Development of a rapamycin-inducible protein-knockdown system in the unicellular red alga Cyanidioschyzon merolae.

An inducible protein-knockdown system is highly effective for investigating the functions of proteins and mechanisms essential for the survival and growth of organisms. However, this technique is not available in photosynthetic eukaryotes. The unicellular red alga Cyanidioschyzon merolae possesses a very simple cellular and genomic architecture and is genetically tractable but lacks RNA interference machinery. In this study, we developed a protein-knockdown system in this alga. The constitutive system utilizes the destabilizing activity of the FRB domain of human target of rapamycin (TOR) kinase or its derivatives to knock down target proteins. In the inducible system, rapamycin treatment induces the heterodimerization of the human FKBP12-rapamycin binding (FRB) domain fused to the target proteins with the human FK506-binding protein 12 (FKBP) fused to S-phase kinase associated protein 1 (SKP1) or Cullin 1 (CUL1), subunits of the SCF E3 ubiquitin ligase. This results in the rapid degradation of the target proteins through the ubiquitin-proteasome pathway. With this system, we successfully degraded endogenous essential proteins such as the chloroplast division protein Dynamin related protein 5B (DRP5B) and E2 transcription factor (E2F), a regulator of the G1/S transition, within 2-3 hours after rapamycin administration, enabling the assessment of resulting phenotypes. This rapamycin-inducible protein-knockdown system contributes to the functional analysis of genes whose disruption leads to lethality.

Investigation of bionano additives in red algae Cyanidioschyzon merolae ultrasonified MgO/MWCNT catalyzed biodiesel in optimized engine performance.

Renewable energy research is burgeoning with the anticipation of finding neat liquid fuel. Ultra sonification assisted biodiesel was derived from red algae Cyanidioschyzon merolae, with biodiesel yield of 98.9%. The results of GC MS of the prepared biodiesel showed higher concentration of methyl palmitate, methyl oleate, and stearate. This composition is appreciable, as this plays significance in desirable pour & cloud point properties. NMR spectrum revealed the ester linkages, presence of olefins, and α methyl position in olefins. Mixture of 30 wt% of biodiesel in diesel exhibited work efficiency, and also exhibited low pour point and, lower viscosity values. CeO2 and Fe2O3 nano particles were bio reduced, and were added as nano additive in biodiesel. 1:1 ratio of CeO2 and Fe2O3 added to biodiesel maximised the combustion ability of fuel owing to the oxygen storage capacity of CeO2. Further, this combination produced a satisfactory calorific value. Imbalanced ratios disrupted the catalytic and oxygen storage effects, reduced the overall energy release and calorific value of the biodiesel blend. Pour point and cetane number value of biodiesel blend ultrasonifacted with 1:1 mass ratio of Fe2O3 and CeO2 was observed to be around -7 °C and 53 °C respectively, and was better than other compositions. 1:1 mass ratio of NPS blended with 30 wt% BD in diesel showed tremendous increase in brake thermal efficiency, torque, and power. HC, NOX, and SOX emissions were reduced by 42.8%, 19.3%, and 57% respectively with 1:1 Fe2O3 and CeO2 mixed biodiesel blend. CeO2 favourably improved the oxygen storage capacity of the fuel, whereas Fe2O3 showed decrease in formation of gums and sediments in biodiesel.

Study of Dispersed Repeats in the Cyanidioschyzon merolae Genome.

In this study, we applied the iterative procedure (IP) method to search for families of highly diverged dispersed repeats in the genome of Cyanidioschyzon merolae, which contains over 16 million bases. The algorithm included the construction of position weight matrices (PWMs) for repeat families and the identification of more dispersed repeats based on the PWMs using dynamic programming. The results showed that the C. merolae genome contained 20 repeat families comprising a total of 33,938 dispersed repeats, which is significantly more than has been previously found using other methods. The repeats varied in length from 108 to 600 bp (522.54 bp in average) and occupied more than 72% of the C. merolae genome, whereas previously identified repeats, including tandem repeats, have been shown to constitute only about 28%. The high genomic content of dispersed repeats and their location in the coding regions suggest a significant role in the regulation of the functional activity of the genome.

Immobilization of fumarase from thermophilic eukaryotic red alga Cyanidioschyzon merolae on ceramic carrier.

