The Phytophthora parasitica effector AVH195 interacts with ATG8, attenuates host autophagy, and promotes biotrophic infection.

BACKGROUND: Plant pathogens secrete effector proteins into host cells to suppress immune responses and manipulate fundamental cellular processes. One of these processes is autophagy, an essential recycling mechanism in eukaryotic cells that coordinates the turnover of cellular components and contributes to the decision on cell death or survival. RESULTS: We report the characterization of AVH195, an effector from the broad-spectrum oomycete plant pathogen, Phytophthora parasitica. We show that P. parasitica expresses AVH195 during the biotrophic phase of plant infection, i.e., the initial phase in which host cells are maintained alive. In tobacco, the effector prevents the initiation of cell death, which is caused by two pathogen-derived effectors and the proapoptotic BAX protein. AVH195 associates with the plant vacuolar membrane system and interacts with Autophagy-related protein 8 (ATG8) isoforms/paralogs. When expressed in cells from the green alga, Chlamydomonas reinhardtii, the effector delays vacuolar fusion and cargo turnover upon stimulation of autophagy, but does not affect algal viability. In Arabidopsis thaliana, AVH195 delays the turnover of ATG8 from endomembranes and promotes plant susceptibility to P. parasitica and the obligate biotrophic oomycete pathogen Hyaloperonospora arabidopsidis. CONCLUSIONS: Taken together, our observations suggest that AVH195 targets ATG8 to attenuate autophagy and prevent associated host cell death, thereby favoring biotrophy during the early stages of the infection process.

Engineering Co-N-Cr Cross-Interfacial Electron Bridges to Break Activity-Stability Trade-Off for Superdurable Bifunctional Single Atom Oxygen Electrocatalysts.

Atomically dispersed metal-nitrogen-carbon (M-N-C) catalysts have exhibited encouraging oxygen reduction reaction (ORR) activity. Nevertheless, the insufficient long-term stability remains a widespread concern owing to the inevitable 2-electron byproducts, H2O2. Here, we construct Co-N-Cr cross-interfacial electron bridges (CIEBs) via the interfacial electronic coupling between Cr2O3 and Co-N-C, breaking the activity-stability trade-off. The partially occupied Cr 3d-orbitals of Co-N-Cr CIEBs induce the electron rearrangement of CoN4 sites, lowering the Co-OOH* antibonding orbital occupancy and accelerating the adsorption of intermediates. Consequently, the Co-N-Cr CIEBs suppress the two-electron ORR process and approach the apex of Sabatier volcano plot for four-electron pathway simultaneously. As a proof-of-concept, the Co-N-Cr CIEBs is synthesized by the molten salt template method, exhibiting dominant 4-electron selectively and extremely low H2O2 yield confirmed by Damjanovic kinetic analysis. The Co-N-Cr CIEBs demonstrates impressive bifunctional oxygen catalytic activity (▵E=0.70 V) and breakthrough durability including 100 % current retention after 10 h continuous operation and cycling performance over 1500 h for Zn-air battery. The hybrid interfacial configuration and the understanding of the electronic coupling mechanism reported here could shed new light on the design of superdurable M-N-C catalysts.

Enhanced triacyclglycerols and starch synthesis in Chlamydomonas stimulated by the engineered biodegradable nanoparticles.

Microalgae are promising feedstock for renewable fuels. The accumulation of oils in microalgae can be enhanced by nanoparticle exposure. However, the nanoparticles employed in previous studies are mostly non-biodegradable, which hinders nanoparticles developing as promising approach for biofuel production. We recently reported the engineered resin nanoparticles (iBCA-NPs), which were found to be biodegradable in this study. When the cells of green microalga Chlamydomonas reinhardtii were exposed to the iBCA-NPs for 1 h, the cellular triacyclglycerols (TAG) and starch contents increased by 520% and 60% than that in the control. The TAG production improved by 1.8-fold compared to the control without compromised starch production. Additionally, the content of total fatty acids increased by 1.3-fold than that in control. Furthermore, we found that the iBCA-NPs addition resulted in increased cellular reactive oxygen species (ROS) content and upregulated the activities of antioxidant enzymes. The relative expressions of the key genes involved in TAG and starch biosynthesis were also upregulated. Overall, our results showed that short exposure of the iBCA-NPs dramatically enhances TAG and starch accumulation in Chlamydomonas, which probably resulted from prompt upregulated expression of the key genes in lipid and starch metabolic pathways that were triggered by over-accumulated ROS. This study reported a useful approach to enhance energy-rich reserve accumulation in microalgae. KEY POINTS: 1. The first attempt to increase oil and starch in microalgae by biodegradable NPs. 2. The biodegradability of iBCA-NPs by the BOD test was more than 50% after 28 days. 3. The iBCA-NPs induce more energy reserves than that of previously reported NPs.

