Advances in algal lipid metabolism and their use to improve oil content.

Microalgae are eukaryotic photosynthetic micro-organisms that convert CO2 into carbohydrates, lipids, and other valuable metabolites. They are considered promising chassis for the production of various bioproducts, including fatty acid-derived biofuels. However, algae-based biofuels are not yet commercially available, mainly because of their low yields and high production cost. Optimizing strains to improve lipid productivity using the principles of synthetic biology should help move forward. This necessitates developments in the following areas: (1) identification of molecular bricks (enzymes, transcription factors, regulatory proteins etc.); (2) development of genetic tools; and (3) availability of high-throughput phenotyping methods. Here, we highlight the most recent developments in some of these areas and provide examples of the use of genome editing tools to improve oil content.

Fatty acid export (FAX) proteins contribute to oil production in the green microalga Chlamydomonas reinhardtii.

In algae and land plants, transport of fatty acids (FAs) from their site of synthesis in the plastid stroma to the endoplasmic reticulum (ER) for assembly into acyl lipids is crucial for cellular lipid homeostasis, including the biosynthesis of triacylglycerol (TAG) for energy storage. In the unicellular green alga Chlamydomonas reinhardtii, understanding and engineering of these processes is of particular interest for microalga-based biofuel and biomaterial production. Whereas in the model plant Arabidopsis thaliana, FAX (fatty acid export) proteins have been associated with a function in plastid FA-export and hence TAG synthesis in the ER, the knowledge on the function and subcellular localization of this protein family in Chlamydomonas is still scarce. Among the four FAX proteins encoded in the Chlamydomonas genome, we found Cr-FAX1 and Cr-FAX5 to be involved in TAG production by functioning in chloroplast and ER membranes, respectively. By in situ immunolocalization, we show that Cr-FAX1 inserts into the chloroplast envelope, while Cr-FAX5 is located in ER membranes. Severe reduction of Cr-FAX1 or Cr-FAX5 proteins by an artificial microRNA approach results in a strong decrease of the TAG content in the mutant strains. Further, overexpression of chloroplast Cr-FAX1, but not of ER-intrinsic Cr-FAX5, doubled the content of TAG in Chlamydomonas cells. We therefore propose that Cr-FAX1 in chloroplast envelopes and Cr-FAX5 in ER membranes represent a basic set of FAX proteins to ensure shuttling of FAs from chloroplasts to the ER and are crucial for oil production in Chlamydomonas.

Chlamydomonas cell cycle mutant crcdc5 over-accumulates starch and oil.

The use of algal biomass for biofuel production requires improvements in both biomass productivity and its energy density. Green microalgae store starch and oil as two major forms of carbon reserves. Current strategies to increase the amount of carbon reserves often compromise algal growth. To better understand the cellular mechanisms connecting cell division to carbon storage, we examined starch and oil accumulation in two Chlamydomonas mutants deficient in a gene encoding a homolog of the Arabidopsis Cell Division Cycle 5 (CDC5), a MYB DNA binding protein known to be involved in cell cycle in higher plants. The two crcdc5 mutants (crcdc5-1 and crcdc5-2) were found to accumulate significantly higher amount of starch and oil than their corresponding parental lines. Flow cytometry analysis on synchronized cultures cultivated in a diurnal light/dark cycle revealed an abnormal division of the two mutants, characterized by a prolonged S/M phase, therefore demonstrating its implication in cell cycle in Chlamydomonas. Taken together, these results suggest that the energy saved by a slowdown in cell division is used for the synthesis of reserve compounds. This work highlights the importance in understanding the interplay between cell cycle and starch/oil homeostasis, which should have a critical impact on improving lipid/starch productivity.

Subcellular Energetics and Carbon Storage in Chlamydomonas.

Microalgae have emerged as a promising platform for production of carbon- and energy- rich molecules, notably starch and oil. Establishing an economically viable algal biotechnology sector requires a holistic understanding of algal photosynthesis, physiology, cell cycle and metabolism. Starch/oil productivity is a combined effect of their cellular content and cell division activities. Cell growth, starch and fatty acid synthesis all require carbon building blocks and a source of energy in the form of ATP and NADPH, but with a different requirement in ATP/NADPH ratio. Thus, several cellular mechanisms have been developed by microalgae to balance ATP and NADPH supply which are essentially produced by photosynthesis. Major energy management mechanisms include ATP production by the chloroplast-based cyclic electron flow and NADPH removal by water-water cycles. Furthermore, energetic coupling between chloroplast and other cellular compartments, mitochondria and peroxisome, is increasingly recognized as an important process involved in the chloroplast redox poise. Emerging literature suggests that alterations of energy management pathways affect not only cell fitness and survival, but also influence biomass content and composition. These emerging discoveries are important steps towards diverting algal photosynthetic energy to useful products for biotechnological applications.

