Giorgio Perin

Light Remodels Lipid Biosynthesis in Nannochloropsis gaditana by Modulating Carbon Partitioning between Organelles

Light availability remodels carbon partitioning between organelles toward a greater TAG accumulation in Nannochloropsis gaditana. The seawater microalga Nannochloropsis gaditana is capable of accumulating a large fraction of reduced carbon as lipids. To clarify the molecular bases of this metabolic feature, we investigated light-driven lipid biosynthesis in Nannochloropsis gaditana cultures combining the analysis of photosynthetic functionality with transcriptomic, lipidomic and metabolomic approaches. Light-dependent alterations are observed in amino acid, isoprenoid, nucleic acid, and vitamin biosynthesis, suggesting a deep remodeling in the microalgal metabolism triggered by photoadaptation. In particular, high light intensity is shown to affect lipid biosynthesis, inducing the accumulation of diacylglyceryl-N,N,N-trimethylhomo-Ser and triacylglycerols, together with the up-regulation of genes involved in their biosynthesis. Chloroplast polar lipids are instead decreased. This situation correlates with the induction of genes coding for a putative cytosolic fatty acid synthase of type 1 (FAS1) and polyketide synthase (PKS) and the down-regulation of the chloroplast fatty acid synthase of type 2 (FAS2). Lipid accumulation is accompanied by the regulation of triose phosphate/inorganic phosphate transport across the chloroplast membranes, tuning the carbon metabolic allocation between cell compartments, favoring the cytoplasm, mitochondrion, and endoplasmic reticulum at the expense of the chloroplast. These results highlight the high flexibility of lipid biosynthesis in N. gaditana and lay the foundations for a hypothetical mechanism of regulation of primary carbon partitioning by controlling metabolite allocation at the subcellular level.

Generation of random mutants to improve light-use efficiency of Nannochloropsis gaditana cultures for biofuel production

BackgroundThe productivity of an algal culture depends on how efficiently it converts sunlight into biomass and lipids. Wild-type algae in their natural environment evolved to compete for light energy and maximize individual cell growth; however, in a photobioreactor, global productivity should be maximized. Improving light use efficiency is one of the primary aims of algae biotechnological research, and genetic engineering can play a major role in attaining this goal.ResultsIn this work, we generated a collection of Nannochloropsis gaditana mutant strains and screened them for alterations in the photosynthetic apparatus. The selected mutant strains exhibited diverse phenotypes, some of which are potentially beneficial under the specific artificial conditions of a photobioreactor. Particular attention was given to strains showing reduced cellular pigment contents, and further characterization revealed that some of the selected strains exhibited improved photosynthetic activity; in at least one case, this trait corresponded to improved biomass productivity in lab-scale cultures.ConclusionsThis work demonstrates that genetic modification of N. gaditana has the potential to generate strains with improved biomass productivity when cultivated under the artificial conditions of a photobioreactor.

A Palmitic Acid Elongase Affects Eicosapentaenoic Acid and Plastidial Monogalactosyldiacylglycerol Levels in Nannochloropsis

A heterokont palmitic acid desaturase initiates the production of eicosapentaenoic acid in the endoplasmic reticulum of Nannochloropsis, which is specifically used for monogalactosyldiacylglycerol assembly in the secondary plastid. Nannochloropsis species are oleaginous eukaryotes containing a plastid limited by four membranes, deriving from a secondary endosymbiosis. In Nannochloropsis, thylakoid lipids, including monogalactosyldiacylglycerol (MGDG), are enriched in eicosapentaenoic acid (EPA). The need for EPA in MGDG is not understood. Fatty acids are de novo synthesized in the stroma, then converted into very-long-chain polyunsaturated fatty acids (FAs) at the endoplasmic reticulum (ER). The production of MGDG relies therefore on an EPA supply from the ER to the plastid, following an unknown process. We identified seven elongases and five desaturases possibly involved in EPA production in Nannochloropsis gaditana. Among the six heterokont-specific saturated FA elongases possibly acting upstream in this pathway, we characterized the highly expressed isoform Δ0-ELO1. Heterologous expression in yeast (Saccharomyces cerevisiae) showed that NgΔ0-ELO1 could elongate palmitic acid. Nannochloropsis Δ0-elo1 mutants exhibited a reduced EPA level and a specific decrease in MGDG. In NgΔ0-elo1 lines, the impairment of photosynthesis is consistent with a role of EPA-rich MGDG in nonphotochemical quenching control, possibly providing an appropriate MGDG platform for the xanthophyll cycle. Concomitantly with MGDG decrease, the level of triacylglycerol (TAG) containing medium chain FAs increased. In Nannochloropsis, part of EPA used for MGDG production is therefore biosynthesized by a channeled process initiated at the elongation step of palmitic acid by Δ0-ELO1, thus acting as a committing enzyme for galactolipid production. Based on the MGDG/TAG balance controlled by Δ0-ELO1, this study also provides novel prospects for the engineering of oleaginous microalgae for biotechnological applications.