The phosphatidylethanolamine-binding protein DTH1 mediates degradation of lipid droplets in Chlamydomonas reinhardtii.

Lipid droplets (LDs) are intracellular organelles found in a wide range of organisms and play important roles in stress tolerance. During nitrogen (N) starvation, Chlamydomonas reinhardtii stores large amounts of triacylglycerols (TAGs) inside LDs. When N is resupplied, the LDs disappear and the TAGs are degraded, presumably providing carbon and energy for regrowth. The mechanism by which cells degrade LDs is poorly understood. Here, we isolated a mutant (dth1-1, Delayed in TAG Hydrolysis 1) in which TAG degradation during recovery from N starvation was compromised. Consequently, the dth1-1 mutant grew poorly compared to its parental line during N recovery. Two additional independent loss-of-function mutants (dth1-2 and dth1-3) also exhibited delayed TAG remobilization. DTH1 transcript levels increased sevenfold upon N resupply, and DTH1 protein was localized to LDs. DTH1 contains a putative lipid-binding domain (DTH1LBD) with alpha helices predicted to be structurally similar to those in apolipoproteins E and A-I. Recombinant DTH1LBD bound specifically to phosphatidylethanolamine (PE), a major phospholipid coating the LD surface. Overexpression of DTH1LBD in Chlamydomonas phenocopied the dth1 mutant's defective TAG degradation, suggesting that the function of DTH1 depends on its ability to bind PE. Together, our results demonstrate that the lipid-binding DTH1 plays an essential role in LD degradation and provide insight into the molecular mechanism of protein anchorage to LDs at the LD surface in photosynthetic cells.

CoW Bimetallic Carbide Nanocatalysts: Computational Exploration, Confined Disassembly-Assembly Synthesis and Alkaline/Seawater Hydrogen Evolution.

Earth-abundant tungsten carbide exhibits potential hydrogen evolution reaction (HER) catalytic activity owing to its Pt-like d-band electronic structure, which, unfortunately, suffers from the relatively strong tungsten-hydrogen binding, deteriorating its HER performance. Herein, a catalyst design concept of incorporating late transition metal into early transition metal carbide is proposed for regulating the metal-H bonding strength and largely enhancing the HER performance, which is employed to synthesize CoW bi-metallic carbide Co6 W6 C by a "disassembly-assembly" approach in a confined environment. Such synthesized Co6 W6 C nanocatalyst features the optimal Gibbs free energy of *H intermediate and dissociation barrier energy of H2 O molecules as well by taking advantage of the electron complementary effect between Co and W species, which endows the electrocatalyst with excellent HER performance in both alkaline and seawater/alkaline electrolytes featuring especially low overpotentials, elevated current densities, and much-enhanced operation durability in comparison to commercial Pt/C catalyst. Moreover, a proof-of-concept Mg/seawater battery equipped with Co6 W6 C-2-600 as cathode offers a peak power density of 9.1 mW cm-2 and an open-circuit voltage of ≈1.71 V, concurrently realizing hydrogen production and electricity output.

The Chlamydomonas transcription factor MYB1 mediates lipid accumulation under nitrogen depletion.