Molecular Genetic Tools and Emerging Synthetic Biology Strategies to Increase Cellular Oil Content in Chlamydomonas reinhardtii.

Microalgae constitute a highly diverse group of eukaryotic and photosynthetic microorganisms that have developed extremely efficient systems for harvesting and transforming solar energy into energy-rich molecules such as lipids. Although microalgae are considered to be one of the most promising platforms for the sustainable production of liquid oil, the oil content of these organisms is naturally low, and algal oil production is currently not economically viable. Chlamydomonas reinhardtii (Chlamydomonas) is an established algal model due to its fast growth, high transformation efficiency, and well-understood physiology and to the availability of detailed genome information and versatile molecular tools for this organism. In this review, we summarize recent advances in the development of genetic manipulation tools for Chlamydomonas, from gene delivery methods to state-of-the-art genome-editing technologies and fluorescent dye-based high-throughput mutant screening approaches. Furthermore, we discuss practical strategies and toolkits that enhance transgene expression, such as choice of expression vector and background strain. We then provide examples of how advanced genetic tools have been used to increase oil content in Chlamydomonas. Collectively, the current literature indicates that microalgal oil content can be increased by overexpressing key enzymes that catalyze lipid biosynthesis, blocking lipid degradation, silencing metabolic pathways that compete with lipid biosynthesis and modulating redox state. The tools and knowledge generated through metabolic engineering studies should pave the way for developing a synthetic biological approach to enhance lipid productivity in microalgae.

Seed-Specific Overexpression of the Pyruvate Transporter BASS2 Increases Oil Content in Arabidopsis Seeds.

Seed oil is important not only for human and animal nutrition, but also for various industrial applications. Numerous genetic engineering strategies have been attempted to increase the oil content per seed, but few of these strategies have involved manipulating the transporters. Pyruvate is a major source of carbon for de novo fatty acid biosynthesis in plastids, and the embryo's demand for pyruvate is reported to increase during active oil accumulation. In this study, we tested our hypothesis that oil biosynthesis could be boosted by increasing pyruvate flux into plastids. We expressed the known plastid-localized pyruvate transporter BILE ACID:SODIUM SYMPORTER FAMILY PROTEIN 2 (BASS2) under the control of a seed-specific soybean (Glycine max) glycinin-1 promoter in Arabidopsis thaliana. The resultant transgenic Arabidopsis plants (OEs), which expressed high levels of BASS2, produced seeds that were larger and heavier and contained 10-37% more oil than those of the wild type (WT), but were comparable to the WT seeds in terms of protein and carbohydrate contents. The total seed number did not differ significantly between the WT and OEs. Therefore, oil yield per plant was increased by 24-43% in the OE lines compared to WT. Taken together, our results demonstrate that seed-specific overexpression of the pyruvate transporter BASS2 promotes oil production in Arabidopsis seeds. Thus, manipulating the level of specific transporters is a feasible approach for increasing the seed oil content.

Saturating Light Induces Sustained Accumulation of Oil in Plastidal Lipid Droplets in Chlamydomonas reinhardtii.

Enriching algal biomass in energy density is an important goal in algal biotechnology. Nitrogen (N) starvation is considered the most potent trigger of oil accumulation in microalgae and has been thoroughly investigated. However, N starvation causes the slow down and eventually the arrest of biomass growth. In this study, we show that exposing a Chlamydomonas reinhardtii culture to saturating light (SL) under a nonlimiting CO2 concentration in turbidostatic photobioreactors induces a sustained accumulation of lipid droplets (LDs) without compromising growth, which results in much higher oil productivity than N starvation. We also show that the polar membrane lipid fraction of SL-induced LDs is rich in plastidial lipids (approximately 70%), in contrast to N starvation-induced LDs, which contain approximately 60% lipids of endoplasmic reticulum origin. Proteomic analysis of LDs isolated from SL-exposed cells identified more than 200 proteins, including known proteins of lipid metabolism, as well as 74 proteins uniquely present in SL-induced LDs. LDs induced by SL and N depletion thus differ in protein and lipid contents. Taken together, lipidomic and proteomic data thus show that a large part of the sustained oil accumulation occurring under SL is likely due to the formation of plastidial LDs. We discuss our data in relation to the different metabolic routes used by microalgae to accumulate oil reserves depending on cultivation conditions. Finally, we propose a model in which oil accumulation is governed by an imbalance between photosynthesis and growth, which can be achieved by impairing growth or by boosting photosynthetic carbon fixation, with the latter resulting in higher oil productivity.

Identification of a Chlamydomonas plastidial 2-lysophosphatidic acid acyltransferase and its use to engineer microalgae with increased oil content.