Fumarase is an enzyme catalyzing reversible reaction between fumarate and L-malate in the citric acid cycle. Fumarase is used in the industrial production of L-malate, and its immobilization is required for reuse of the fumarases to reduce the cost. Accordingly, understanding the properties of immobilized fumarase is crucial, and several groups report on the storage stability and kinetic parameters of immobilized fumarase. Here we have immobilized fumarase from the thermophilic red alga Cyanidioschyzon merolae (CmFUM) on ceramic beads and investigated its biochemical and physical properties. CmFUM demonstrated sufficient stability and reusability for industry use after immobilization. Notably, the thermostability was dramatically enhanced through immobilization. The Km value and kcat of immobilized CmFUM for fumarate were 1.7 mM and 22.7 s-1 respectively. The Km value for fumarate was lower than that of other reported immobilized fumarases, indicating a high substrate affinity of immobilized CmFUM. Furthermore, the enhanced stability resulting from immobilization partially compensated for the decrease in activity. The high affinity towards fumarate and good thermostability of immobilized CmFUM revealed in this study are advantageous traits for improving enzyme-mediated isomer-specific L-malate production.

Dynamic adaptation of the extremophilic red microalga Cyanidioschyzon merolae to high nickel stress.

The order of Cyanidiales comprises seven acido-thermophilic red microalgal species thriving in hot springs of volcanic origin characterized by extremely low pH, moderately high temperatures and the presence of high concentrations of sulphites and heavy metals that are prohibitive for most other organisms. Little is known about the physiological processes underlying the long-term adaptation of these extremophiles to such hostile environments. Here, we investigated the long-term adaptive responses of a red microalga Cyanidioschyzon merolae, a representative of Cyanidiales, to extremely high nickel concentrations. By the comprehensive physiological, microscopic and elemental analyses we dissected the key physiological processes underlying the long-term adaptation of this model extremophile to high Ni exposure. These include: (i) prevention of significant Ni accumulation inside the cells; (ii) activation of the photoprotective response of non-photochemical quenching; (iii) significant changes of the chloroplast ultrastructure associated with the formation of prolamellar bodies and plastoglobuli together with loosening of the thylakoid membranes; (iv) activation of ROS amelioration machinery; and (v) maintaining the efficient respiratory chain functionality. The dynamically regulated processes identified in this study are discussed in the context of the mechanisms driving the remarkable adaptability of C. merolae to extremely high Ni levels exceeding by several orders of magnitude those found in the natural environment of the microalga. The processes identified in this study provide a solid basis for the future investigation of the specific molecular components and pathways involved in the adaptation of Cyanidiales to the extremely high Ni concentrations.

Expression of bacterial phosphite dehydrogenase confers phosphite availability in a unicellular red alga Cyanidioschyzon merolae.

　Microalgae are promising cell factories for producing value-added products. Large-scale microalgal cultivation suffers from invasion by contaminating microorganisms. Since most contaminating organisms cannot utilize phosphite as a unique phosphorus source, phosphite-utilizing ability may provide a growth advantage against contaminating organisms and solve this problem. Studies showed that microorganisms, typically unable to metabolize phosphite, can utilize phosphite by expressing exogenous phosphite dehydrogenase. Here, we constructed Cyanidioschyzon merolae strains introduced with the phosphite dehydrogenase gene, ptxD, from Ralstonia sp. 4506. The ptxD-introduced strains grew in a phosphite-dependent manner, with the phosphite-related growth rate almost matching that with phosphate as sole phosphorus source.

Factors affecting light harvesting in the red alga Cyanidioschyzon merolae.

The phycobilisome antennas, which contain phycobilin pigments instead of chlorophyll, are crucial for the photosynthetic activity of Cyanidioschyzon merolae cells, which thrive in an acidic and hot water environment. The accessible light intensity and quality, temperature, acidity, and other factors in this environment are quite different from those in the air available for terrestrial plants. Under these conditions, adaptation to the intensity and quality of light, as well as temperature, which are key factors in photosynthesis of higher plants, also affects this process in Cyanidioschyzon merolae cells. Adaptation to varying light conditions requires fast remodeling and re-tuning of their light-harvesting antennas (phycobilisomes) at multiple levels, from regulation of gene expression to structural reorganization of protein-pigment complexes. This review presents selected data on the structure of phycobilisomes, the genetic engineering of the constituent proteins, and the latest results and opinions on the adaptation of phycobilisomes to light intensity and quality, and temperature to photosynthetic activities. We pay special attention to the latest results of the C. merolae research.

