essential macromolecules for photosynthetic growth.

Subunit Asa3 ensures the attachment of the peripheral stalk to the membrane sector of the dimeric ATP synthase of Polytomella sp.

The mitochondrial ATP synthase of Polytomella exhibits a peripheral stalk and a dimerization domain built by the Asa subunits, unique to chlorophycean algae. The topology of these subunits has been extensively studied. Here we explored the interactions of subunit Asa3 using Far Western blotting and subcomplex reconstitution, and found it associates with Asa1 and Asa8. We also identified the novel interactions Asa1-Asa2 and Asa1-Asa7. In silico analyses of Asa3 revealed that it adopts a HEAT repeat-like structure that points to its location within the enzyme based on the available 3D-map of the algal ATP synthase. We suggest that subunit Asa3 is instrumental in securing the attachment of the peripheral stalk to the membrane sector, thus stabilizing the dimeric mitochondrial ATP synthase.

Establishment of an efficient genetic transformation method in Dunaliella tertiolecta mediated by Agrobacterium tumefaciens.

Microalgae are photosynthetic microorganisms widely used for the production of highly valued compounds, and recently they have been shown to be promising as a system for the heterologous expression of proteins. Several transformation methods have been successfully developed, from which the Agrobacterium tumefaciens-mediated method remains the most promising. However, microalgae transformation efficiency by A. tumefaciens is shown to vary depending on several transformation conditions. The present study aimed to establish an efficient genetic transformation system in the green microalgae Dunaliella tertiolecta using the A. tumefaciens method. The parameters assessed were the infection medium, the concentration of the A. tumefaciens and co-culture time. As a preliminary screening, the expression of the gusA gene and the viability of transformed cells were evaluated and used to calculate a novel parameter called Transformation Efficiency Index (TEI). The statistical analysis of TEI values showed five treatments with the highest gusA gene expression. To ensure stable transformation, transformed colonies were cultured on selective medium using hygromycin B and the DNA of resistant colonies were extracted after five subcultures and molecularly analyzed by PCR. Results revealed that treatments which use solid infection medium, A. tumefaciens OD600 = 0.5 and co-culture times of 72 h exhibited the highest percentage of stable gusA expression. Overall, this study established an efficient, optimized A. tumefaciens-mediated genetic transformation of D. tertiolecta, which represents a relatively easy procedure with no expensive equipment required. This simple and efficient protocol opens the possibility for further genetic manipulation of this commercially-important microalgae for biotechnological applications.