A novel Sphingobium yanoikuyae strain, CC4533, was isolated from a contaminated tris-acetate-phosphate medium culture plate of a green microalga, Chlamydomonas reinhardtii. In this study, we present the complete genome sequence of this beta-carotene-synthesizing, xenobiotic-degrading, and heavy metal-tolerant strain, providing insights into its metabolic capabilities and phylogenetic relationships.

Aquatic photosynthetic systems account for approximately one-half of all global carbon assimilation and could be a significant source of renewable fuels and feedstocks. However, rapid growth and biomass production in algae have not always translated into high product yields, partly because central metabolism is context specific, with metabolic fluxes being influenced by nutrient conditions and other environmental factors. In the green microalga Chlamydomonas reinhardtii (Chlamydomonas), mixotrophic cultures (acetate + light) grow far faster than phototrophic (light only) or heterotrophic (acetate + dark) cultures, even though acetate partially suppresses photosynthesis. Here, an isotopic dilution strategy with unlabeled acetate was combined with 13CO2 transient labeling to perform isotopically nonstationary metabolic flux analysis (INST-MFA) and to directly compare autotrophic and mixotrophic metabolism in Chlamydomonas supported by data from transcriptomics, proteomics, and metabolomics. INST-MFA indicated that acetate induces a synergistic rewiring of metabolism, conserving carbon by using the glyoxylate cycle and suppressing gluconeogenesis, the latter of which was discordant with omics results and prior models. Additionally, our data provide a plausible rationale for the well-known suppression of photosynthesis by acetate. We propose that reduced total protein content in mixotrophic versus phototrophic cells, much of which is attributed to reduced levels of photosynthetic proteins, decreases the costly metabolic burden of protein synthesis and represents a growth rate optimization strategy.

Investigation of the DNA Binding Ability of CIA5 in Chlamydomonas reinhardtii

Lysine succinylation is an evolutionarily conserved post-translational modification (PTM) involved in cellular metabolic regulation. Chlamydomonas reinhardtii, a GRAS-certified microalga and emerging chassis for biofuel and biobased production, has not yet been systematically characterized for succinylation. Here, we present a comprehensive proteome-wide lysine succinylome map of Chlamydomonas and identify 2806 high-confidence succinylation sites across 791 proteins. These proteins were predominantly distributed in chloroplasts, cytoplasm, and mitochondria and were significantly enriched in pathways central to carbon and lipid metabolism. Comparative analyses revealed extensive crosstalk between succinylation and acetylation with distinct sequence patterns and functional preferences. Site-directed mutagenesis and enzymatic assays revealed that succinylation at K597 of Acetyl-CoA synthetase 3 (ACS3) markedly reduced enzymatic activity, with an inhibitory effect stronger than that of acetylation. These findings establish succinylation as a key metabolic regulator and provide a valuable resource for advancing metabolic engineering in algae.

Cross-kingdom microbial symbioses, such as those between algae and bacteria, are key players in biogeochemical cycles. The molecular changes during initiation and establishment of symbiosis are of great interest, but quantitatively monitoring such changes can be challenging, particularly when the microorganisms differ greatly in size or are intimately associated. Here, we analyze output from label-free, data-dependent acquisition (DDA) LC-MS/MS proteomics experiments investigating the well-studied interaction between the alga Chlamydomonas reinhardtii and the heterotrophic bacterium Mesorhizobium japonicum. We found that detection of bacterial proteins decreased in coculture by 50% proteome-wide due to the abundance of algal proteins. As a result, standard differential expression analysis led to numerous false-positive reports of significantly downregulated proteins, where it was not possible to distinguish meaningful biological responses to symbiosis from artifacts of the reduced protein detection in coculture relative to monoculture. We show that data normalization alone does not eliminate the impact of altered detection on differential expression analysis of the cross-kingdom symbiosis. We assessed two additional strategies to overcome this methodological artifact inherent to DDA proteomics. In the first, we combined algal and bacterial monocultures at a relative abundance that mimicked the coculture, creating a “mono-mix” control to which the coculture could be compared. This approach enabled comparable detection of bacterial proteins in the coculture and the monoculture control. In the second strategy, we enhanced detection of lowly abundant bacterial proteins by using sample fractionation upstream of LC-MS/MS analysis. When these simple approaches were combined, they allowed for meaningful comparisons of nearly 10,000 algal proteins and over 4,000 bacterial proteins in response to symbiosis by DDA. They successfully recovered expected changes in the bacterial proteome in response to algal coculture, including upregulation of sugar-binding proteins and transporters. They also revealed novel proteomic responses to coculture that guide hypotheses about algal-bacterial interactions.