Microalgae accumulate high levels of oil under stress, but the underlying biosynthetic pathways are not fully understood. We sought to identify key regulators of lipid metabolism under stress conditions. We found that the Chlamydomonas reinhardtii gene encoding the MYB-type transcription factor MYB1 is highly induced under stress conditions. Two myb1 mutants accumulated less total fatty acids and storage lipids than their parental strain upon nitrogen (N) depletion. Transcriptome analysis revealed that genes involved in lipid metabolism are highly enriched in the wild-type but not in the myb1-1 mutant after 4 h of N depletion. Among these genes were several involved in the transport of fatty acids from the chloroplast to the endoplasmic reticulum (ER): acyl-ACP thioesterase (FAT1), Fatty Acid EXporters (FAX1, FAX2), and long-chain acyl-CoA synthetase1 (LACS1). Furthermore, overexpression of FAT1 in the chloroplast increased lipid production. These results suggest that, upon N depletion, MYB1 promotes lipid accumulation by facilitating fatty acid transport from the chloroplast to the ER. This study identifies MYB1 as an important positive regulator of lipid accumulation in C. reinhardtii upon N depletion, adding another player to the established regulators of this process, including NITROGEN RESPONSE REGULATOR 1 (NRR1) and TRIACYLGLYCEROL ACCUMULATION REGULATOR 1 (TAR1).

The bZIP1 Transcription Factor Regulates Lipid Remodeling and Contributes to ER Stress Management in Chlamydomonas reinhardtii.

Endoplasmic reticulum (ER) stress is caused by the stress-induced accumulation of unfolded proteins in the ER. Here, we identified proteins and lipids that function downstream of the ER stress sensor INOSITOL-REQUIRING ENZYME1 (CrIRE1) that contributes to ER stress tolerance in Chlamydomonas (Chlamydomonas reinhardtii). Treatment with the ER stress inducer tunicamycin resulted in the splicing of a 32-nucleotide fragment of a basic leucine zipper 1 (bZIP1) transcription factor (CrbZIP1) mRNA by CrIRE1 that, in turn, resulted in the loss of the transmembrane domain in CrbZIP1, and the translocation of CrbZIP1 from the ER to the nucleus. Mutants deficient in CrbZIP1 failed to induce the expression of the unfolded protein response genes and grew poorly under ER stress. Levels of diacylglyceryltrimethylhomoserine (DGTS) and pinolenic acid (18:3Δ5,9,12) increased in the parental strains but decreased in the crbzip1 mutants under ER stress. A yeast one-hybrid assay revealed that CrbZIP1 activated the expression of enzymes catalyzing the biosynthesis of DGTS and pinolenic acid. Moreover, two lines harboring independent mutant alleles of Chlamydomonas desaturase (CrDES) failed to synthesize pinolenic acid and were more sensitive to ER stress than were their parental lines. Together, these results indicate that CrbZIP1 is a critical component of the ER stress response mediated by CrIRE1 in Chlamydomonas that acts via lipid remodeling.

N-Doped Carbon Electrocatalyst: Marked ORR Activity in Acidic Media without the Contribution from Metal Sites?

Fe-N-C electrocatalysts have been demonstrated to be the most promising substitutes for benchmark Pt/C catalysts for the oxygen reduction reaction (ORR). Herein, we report that N-doped carbon materials with trace amounts of iron (0-0.08 wt. %) show excellent ORR activity and durability comparable and even superior to those of Pt/C in both alkaline and acidic media without significant contribution by the metal sites. Such an N-doped carbon (denoted as N-HPCs) features a hollow and hierarchically porous architecture, and more importantly, a noncovalently bonded N-deficient/N-rich heterostructure providing the active sites for oxygen adsorption and activation owing to the efficient electron transfer between the layers. The primary Zn-air battery using N-HPCs as the cathode delivers a much higher power density of 158 mW cm-2 , and the maximum power density in the H2 -O2 fuel cell reaches 486 mW cm-2 , which is comparable to and even better than those using conventional Fe-N-C catalysts at cathodes.

Fe2+ /Fe3+ Cycling for Coupling Self-Powered Hydrogen Evolution and Preparation of Electrode Catalysts.