The Polycomb repressive complex 2 deposits H3K27me3 and represses transposable elements in a broad range of eukaryotes.

The mobility of transposable elements (TEs) contributes to evolution of genomes. Their uncontrolled activity causes genomic instability; therefore, expression of TEs is silenced by host genomes. TEs are marked with DNA and H3K9 methylation, which are associated with silencing in flowering plants, animals, and fungi. However, in distantly related groups of eukaryotes, TEs are marked by H3K27me3 deposited by the Polycomb repressive complex 2 (PRC2), an epigenetic mark associated with gene silencing in flowering plants and animals. The direct silencing of TEs by PRC2 has so far only been shown in one species of ciliates. To test if PRC2 silences TEs in a broader range of eukaryotes, we generated mutants with reduced PRC2 activity and analyzed the role of PRC2 in extant species along the lineage of Archaeplastida and in the diatom P. tricornutum. In this diatom and the red alga C. merolae, a greater proportion of TEs than genes were repressed by PRC2, whereas a greater proportion of genes than TEs were repressed by PRC2 in bryophytes. In flowering plants, TEs contained potential cis-elements recognized by transcription factors and associated with neighbor genes as transcriptional units repressed by PRC2. Thus, silencing of TEs by PRC2 is observed not only in Archaeplastida but also in diatoms and ciliates, suggesting that PRC2 deposited H3K27me3 to silence TEs in the last common ancestor of eukaryotes. We hypothesize that during the evolution of Archaeplastida, TE fragments marked with H3K27me3 were selected to shape transcriptional regulation, controlling networks of genes regulated by PRC2.

How Light Modulates the Growth of Cyanidioschyzon merolae Cells by Changing the Function of Phycobilisomes.

The aim of this study was to examine how light intensity and quality affect the photosynthetic apparatus of Cyanidioschyzon merolae cells by modulating the structure and function of phycobilisomes. Cells were grown in equal amounts of white, blue, red, and yellow light of low (LL) and high (HL) intensity. Biochemical characterization, fluorescence emission, and oxygen exchange were used to investigate selected cellular physiological parameters. It was found that the allophycocyanin content was sensitive only to light intensity, whereas the phycocynin content was also sensitive to light quality. Furthermore, the concentration of the PSI core protein was not affected by the intensity or quality of the growth light, but the concentration of the PSII core D1 protein was. Finally, the amount of ATP and ADP was lower in HL than LL. In our opinion, both light intensity and quality are main factors that play an important regulatory role in acclimatization/adaptation of C. merolae to environmental changes, and this is achieved by balancing the amounts of thylakoid membrane and phycobilisome proteins, the energy level, and the photosynthetic and respiratory activity. This understanding contributes to the development of a mix of cultivation techniques and genetic changes for a future large-scale synthesis of desirable biomolecules.

Cultivation of the polyextremophile Cyanidioschyzon merolae 10D during summer conditions on the coast of the Red Sea and its adaptation to hypersaline sea water.

The west coast of the Arabian Peninsula borders the Red Sea, a water body which maintains high average temperatures and increased salinity compared to other seas or oceans. This geography has many resources which could be used to support algal biotechnology efforts in bio-resource circularity. However, summer conditions in this region may exceed the temperature tolerance of most currently cultivated microalgae. The Cyanidiophyceae are a class of polyextremophilic red algae that natively inhabit acidic hot springs. C. merolae 10D has recently emerged as an interesting model organism capable of high-cell density cultivation on pure CO2 with optimal growth at elevated temperatures and acidic pH. C. merolae biomass has an interesting macromolecular composition, is protein rich, and contains valuable bio-products like heat-stable phycocyanin, carotenoids, ß-glucan, and starch. Here, photobioreactors were used to model C. merolae 10D growth performance in simulated environmental conditions of the mid-Red Sea coast across four seasons, it was then grown at various scales outdoors in Thuwal, Saudi Arabia during the Summer of 2022. We show that C. merolae 10D is amenable to cultivation with industrial-grade nutrient and CO2 inputs outdoors in this location and that its biomass is relatively constant in biochemical composition across culture conditions. We also show the adaptation of C. merolae 10D to high salinity levels of those found in Red Sea waters and conducted further modeled cultivations in nutrient enriched local sea water. It was determined that salt-water adapted C. merolae 10D could be cultivated with reduced nutrient inputs in local conditions. The results presented here indicate this may be a promising alternative species for algal bioprocesses in outdoor conditions in extreme coastal desert summer environments.