Cross-kingdom microbial symbioses, such as those between algae and bacteria, are key players in biogeochemical cycles. The molecular changes during initiation and establishment of symbiosis are of great interest, but quantitatively monitoring such changes can be challenging, particularly when the microorganisms differ greatly in size or are intimately associated. Here, we analyze output from label-free, data-dependent acquisition (DDA) LC-MS/MS proteomics experiments investigating the well-studied interaction between the alga Chlamydomonas reinhardtii and the heterotrophic bacterium Mesorhizobium japonicum. We found that detection of bacterial proteins decreased in coculture by 50% proteome-wide due to the abundance of algal proteins. As a result, standard differential expression analysis led to numerous false-positive reports of significantly downregulated proteins, where it was not possible to distinguish meaningful biological responses to symbiosis from artifacts of the reduced protein detection in coculture relative to monoculture. We show that data normalization alone does not eliminate the impact of altered detection on differential expression analysis of the cross-kingdom symbiosis. We assessed two additional strategies to overcome this methodological artifact inherent to DDA proteomics. In the first, we combined algal and bacterial monocultures at a relative abundance that mimicked the coculture, creating a "mono-mix" control to which the coculture could be compared. This approach enabled comparable detection of bacterial proteins in the coculture and the monoculture control. In the second strategy, we enhanced detection of lowly abundant bacterial proteins by using sample fractionation upstream of LC-MS/MS analysis. When these simple approaches were combined, they allowed for meaningful comparisons of nearly 10,000 algal proteins and over 4,000 bacterial proteins in response to symbiosis by DDA. They successfully recovered expected changes in the bacterial proteome in response to algal coculture, including upregulation of sugar-binding proteins and transporters. They also revealed novel proteomic responses to coculture that guide hypotheses about algal-bacterial interactions.

Plant-type ferredoxins (Fd) comprise small, soluble protein families that distribute electrons from photosystem I to various client proteins within the chloroplast stroma. In Chlamydomonas reinhardtii, the major, constitutively expressed FDX1/PetF supports Fd-NADP+ reductase (FNR) in NADPH production. The highly similar FDX2 is present only when its preferred nitrogen (N) source ammonium is absent, supplying Fd-dependent nitrite reductase (NiR) for nitrate/nitrite assimilation. Surprisingly, despite accumulating to ~10% of FDX1 abundance and preferential interaction with NiR, fdx2 mutants are asymptomatic when grown on nitrate, requiring to additionally deplete FDX1 for growth to be halted. A fdx1 knockout itself appears lethal, severe fdx1 knockdowns have reduced growth rates both in phototrophic and photoheterotrophic conditions, independent of the N source. Transcriptome analyses of fdx1 mutants revealed expression patterns similar to sulfur (S) deficient algae, and fdx1 strains have a reduced total cellular S content. S assimilation requires Fd-dependent sulfite reductase (SiR) activity, an enzyme distantly related to FDX2 client NiR. Expression defects are partially alleviated; growth and S content are less impacted with FDX2 expression. Our mutant analysis shows the two major Fds in Chlamydomonas focus on a specific subset of Fd-dependent metabolism, mostly supplying Fd-dependent enzymes involved in macronutrient assimilation (C/N/S).

Genome scale engineering has enabled codon compression of the universal genetic code to eliminate seven codons in Escherichia coli, but to allow more radical schemes for codon compression and reassignment to be tested at genome scale, while avoiding significant technical challenges, smaller, simpler genetic systems are needed. Here, we report a recoding scheme for the 205 kb Chlamydomonas reinhardtii chloroplast genome, in which two stop codons and one or more of the codons for arginine, glycine, isoleucine, leucine, and serine, all of which have two cognate transfer RNAs (tRNAs), are absent, compressing the genetic code to 51 codons. Several recoding strategies were tested on the essential rpoA gene, encoding a subunit of the chloroplast RNA polymerase. A defined compression scheme, which relied on swapping the target codons with the permitted frequent codons, could replace the native sequence without affecting expression of a reporter protein or strain fitness under standard laboratory conditions. The same strategy was successfully used for codon compression of ycf1, encoding a subunit of the chloroplast translocon, psaA and psbA, intron-containing highly expressed genes encoding reaction center subunits of both photosystems, and an 8.5 kb operon encoding essential and nonessential genes. Finally, we tested degeneracy of the 51-codon genetic code by exploring the combinatorial design for the large subunit of Rubisco, relying on restoration of photosynthesis in an rbcL mutant strain. More than 70 functional sequences with diverse codons were recovered. For all recoded genes, viable homoplasmic lines were obtained, showing the efficacy of our codon compression scheme.