A novel Zn-Fe flow battery featuring an Fe3+ reduction reaction (Fe3+ RR)-coupled zinc oxidation, and an Fe2+ oxidation reaction (Fe2+ OR)-coupled hydrogen evolution reaction (HER) system as well, was established. This battery is capable of driving two Fe2+ OR-coupled HER systems in series based on the above Fe2+ /Fe3+ cycling, for efficient self-powered hydrogen evolution. Meanwhile, this Fe2+ /Fe3+ cycling enables the preparation of a multifunctional catalyst, Pt-3@SXNS (siloxene nanosheet), by the Fe2+ OR-promoted dispersion of Pt nanoparticles on SXNS; alternatively, this support could be obtained by Fe3+ RR-assisted exfoliation using Fe3+ from the anolyte of Fe2+ OR-coupled HER. The Pt-3@SXNS catalyst exhibits excellent catalytic activities toward Fe3+ RR in the Zn-Fe flow battery, HER, and Fe2+ OR in the electrolyzer, which is attributed to the strong electronic interaction between Pt and Si. This work offers a new strategy for energy storage and low-cost hydrogen production from acidic wastewater.

The disassembly of lipid droplets in Chlamydomonas.

Lipid droplets (LDs) are ubiquitous and specialized organelles in eukaryotic cells. Consisting of a triacylglycerol core surrounded by a monolayer of membrane lipids, LDs are decorated with proteins and have myriad functions, from carbon/energy storage to membrane lipid remodeling and signal transduction. The biogenesis and turnover of LDs are therefore tightly coordinated with cellular metabolic needs in a fluctuating environment. Lipid droplet turnover requires remodeling of the protein coat, lipolysis, autophagy and fatty acid ß-oxidation. Several key components of these processes have been identified in Chlamydomonas (Chlamydomonas reinhardtii), including the major lipid droplet protein, a CXC-domain containing regulatory protein, the phosphatidylethanolamine-binding DTH1 (DELAYED IN TAG HYDROLYSIS1), two lipases and two enzymes involved in fatty acid ß-oxidation. Here, we review LD turnover and discuss its physiological significance in Chlamydomonas, a major model green microalga in research on algal oil.

Interorganelle Communication: Peroxisomal MALATE DEHYDROGENASE2 Connects Lipid Catabolism to Photosynthesis through Redox Coupling in Chlamydomonas.

Plants and algae must tightly coordinate photosynthetic electron transport and metabolic activities given that they often face fluctuating light and nutrient conditions. The exchange of metabolites and signaling molecules between organelles is thought to be central to this regulation but evidence for this is still fragmentary. Here, we show that knocking out the peroxisome-located MALATE DEHYDROGENASE2 (MDH2) of Chlamydomonas reinhardtii results in dramatic alterations not only in peroxisomal fatty acid breakdown but also in chloroplast starch metabolism and photosynthesis. mdh2 mutants accumulated 50% more storage lipid and 2-fold more starch than the wild type during nitrogen deprivation. In parallel, mdh2 showed increased photosystem II yield and photosynthetic CO2 fixation. Metabolite analyses revealed a >60% reduction in malate, together with increased levels of NADPH and H2O2 in mdh2 Similar phenotypes were found upon high light exposure. Furthermore, based on the lack of starch accumulation in a knockout mutant of the H2O2-producing peroxisomal ACYL-COA OXIDASE2 and on the effects of H2O2 supplementation, we propose that peroxisome-derived H2O2 acts as a regulator of chloroplast metabolism. We conclude that peroxisomal MDH2 helps photoautotrophs cope with nitrogen scarcity and high light by transmitting the redox state of the peroxisome to the chloroplast by means of malate shuttle- and H2O2-based redox signaling.

Glowing plants can light up the night sky? A review.

Luminescence, a physical phenomenon that producing cool light in vivo, has been found in bacteria, fungi, and animals but not yet in terrestrial higher plants. Through genetic engineering, it is feasible to introduce luminescence systems into living plant cells as biomarkers. Recently, some plants transformed with luminescent systems can glimmer in darkness, which can be observed by our naked eyes and provides a novel lighting resource. In this review, we summarized the bioassay development of luminescence in plant cells, followed by exampling the successful cases of glowing plants transformed with diverse luminescent systems. The potential key factors to design or optimize a glowing plant were also discussed. Our review is useful for the creation of the optimized glowing plants, which can be used not only in scientific research, but also as promising substitutes of artificial light sources in the future.

Galactolipid DGDG and Betaine Lipid DGTS Direct De Novo Synthesized Linolenate into Triacylglycerol in a Stress-Induced Starchless Mutant of Chlamydomonas reinhardtii.