Despite a strong interest in microalgal oil production, our understanding of the biosynthetic pathways that produce algal lipids and the genes involved in the biosynthetic processes remains incomplete. Here, we report that Chlamydomonas reinhardtii Cre09.g398289 encodes a plastid-targeted 2-lysophosphatidic acid acyltransferase (CrLPAAT1) that acylates the sn-2 position of a 2-lysophosphatidic acid to form phosphatidic acid, the first common precursor of membrane and storage lipids. In vitro enzyme assays showed that CrLPAAT1 prefers 16:0-CoA to 18:1-CoA as an acyl donor. Fluorescent protein-tagged CrLPAAT1 was localized to the plastid membrane in C. reinhardtii cells. Furthermore, expression of CrLPAAT1 in plastids led to a > 20% increase in oil content under nitrogen-deficient conditions. Taken together, these results demonstrate that CrLPAAT1 is an authentic plastid-targeted LPAAT in C. reinhardtii, and that it may be used as a molecular tool to genetically increase oil content in microalgae.

Hyper-accumulation of starch and oil in a Chlamydomonas mutant affected in a plant-specific DYRK kinase.

BACKGROUND: Because of their high biomass productivity and their ability to accumulate high levels of energy-rich reserve compounds such as oils or starch, microalgae represent a promising feedstock for the production of biofuel. Accumulation of reserve compounds takes place when microalgae face adverse situations such as nutrient shortage, conditions which also provoke a stop in cell division, and down-regulation of photosynthesis. Despite growing interest in microalgal biofuels, little is known about molecular mechanisms controlling carbon reserve formation. In order to discover new regulatory mechanisms, and identify genes of interest to boost the potential of microalgae for biofuel production, we developed a forward genetic approach in the model microalga Chlamydomonas reinhardtii. RESULTS: By screening an insertional mutant library on the ability of mutants to accumulate and re-mobilize reserve compounds, we isolated a Chlamydomonas mutant (starch degradation 1, std1) deficient for a dual-specificity tyrosine-phosphorylation-regulated kinase (DYRK). The std1 mutant accumulates higher levels of starch and oil than wild-type and maintains a higher photosynthetic activity under nitrogen starvation. Phylogenetic analysis revealed that this kinase (named DYRKP) belongs to a plant-specific subgroup of the evolutionarily conserved DYRK kinase family. Furthermore, hyper-accumulation of storage compounds occurs in std1 mostly under low light in photoautotrophic condition, suggesting that the kinase normally acts under conditions of low energy status to limit reserve accumulation. CONCLUSIONS: The DYRKP kinase is proposed to act as a negative regulator of the sink capacity of photosynthetic cells that integrates nutrient and energy signals. Inactivation of the kinase strongly boosts accumulation of reserve compounds under photoautotrophic nitrogen deprivation and allows maintaining high photosynthetic activity. The DYRKP kinase therefore represents an attractive target for improving the energy density of microalgae or crop plants.

Development of a forward genetic screen to isolate oil mutants in the green microalga Chlamydomonas reinhardtii.

BACKGROUND: Oils produced by microalgae are precursors to biodiesel. To achieve a profitable production of biodiesel from microalgae, identification of factors governing oil synthesis and turnover is desirable. The green microalga Chlamydomonas reinhardtii is amenable to genetic analyses and has recently emerged as a model to study oil metabolism. However, a detailed method to isolate various types of oil mutants that is adapted to Chlamydomonas has not been reported. RESULTS: We describe here a forward genetic approach to isolate mutants altered in oil synthesis and turnover from C. reinhardtii. It consists of a three-step screening procedure: a primary screen by flow cytometry of Nile red stained transformants grown in 96-deep-well plates under three sequential conditions (presence of nitrogen, then absence of nitrogen, followed by oil remobilization); a confirmation step using Nile red stained biological triplicates; and a validation step consisting of the quantification by thin layer chromatography of oil content of selected strains. Thirty-one mutants were isolated by screening 1,800 transformants generated by random insertional mutagenesis (1.7%). Five showed increased oil accumulation under the nitrogen-replete condition and 13 had altered oil content under nitrogen-depletion. All mutants were affected in oil remobilization. CONCLUSION: This study demonstrates that various types of oil mutants can be isolated in Chlamydomonas based on the method set-up here, including mutants accumulating oil under optimal biomass growth. The strategy conceived and the protocol set-up should be applicable to other microalgal species such as Nannochloropsis and Chlorella, thus serving as a useful tool in Chlamydomonas oil research and algal biotechnology.

Proteomic profiling of oil bodies isolated from the unicellular green microalga Chlamydomonas reinhardtii: with focus on proteins involved in lipid metabolism.