The growing threat of antibiotic resistance underscores the urgent need for novel therapeutic agents. Antimicrobial peptides (AMPs), such as human defensins, are promising candidates due to their broad-spectrum activities, but their widespread application is severely hindered by high production costs and limited stability. Among these, human neutrophil peptide-2 (HNP2), a member of the α-defensin family, is a particularly potent AMP that kills microorganisms by disrupting their membrane integrity. The eukaryotic microalga Chlamydomonas reinhardtii (C. reinhardtii) has emerged as a low-cost and efficient bioreactor for exogenous protein production, yet its potential for expressing human defensins remains largely unexplored. This study aimed to express a tandem trimer of the HNP2 mature peptide (3×HNP2) in C. reinhardtii and to characterize its stability, safety, and antibacterial functions. In this study, a gene encoding 3×HNP2 was successfully expressed in the C. reinhardtii strain CC-5325, yielding a fusion protein of approximately 22kDa. The recombinant protein's expression remained stable for over five months of continuous subculturing. Obtained via fermentation technology and affinity purification, the purified 3×HNP2 demonstrated potent antibacterial activity against the Gram-negative pathogens Pseudomonas aeruginosa (P. aeruginosa) and Escherichia coli (E. coli). The protein exhibited high thermal stability (up to 90°C), broad pH tolerance (pH 2-10), and resistance to degradation by proteinase K and papain. Mechanistic investigations using propidium iodide (PI) staining and scanning electron microscopy (SEM) confirmed that 3×HNP2 acts by disrupting bacterial membrane integrity. Furthermore, qRT-PCR analysis revealed that 3×HNP2 significantly downregulated the expression of key virulence-associated genes (lecA, phzA2, csgA, and rpoS). Biosafety assays showed that the peptide had minimal hemolytic activity and low cytotoxicity against mammalian cell lines. This work establishes a green platform for producing a functional human defensin in C. reinhardtii. It demonstrates that algae-derived 3×HNP2 is a stable, safe, and effective antimicrobial agent, suggesting its potential as a candidate for further development in antimicrobial therapeutics or food safety applications.

Chloroplasts offer significant potential for multigene engineering in microalgae, but the lack of well-characterized regulatory elements and limited understanding of plastid transcriptional mechanisms have hindered progress. Here, through a comparative conservation analysis across fifteen species of microalgae and higher plants, we identified bidirectional promoter (BDP) intergenic regions (IRs) showing diverse evolutionary trajectories, from lineage-specific rearrangements of atpA/rbcL (BDP1) and chlL/petB (BDP2), to strict conservation of the psbH/psbN IR (BDP3), and complete loss of rpoB-1/psbF (BDP4). Based on promoter signature analysis, we selected three candidate regions (BDP1, BDP2, and BDP3) from the Chlamydomonas reinhardtii chloroplast genome for functional characterization. A semi-rational screen revealed that BDP1 supports expression of two transgenes, mVenus and tdTomato in opposing orientations; BDP2 drives balanced expression, but low protein accumulation; and BDP3 exhibits minimal activity, suggesting UTR-dependent post-transcriptional regulation. Strikingly, methyl-jasmonate selectively enhanced tdTomato expression from BDP1, offering a chemical method to regulate chloroplast transgene expression. Collectively, these results underscore the evolutionary diversity and functional potential of natural BDPs, particularly BDP1, as powerful tools for multigene engineering and chemical modulation in microalgae and higher plants. This study also provides fundamental insights into chloroplast transcription, establishing a basis for future investigations into its regulatory mechanisms.