The increasing demand for triacylglycerol (TAG) enriching polyunsaturated fatty acids (PUFAs) has led to a surge of interest in microalgal TAG metabolism. Polar membrane lipids serve as the desaturation carrier for PUFA, and the functional group of PUFA can be incorporated into TAG. Monogalactoglycerolipid has been found to provide the de novo synthesized oleate acyl group or the nascent polyunsaturated diacylglycerol backbone for TAG biosynthesis in the model green alga, Chlamydomonas reinhardtii. However, whether other membrane lipids take part in the formation of PUFA-attached TAG has not been clearly discovered. A time course study of glycerolipidomics in the starchless mutant of C. reinhardtii, BAFJ5, which hyper-accumulates TAG, revealed that digalactosyldiacylglycerol (DGDG) and diacylglycerol-N,N,N-trimethylhomoserine (DGTS) turned into the main components of membrane lipids, accounting for 62% of the total polar lipids, under nitrogen deprivation combined with high light conditions. In addition, the membrane lipid molecules DGDG 18:3n3/16:0 and DGTS 16:0/18:3n6 were presumed to be involved in the consecutive integration of the de novo synthesized linolenates into TAG. Based on the stoichiometry calculation, DGDG and DGTS were demonstrated to provide a major contribution to the accumulation of linolenate-attached TAG. Our study gives insights into the potential PUFA-attached TAG formation pathway mediated by the turnover of de novo synthesized DGDG and DGTS in the starchless mutant of Chlamydomonas.

Branched-Chain Amino Acid Catabolism Impacts Triacylglycerol Homeostasis in Chlamydomonas reinhardtii.

Nitrogen (N) starvation-induced triacylglycerol (TAG) synthesis, and its complex relationship with starch metabolism in algal cells, has been intensively studied; however, few studies have examined the interaction between amino acid metabolism and TAG biosynthesis. Here, via a forward genetic screen for TAG homeostasis, we isolated a Chlamydomonas (Chlamydomonas reinhardtii) mutant (bkdE1α) that is deficient in the E1α subunit of the branched-chain ketoacid dehydrogenase (BCKDH) complex. Metabolomics analysis revealed a defect in the catabolism of branched-chain amino acids in bkdE1α Furthermore, this mutant accumulated 30% less TAG than the parental strain during N starvation and was compromised in TAG remobilization upon N resupply. Intriguingly, the rate of mitochondrial respiration was 20% to 35% lower in bkdE1α compared with the parental strains. Three additional knockout mutants of the other components of the BCKDH complex exhibited phenotypes similar to that of bkdE1α Transcriptional responses of BCKDH to different N status were consistent with its role in TAG homeostasis. Collectively, these results indicate that branched-chain amino acid catabolism contributes to TAG metabolism by providing carbon precursors and ATP, thus highlighting the complex interplay between distinct subcellular metabolisms for oil storage in green microalgae.

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.

Progress on the development of floating photobioreactor for microalgae cultivation and its application potential.

Microalgae present great potential to replace land crops for the efficient production of large volumes of biomass for food, feed, fuels, and chemicals, as well as to treat wastewater and capture carbon. However, the commercialization of these technologies for bulk commodities requires a great reduction in the current microalgal biomass production cost. The bioreactor is the core of bioprocess engineering and is the premise for the commercial application of certain types of biotechnology. The challenges of phototrophic cultivation are completely different from those of heterotrophic processes because the efficiency of phototrophic cultivation is limited by the energy density of the input sunlight and the inorganic carbon supply. Thus, the development of microalgae cultivation technologies with low manufacturing and operating costs is key to addressing this problem, and floating photobioreactors (PBRs) are a promising solution. PBRs are deployed on the water surface without any land requirements, and wave energy provides free mixing energy. Additionally, the surrounding water can be used to control the culture temperature and to supply nutrients for microalgae growth. In this mini-review, the development of floating PBRs and their recent progress are presented. The effect of the carbon supply approach on the mixing and scaling-up of floating PBRs are critically discussed. The limitations and challenges in commercial applications of floating PBRs are analysed, and the need for future research is proposed. Finally, it is noted that microalgae farming on the ocean is a promising solution for human society to address the challenge of land space exhaustion due to the global population boom.