Oil bodies are sites of energy and carbon storage in many organisms including microalgae. As a step toward deciphering oil accumulation mechanisms in algae, we used proteomics to analyze purified oil bodies from the model microalga Chlamydomonas reinhardtii grown under nitrogen deprivation. Among the 248 proteins (≥ 2 peptides) identified by LC-MS/MS, 33 were putatively involved in the metabolism of lipids (mostly acyl-lipids and sterols). Compared with a recently reported Chlamydomonas oil body proteome, 19 new proteins of lipid metabolism were identified, spanning the key steps of the triacylglycerol synthesis pathway and including a glycerol-3-phosphate acyltransferase (GPAT), a lysophosphatidic acid acyltransferase (LPAT) and a putative phospholipid:diacylglycerol acyltransferase (PDAT). In addition, proteins putatively involved in deacylation/reacylation, sterol synthesis, lipid signaling and lipid trafficking were found to be associated with the oil body fraction. This data set thus provides evidence that Chlamydomonas oil bodies are not only storage compartments but also are dynamic structures likely to be involved in processes such as oil synthesis, degradation and lipid homeostasis. The proteins identified here should provide useful targets for genetic studies aiming at increasing our understanding of triacyglycerol synthesis and the role of oil bodies in microalgal cell functions.

Oil accumulation in the model green alga Chlamydomonas reinhardtii: characterization, variability between common laboratory strains and relationship with starch reserves.

BACKGROUND: When cultivated under stress conditions, many microalgae species accumulate both starch and oil (triacylglycerols). The model green microalga Chlamydomonas reinhardtii has recently emerged as a model to test genetic engineering or cultivation strategies aiming at increasing lipid yields for biodiesel production. Blocking starch synthesis has been suggested as a way to boost oil accumulation. Here, we characterize the triacylglycerol (TAG) accumulation process in Chlamydomonas and quantify TAGs in various wild-type and starchless strains. RESULTS: In response to nitrogen deficiency, Chlamydomonas reinhardtii produced TAGs enriched in palmitic, oleic and linoleic acids that accumulated in oil-bodies. Oil synthesis was maximal between 2 and 3 days following nitrogen depletion and reached a plateau around day 5. In the first 48 hours of oil deposition, a ~80% reduction in the major plastidial membrane lipids occurred. Upon nitrogen re-supply, mobilization of TAGs started after starch degradation but was completed within 24 hours. Comparison of oil content in five common laboratory strains (CC124, CC125, cw15, CC1690 and 11-32A) revealed a high variability, from 2 µg TAG per million cell in CC124 to 11 µg in 11-32A. Quantification of TAGs on a cell basis in three mutants affected in starch synthesis (cw15sta1-2, cw15sta6 and cw15sta7-1) showed that blocking starch synthesis did not result in TAG over-accumulation compared to their direct progenitor, the arginine auxotroph strain 330. Moreover, no significant correlation was found between cellular oil and starch levels among the twenty wild-type, mutants and complemented strains tested. By contrast, cellular oil content was found to increase steeply with salt concentration in the growth medium. At 100 mM NaCl, oil level similar to nitrogen depletion conditions could be reached in CC124 strain. CONCLUSION: A reference basis for future genetic studies of oil metabolism in Chlamydomonas is provided. Results highlight the importance of using direct progenitors as control strains when assessing the effect of mutations on oil content. They also suggest the existence in Chlamydomonas of complex interplays between oil synthesis, genetic background and stress conditions. Optimization of such interactions is an alternative to targeted metabolic engineering strategies in the search for high oil yields.

Cloning and molecular characterization of a glycerol-3-phosphate O-acyltransferase (GPAT) gene from Echium (Boraginaceae) involved in the biosynthesis of cutin polyesters.

The glycerol-based lipid polyester called cutin is a main component of cuticle, the protective interface of aerial plant organs also controlling compound exchange with the environment. Though recent progress towards understanding of cutin biosynthesis has been made in Arabidopsis thaliana, little is known in other plants. One key step in this process is the acyl transfer reaction to the glycerol backbone. Here we report the cloning and molecular characterization of EpGPAT1, a gene encoding a glycerol-3-phosphate O-acyltransferase (GPAT) from Echium pitardii (Boraginaceae) with high similarity to the AtGPAT4/AtGPAT8 of Arabidopsis. Quantitative analysis by qRT-PCR showed highest expression of EpGPAT1 in seeds, roots, young leaves and flowers. Acyltransferase activity of EpGPAT1 was evidenced by heterologous expression in yeast. Ectopic expression in leaves of tobacco plants lead to an increase of C16 and C18 hydroxyacids and alpha,omega-diacids in the cell wall fraction, indicating a role in the biosynthesis of polyesters. Analysis of the genomic organization in Echium revealed the presence of EpGPAT2, a closely related gene which was found to be mostly expressed in developing leaves and flowers. The presence of a conserved HAD-like domain at the N-terminal moiety of GPATs from Echium, Arabidopsis and other plant species suggests a possible phosphohydrolase activity in addition to the reported acyltransferase activity. Evolutive implications of this finding are discussed.