Chlamydomonas acclimates to repeated low (LL) or high light (HL) days by changing the abundance of photosynthetic complexes and the ultrastructure of its thylakoid membranes. These phenotypes persist through the night phases, suggesting a readiness for the daylight environment that is routinely experienced despite the intervening dark periods [S. Dupuis et al., Plant Cell 37, koaf086 (2025), 10.1093/plcell/koaf086]. Here, we investigate how prior acclimation impacts algal fitness upon a change in daylight intensity and how quickly Chlamydomonas can reprogram its photoprotective strategy in a diurnal context. We performed a systems analysis of synchronized populations acclimated to diurnal LL when subjected to HL days and of populations acclimated to diurnal HL when subjected to LL days. In the latter case, diurnal photoacclimation decreased fitness during the first day at a new light intensity: HL-acclimated cells barely increased in size over the first LL period, and they failed to complete a cell cycle. However, although LL-acclimated cells showed severe photodamage after 6 h of HL, they recovered chloroplast form and function later that afternoon and successfully divided at nightfall. These cells rapidly altered their thylakoid membrane ultrastructure, increased their photoprotective quenching capacity, and decreased their inventory of photosystem and antenna proteins by the end of the first HL day. Transcriptomic and proteomic analyses revealed rapid induction of thousands of genes, including those encoding proteases, chaperones, and other proteins involved in the chloroplast unfolded protein response. These results show that the alga is highly flexible and competent to rapidly acclimate to changes in diurnal light intensity.

In situ cryo-electron tomography (cryo-ET) has emerged as the method of choice to investigate the structures of biomolecules in their native context. However, challenges remain for the efficient production and sharing of large-scale cryo-ET datasets. Here, we combined cryogenic plasma-based focused ion beam (cryo-PFIB) milling with recent advances in cryo-ET acquisition and processing to generate a dataset of 1,829 annotated tomograms of the green alga Chlamydomonas reinhardtii, which we provide as a community resource to drive method development and inspire biological discovery. To assay data quality, we performed subtomogram averaging of both soluble and membrane-bound complexes ranging in size from >3 MDa to ∼200 kDa, including 80S ribosomes, Rubisco, nucleosomes, microtubules, clathrin, photosystem II, and mitochondrial ATP synthase. The majority of these density maps reached sub-nanometer resolution, demonstrating the potential of this C. reinhardtii dataset as well as the promise of modern cryo-ET workflows and open data sharing to empower visual proteomics.

Circular RNAs in microalgae: Uncovering their biological significance.

DNA epigenetic modifications play crucial roles in regulating gene expression and cellular function across diverse organisms. Among them, 5-glyceryl-methylcytosine (5gmC), a unique DNA modification first discovered in Chlamydomonas reinhardtii, represents a novel link between redox metabolism and epigenetic regulation. Accurate genome-wide detection of 5gmC is essential for investigating its biological functions, yet no streamlined method has been available. Here, we present deaminase-assisted sequencing (DEA-seq), a simple and robust approach for base-resolution mapping of 5gmC. DEA-seq employs a single DNA deaminase that efficiently converts unmodified cytosines (C) and 5-methylcytosine (5mC) into uracils or thymines, while leaving 5gmC intact. This selective resistance generates a clear sequence signature that enables precise identification of 5gmC sites across the genome. The method operates under mild reaction conditions and is compatible with low-input DNA, minimizing sample loss and improving detection sensitivity. Overall, DEA-seq provides an accessible, efficient, and highly accurate protocol for profiling 5gmC, offering clear advantages in workflow simplicity, DNA integrity, and analytical performance. Key features • A commercial deaminase mix (DEA) efficiently converts unmodified cytosines and 5-methylcytosines into uracils or thymines. • 5gmC specifically resists DEA-mediated deamination, enabling its identification at single-base resolution. • DEA-seq requires only minimal DNA input and supports high-sensitivity detection from nanogram-level samples.

Cryo-EM structural analyses of chlorophyll b-enriched PSI-LHC and PSII-LHC supercomplexes of the siphonous green alga Bryopsis corticulans.