Citrate synthase from Cyanidioschyzon merolae exhibits high oxaloacetate and acetyl-CoA catalytic efficiency.

Citrate synthase (CS) catalyzes the reaction that produces citrate and CoA from oxaloacetate and acetyl-CoA in the tricarboxylic acid (TCA) cycle. All TCA cycle enzymes are localized to the mitochondria in the model organism, the red alga Cyanidioschyzon merolae. The biochemical properties of CS have been studied in some eukaryotes, but the biochemical properties of CS in algae, including C. merolae, have not been studied. We then performed the biochemical analysis of CS from C. merolae mitochondria (CmCS4). The results showed that the kcat/Km of CmCS4 for oxaloacetate and acetyl-CoA were higher than those of the cyanobacteria, such as Synechocystis sp. PCC 6803, Microcystis aeruginosa PCC 7806 and Anabaena sp. PCC 7120. Monovalent and divalent cations inhibited CmCS4, and in the presence of KCl, the Km of CmCS4 for oxaloacetate and acetyl-CoA was higher in the presence of MgCl2, the Km of CmCS4 for oxaloacetate and acetyl-CoA was higher and kcat lower. However, in the presence of KCl and MgCl2, the kcat/Km of CmCS4 was higher than those of the three cyanobacteria species. The high catalytic efficiency of CmCS4 for oxaloacetate and acetyl-CoA may be a factor in the increased carbon flow into the TCA cycle in C. merolae.

Spliceosome assembly and regulation: insights from analysis of highly reduced spliceosomes.

Premessenger RNA splicing is catalyzed by the spliceosome, a multimegadalton RNA-protein complex that assembles in a highly regulated process on each intronic substrate. Most studies of splicing and spliceosomes have been carried out in human or S. cerevisiae model systems. There exists, however, a large diversity of spliceosomes, particularly in organisms with reduced genomes, that suggests a means of analyzing the essential elements of spliceosome assembly and regulation. In this review, we characterize changes in spliceosome composition across phyla, describing those that are most frequently observed and highlighting an analysis of the reduced spliceosome of the red alga Cyanidioschyzon merolae We used homology modeling to predict what effect splicing protein loss would have on the spliceosome, based on currently available cryo-EM structures. We observe strongly correlated loss of proteins that function in the same process, for example, in interacting with the U1 snRNP (which is absent in C. merolae), regulation of Brr2, or coupling transcription and splicing. Based on our observations, we predict splicing in C. merolae to be inefficient, inaccurate, and post-transcriptional, consistent with the apparent trend toward its elimination in this lineage. This work highlights the striking flexibility of the splicing pathway and the spliceosome when viewed in the context of eukaryotic diversity.

The carbon-concentrating mechanism of the extremophilic red microalga Cyanidioschyzon merolae.

Cyanidioschyzon merolae is an extremophilic red microalga which grows in low-pH, high-temperature environments. The basis of C. merolae's environmental resilience is not fully characterized, including whether this alga uses a carbon-concentrating mechanism (CCM). To determine if C. merolae uses a CCM, we measured CO2 uptake parameters using an open-path infra-red gas analyzer and compared them to values expected in the absence of a CCM. These measurements and analysis indicated that C. merolae had the gas-exchange characteristics of a CCM-operating organism: low CO2 compensation point, high affinity for external CO2, and minimized rubisco oxygenation. The biomass δ13C of C. merolae was also consistent with a CCM. The apparent presence of a CCM in C. merolae suggests the use of an unusual mechanism for carbon concentration, as C. merolae is thought to lack a pyrenoid and gas-exchange measurements indicated that C. merolae primarily takes up inorganic carbon as carbon dioxide, rather than bicarbonate. We use homology to known CCM components to propose a model of a pH-gradient-based CCM, and we discuss how this CCM can be further investigated.

Cyanidiales as Polyextreme Eukaryotes.