Chlamydomonas carries out fatty acid ß-oxidation in ancestral peroxisomes using a bona fide acyl-CoA oxidase.

Peroxisomes are thought to have played a key role in the evolution of metabolic networks of photosynthetic organisms by connecting oxidative and biosynthetic routes operating in different compartments. While the various oxidative pathways operating in the peroxisomes of higher plants are fairly well characterized, the reactions present in the primitive peroxisomes (microbodies) of algae are poorly understood. Screening of a Chlamydomonas insertional mutant library identified a strain strongly impaired in oil remobilization and defective in Cre05.g232002 (CrACX2), a gene encoding a member of the acyl-CoA oxidase/dehydrogenase superfamily. The purified recombinant CrACX2 expressed in Escherichia coli catalyzed the oxidation of fatty acyl-CoAs into trans-2-enoyl-CoA and produced H2 O2 . This result demonstrated that CrACX2 is a genuine acyl-CoA oxidase, which is responsible for the first step of the peroxisomal fatty acid (FA) ß-oxidation spiral. A fluorescent protein-tagging study pointed to a peroxisomal location of CrACX2. The importance of peroxisomal FA ß-oxidation in algal physiology was shown by the impact of the mutation on FA turnover during day/night cycles. Moreover, under nitrogen depletion the mutant accumulated 20% more oil than the wild type, illustrating the potential of ß-oxidation mutants for algal biotechnology. This study provides experimental evidence that a plant-type FA ß-oxidation involving H2 O2 -producing acyl-CoA oxidation activity has already evolved in the microbodies of the unicellular green alga Chlamydomonas reinhardtii.

Lipid catabolism in microalgae.

Lipid degradation processes are important in microalgae because survival and growth of microalgal cells under fluctuating environmental conditions require permanent remodeling or turnover of membrane lipids as well as rapid mobilization of storage lipids. Lipid catabolism comprises two major spatially and temporarily separated steps, namely lipolysis, which releases fatty acids and head groups and is catalyzed by lipases at membranes or lipid droplets, and degradation of fatty acids to acetyl-CoA, which occurs in peroxisomes through the ß-oxidation pathway in green microalgae, and can sometimes occur in mitochondria in some other algal species. Here we review the current knowledge on the enzymes and regulatory proteins involved in lipolysis and peroxisomal ß-oxidation and highlight gaps in our understanding of lipid degradation pathways in microalgae. Metabolic use of acetyl-CoA products via glyoxylate cycle and gluconeogenesis is also reviewed. We then present the implication of various cellular processes such as vesicle trafficking, cell cycle and autophagy on lipid turnover. Finally, physiological roles and the manipulation of lipid catabolism for biotechnological applications in microalgae are discussed.

UV-mediated Chlamydomonas mutants with enhanced nuclear transgene expression by disruption of DNA methylation-dependent and independent silencing systems.

KEY MESSAGE: In this investigation, we succeeded to generate Chlamydomonas mutants that bear dramatically enhanced ability for transgene expression. To yield these mutants, we utilized DNA methyltransferase deficient strain. These mutants must be useful as a plant cell factory. Chlamydomonas reinhardtii (hereafter Chlamydomonas) is a green freshwater microalga. It is a promising cell factory for the production of recombinant proteins because it rapidly grows in simple salt-based media. However, expression of transgenes integrated into the nuclear genome of Chlamydomonas is very poor, probably because of severe transcriptional silencing irrespective of the genomic position. In this study, we generated Chlamydomonas mutants by ultraviolet (UV)-mediated mutagenesis of maintenance-type DNA methyltransferase gene (MET1)-null mutants to overcome this disadvantage. We obtained several mutants with an enhanced ability to overexpress various transgenes irrespective of their integrated genomic positions. In addition, transformation efficiencies were significantly elevated. Our findings indicate that in addition to mechanisms involving MET1, transgene expression is regulated by a DNA methylation-independent transgene silencing system in Chlamydomonas. This is in agreement with the fact that DNA methylation occurs rarely in this organism. The generated mutants may be useful for the low-cost production of therapeutic proteins and eukaryotic enzymes based on their rapid growth in simple salt-based media.

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.