An Identifiable State Model To Describe Light Intensity Influence on Microalgae Growth

Despite the high potential as feedstock for the production of fuels and chemicals, the industrial cultivation of microalgae still exhibits many issues. Yield in microalgae cultivation systems is limited by the solar energy that can be harvested. The availability of reliable models representing key phenomena affecting algae growth may help designing and optimizing effective production systems at an industrial level. In this work the complex influence of different light regimes on seawater alga Nannochloropsis salina growth is represented by first principles models. Experimental data such as in vivo fluorescence measurements are employed to develop the model. The proposed model allows description of all growth curves and fluorescence data in a reliable way. The model structure is assessed and modified in order to guarantee the model identifiability and the estimation of its parametric set in a robust and reliable way.

Transcriptome and Cell Physiological Analyses in Different Rice Cultivars Provide New Insights Into Adaptive and Salinity Stress Responses

Salinity tolerance has been extensively investigated in recent years due to its agricultural importance. Several features, such as the regulation of ionic transporters and metabolic adjustments, have been identified as salt tolerance hallmarks. Nevertheless, due to the complexity of the trait, the results achieved to date have met with limited success in improving the salt tolerance of rice plants when tested in the field, thus suggesting that a better understanding of the tolerance mechanisms is still required. In this work, differences between two varieties of rice with contrasting salt sensitivities were revealed by the imaging of photosynthetic parameters, ion content analysis and a transcriptomic approach. The transcriptomic analysis conducted on tolerant plants supported the setting up of an adaptive program consisting of sodium distribution preferentially limited to the roots and older leaves, and in the activation of regulatory mechanisms of photosynthesis in the new leaves. As a result, plants resumed grow even under prolonged saline stress. In contrast, in the sensitive variety, RNA-seq analysis revealed a misleading response, ending in senescence and cell death. The physiological response at the cellular level was investigated by measuring the intracellular profile of H2O2 in the roots, using a fluorescent probe. In the roots of tolerant plants, a quick response was observed with an increase in H2O2 production within 5 min after salt treatment. The expression analysis of some of the genes involved in perception, signal transduction and salt stress response confirmed their early induction in the roots of tolerant plants compared to sensitive ones. By inhibiting the synthesis of apoplastic H2O2, a reduction in the expression of these genes was detected. Our results indicate that quick H2O2 signaling in the roots is part of a coordinated response that leads to adaptation instead of senescence in salt-treated rice plants.

Protein and lipid dynamics in photosynthetic thylakoid membranes investigated by in-situ solid-state NMR.

Photosynthetic thylakoid membranes contain the protein machinery to convert sunlight in chemical energy and regulate this process in changing environmental conditions via interplay between lipid, protein and xanthophyll molecular constituents. This work addresses the molecular effects of zeaxanthin accumulation in thylakoids, which occurs in native systems under high light conditions through the conversion of the xanthophyll violaxanthin into zeaxanthin via the so called xanthophyll cycle. We applied biosynthetic isotope labeling and 13C solid-state NMR spectroscopy to simultaneously probe the conformational dynamics of protein, lipid and xanthophyll constituents of thylakoids isolated from wild type (cw15) and npq2 mutant of the green alga Chlamydomonas reinhardtii, that accumulates zeaxanthin constitutively. Results show differential dynamics of wild type and npq2 thylakoids. Ordered-phase lipids have reduced mobility and mobile-phase lipids have enlarged dynamics in npq2 membranes, together spanning a broader dynamical range. The fraction of ordered lipids is much larger than the fraction of mobile lipids, which explains why zeaxanthin appears to cause overall reduction of thylakoid membrane fluidity. In addition to the ordered lipids, also the xanthophylls and a subset of protein sites in npq2 thylakoids have reduced conformational dynamics. Our work demonstrates the applicability of solid-state NMR spectroscopy for obtaining a microscopic picture of different membrane constituents simultaneously, inside native, heterogeneous membranes.

A Mathematical model to guide Genetic Engineering of Photosynthetic Metabolism

The optimization of algae biomass productivity in industrial cultivation systems requires genetic improvement of wild type strains isolated from nature. One of the main factors affecting algae productivity is their efficiency in converting light into chemical energy and this has been a major target of recent genetic efforts. However, photosynthetic productivity in algae cultures depends on many environmental parameters, making the identification of advantageous genotypes complex and the achievement of concrete improvements slow. In this work, we developed a mathematical model to describe the key factors influencing algae photosynthetic productivity in a photobioreactor, using experimental measurements for the WT strain of Nannochloropsis gaditana. The model was then exploited to predict the effect of potential genetic modifications on algae performances in an industrial context, showing the ability to predict the productivity of mutants with specific photosynthetic phenotypes. These results show that a quantitative model can be exploited to identify the genetic modifications with the highest impact on productivity taking into full account the complex influence of environmental conditions, efficiently guiding engineering efforts.