Cyanidiales were named enigmatic microalgae due to their unique polyextreme properties, considered for a very long time unattainable for eukaryotes. Cyanidiales mainly inhabit hot sulfuric springs with high acidity (pH 0-4), temperatures up to 56°C, and ability to survive in the presence of dissolved heavy metals. Owing to the minimal for eukaryotes genome size, Cyanidiales have become one of the most important research objects in plant cell physiology, biochemistry, molecular biology, phylogenomics, and evolutionary biology. They play an important role in studying many aspects of oxygenic photosynthesis and chloroplasts origin. The ability to survive in stressful habitats and the corresponding metabolic pathways were acquired by Cyanidiales from archaea and bacteria via horizontal gene transfer (HGT). Thus, the possibility of gene transfer from prokaryotes to eukaryotes was discovered, which was a new step in understanding of the origin of eukaryotic cell.

Crystal structure of CmABCB1 multi-drug exporter in lipidic mesophase revealed by LCP-SFX.

CmABCB1 is a Cyanidioschyzon merolae homolog of human ABCB1, a well known ATP-binding cassette (ABC) transporter responsible for multi-drug resistance in various cancers. Three-dimensional structures of ABCB1 homologs have revealed the snapshots of inward- and outward-facing states of the transporters in action. However, sufficient information to establish the sequential movements of the open-close cycles of the alternating-access model is still lacking. Serial femtosecond crystallography (SFX) using X-ray free-electron lasers has proven its worth in determining novel structures and recording sequential conformational changes of proteins at room temperature, especially for medically important membrane proteins, but it has never been applied to ABC transporters. In this study, 7.7 mono-acyl-glycerol with cholesterol as the host lipid was used and obtained well diffracting microcrystals of the 130 kDa CmABCB1 dimer. Successful SFX experiments were performed by adjusting the viscosity of the crystal suspension of the sponge phase with hy-droxy-propyl methyl-cellulose and using the high-viscosity sample injector for data collection at the SACLA beamline. An outward-facing structure of CmABCB1 at a maximum resolution of 2.22 Šis reported, determined by SFX experiments with crystals formed in the lipidic cubic phase (LCP-SFX), which has never been applied to ABC transporters. In the type I crystal, CmABCB1 dimers interact with adjacent molecules via not only the nucleotide-binding domains but also the transmembrane domains (TMDs); such an interaction was not observed in the previous type II crystal. Although most parts of the structure are similar to those in the previous type II structure, the substrate-exit region of the TMD adopts a different configuration in the type I structure. This difference between the two types of structures reflects the flexibility of the substrate-exit region of CmABCB1, which might be essential for the smooth release of various substrates from the transporter.

A cotransformation system of the unicellular red alga Cyanidioschyzon merolae with blasticidin S deaminase and chloramphenicol acetyltransferase selectable markers.

BACKGROUND: The unicellular red alga Cyanidioschyzon merolae exhibits a very simple cellular and genomic architecture. In addition, procedures for genetic modifications, such as gene targeting by homologous recombination and inducible/repressible gene expression, have been developed. However, only two markers for selecting transformants, uracil synthase (URA) and chloramphenicol acetyltransferase (CAT), are available in this alga. Therefore, manipulation of two or more different chromosomal loci in the same strain in C. merolae is limited. RESULTS: This study developed a nuclear targeting and transformant selection system using an antibiotics blasticidin S (BS) and the BS deaminase (BSD) selectable marker by homologous recombination in C. merolae. In addition, this study has succeeded in simultaneously modifying two different chromosomal loci by a single-step cotransformation based on the combination of BSD and CAT selectable markers. A C. merolae strain that expresses mitochondrion-targeted mSCARLET (with the BSD marker) and mVENUS (with the CAT marker) from different chromosomal loci was generated with this procedure. CONCLUSIONS: The newly developed BSD selectable marker enables an additional genetic modification to the already generated C. merolae transformants based on the URA or CAT system. Furthermore, the cotransformation system facilitates multiple genetic modifications. These methods and the simple nature of the C. merolae cellular and genomic architecture will facilitate studies on several phenomena common to photosynthetic eukaryotes.

CZON-cutter - a CRISPR-Cas9 system for multiplexed organelle imaging in a simple unicellular alga.

The unicellular alga Cyanidioschyzon merolae has a simple cellular structure; each cell has one nucleus, one mitochondrion, one chloroplast and one peroxisome. This simplicity offers unique advantages for investigating organellar proliferation and the cell cycle. Here, we describe CZON-cutter, an engineered clustered, regularly interspaced, short palindromic repeats (CRISPR)/CRISPR-associated nuclease 9 (Cas9) system for simultaneous genome editing and organellar visualization. We engineered a C. merolae strain expressing a nuclear-localized Cas9-Venus nuclease for targeted editing of any locus defined by a single-guide RNA (sgRNA). We then successfully edited the algal genome and visualized the mitochondrion and peroxisome in transformants using fluorescent protein reporters with different excitation wavelengths. Fluorescent protein labeling of organelles in living transformants allows us to validate phenotypes associated with organellar proliferation and the cell cycle, even when the edited gene is essential. Combined with the exceptional biological features of C. merolae, CZON-cutter will be instrumental for investigating cellular and organellar division in a high-throughput manner. This article has an associated First Person interview with the first author of the paper.

Cis-acting elements involved in the G2/M-phase-specific transcription of the cyclin B gene in the unicellular alga Cyanidioschyzon merolae.

M-specific activator (MSA) cis-acting elements have been determined to be involved in the regulation of G2/M-phase-specific transcription in spermatophytes. In this study, the involvement of MSA-core elements in G2/M-phase-specific transcription was examined in the unicellular red alga Cyanidioschyzon merolae. In the C. merolae genome, MSA-core elements do not accumulate specifically in the upstream of mitosis-specific transcriptional genes. Mutations of the four MSA-core elements of the cyclin B gene, which encodes a central factor of the G2-to-M-phase transition, have resulted in the abolishment of transcription or permission of transcription even in the G1 phase. These results suggest that all four MSA-core elements located in the upstream region of cyclin B are involved in G2/M-phase-specific transcription in C. merolae; however, the nature of the involvement of MSA-core elements in G2/M-phase-specific transcription differed among the four elements. Thus, MSA-core-element-mediated G2/M-phase-specific transcription in C. merolae seems to be regulated by a complex mechanism.

Development of a Novel Nanoarchitecture of the Robust Photosystem I from a Volcanic Microalga Cyanidioschyzon merolae on Single Layer Graphene for Improved Photocurrent Generation.

Here, we report the development of a novel photoactive biomolecular nanoarchitecture based on the genetically engineered extremophilic photosystem I (PSI) biophotocatalyst interfaced with a single layer graphene via pyrene-nitrilotriacetic acid self-assembled monolayer (SAM). For the oriented and stable immobilization of the PSI biophotocatalyst, an His6-tag was genetically engineered at the N-terminus of the stromal PsaD subunit of PSI, allowing for the preferential binding of this photoactive complex with its reducing side towards the graphene monolayer. This approach yielded a novel robust and ordered nanoarchitecture designed to generate an efficient direct electron transfer pathway between graphene, the metal redox center in the organic SAM and the photo-oxidized PSI biocatalyst. The nanosystem yielded an overall current output of 16.5 µA·cm-2 for the nickel- and 17.3 µA·cm-2 for the cobalt-based nanoassemblies, and was stable for at least 1 h of continuous standard illumination. The novel green nanosystem described in this work carries the high potential for future applications due to its robustness, highly ordered and simple architecture characterized by the high biophotocatalyst loading as well as simplicity of manufacturing.

The Unicellular Red Alga Cyanidioschyzon merolae-The Simplest Model of a Photosynthetic Eukaryote.

Several species of unicellular eukaryotic algae exhibit relatively simple genomic and cellular architecture. Laboratory cultures of these algae grow faster than plants and often provide homogeneous cellular populations exposed to an almost equal environment. These characteristics are ideal for conducting experiments at the cellular and subcellular levels. Many microalgal lineages have recently become genetically tractable, which have started to evoke new streams of studies. Among such algae, the unicellular red alga Cyanidioschyzon merolae is the simplest organism; it possesses the minimum number of membranous organelles, only 4,775 protein-coding genes in the nucleus, and its cell cycle progression can be highly synchronized with the diel cycle. These properties facilitate diverse omics analyses of cellular proliferation and structural analyses of the intracellular relationship among organelles. C. merolae cells lack a rigid cell wall and are thus relatively easily disrupted, facilitating biochemical analyses. Multiple chromosomal loci can be edited by highly efficient homologous recombination. The procedures for the inducible/repressive expression of a transgene or an endogenous gene in the nucleus and for chloroplast genome modification have also been developed. Here, we summarize the features and experimental techniques of C. merolae and provide examples of studies using this alga. From these studies, it is clear that C. merolae-either alone or in comparative and combinatory studies with other photosynthetic organisms-can provide significant insights into the biology of photosynthetic eukaryotes.