Microplastics (MPs) are increasingly recognized as emerging pollutants in freshwater systems. Detecting and tracing MP-derived carbon in aquatic food webs, however, remains unresolved, limiting our understanding of ecological impacts. Here, we evaluate the potential and limitations of natural abundance stable carbon isotope measurements (δ13C) as a tool to identify MP signals in freshwater ecosystems. For this purpose, two freshwater algae, Chlorella vulgaris and Chlamydomonas reinhardtii, were exposed under controlled laboratory conditions to one non-biodegradable polymer, low-density polyethylene (LDPE), and two biodegradable polymers, polylactic acid (PLA) and polybutylene adipate-co-terephthalate (PBAT), to assess isotope composition and growth. Laboratory data were complemented by particulate organic carbon (δ13CPOC) measurements from seasonal Danube River campaigns (2023-2024) with modeled predictions based on dissolved organic carbon (δ13CDIC). MP exposure did not inhibit algae growth, but C. vulgaris exhibited significant (p<0.05) δ13C enrichment (+4 to +5‰), whereas C. reinhardtii showed no isotopic response. These shifts were unrelated to polymer isotope values and likely reflect indirect physiological stress rather than assimilation of polymer-derived carbon. Complementary binary mixing experiments further confirmed that measurable isotopic shifts occur only at unrealistically low algae-to-MP ratios (≤10:1), underscoring the limited sensitivity of isotope mass balances. Field surveys revealed pronounced seasonal δ13CPOC variability in the Danube, spanning 7.4‰ annually. Yet deviations from modeled expectations were inconsistent with MP inputs and instead reflected natural drivers such as productivity and remineralization. Overall, while natural abundance δ13C can capture subtle algae responses to MP exposure under laboratory conditions, its diagnostic power for tracing MP-derived carbon in complex freshwater systems appears limited.

Protein S-nitrosylation is a reversible redox-based post-translational modification that plays an important role in cell signaling by modulating protein function and stability. At the molecular level, S-nitrosylation consists of the formation of a nitrosothiol (-SNO) and is primarily induced by the trans-nitrosylating agent nitrosoglutathione (GSNO). Triosephosphate isomerase (TPI), which catalyzes the interconversion of dihydroxyacetone phosphate and glyceraldehyde-3-phosphate, has been identified as a putative target of S-nitrosylation in both plant and non-plant systems. Here we investigate the molecular basis for GSNO-dependent regulation of chloroplast TPI from the model green alga Chlamydomonas reinhardtii (CrTPI). Molecular modelling identified Cys14 and Cys219 as potential sites for interaction with GSNO, though crystallography of GSNO-treated CrTPI revealed S-nitrosylation only at Cys14. To disclose GSNO target sites, we generated and characterized Cys-to-Ser variants for Cys14 and Cys219, identifying Cys219 as a key residue mediating the GSNO-dependent modulation of CrTPI activity. Molecular dynamics simulations further revealed the stabilizing interactions of S-nitrosylated cysteines with their local environments. Overall, our results indicate that CrTPI catalysis is modulated by GSNO through a redox-based mechanism involving Cys219, which highlights a conserved regulatory strategy shared with human TPI.

Beyond protection: How alginate immobilization enhances microalgal capacity for oxytetracycline removal.

The high-value sesquiterpenoid (+)-nootkatone has important applications in food, agriculture, and pharmaceutical industries. Extraction from plant material, however, is technically challenging and inefficient due to inherent low concentrations in native sources. Over the last decade, the eukaryotic green microalga Chlamydomonas reinhardtii has emerged as a powerful alternative for heterologous terpenoid production, due to a natively high carbon flux through its MEP pathway. This study describes strategic fusion protein designs of different valencene and farnesyl pyrophosphate (FPP) synthases, which allowed efficient (+)-valencene biosynthesis in the C. reinhardtii cytosol and also found the algal chloroplast to be highly suitable for heterologous production. Successful co-expression of cytochrome P450 monooxygenases resulted in a two-step oxidation towards (+)-nootkatone at a comparably high conversion rate of 76% and was independent of recombinant reductase activity. In addition, the 1-deoxyxylulose-5-phosphate synthase (DXS) was found to be rate-limiting for increased sesquiterpenoid production. Currently available photobioreactors suffer from limitations in light availability, which can hinder phototrophic growth, especially at higher cell densities. C. reinhardtii harbours the ability to use acetic acid as a carbon source, and fine-tuned cultivation regimes under photo-, mixo-, and heterotrophic conditions were tested to optimize heterologous sesquiterpene production. Customized scale-up cultivations in 2.5L showed efficient volumetric production of 148mg/L under phototrophic conditions and a maximal gravimetric production of 76 mg/gCDW under heterotrophic cultivation regimes, which displays a first industrially relevant (+)-nootkatone production concept in a green cell factory.

Cytochrome P450 (CYP450) monooxygenases are a class of enzymes containing conserved heme-binding functional domain. They contribute to a wide range of biosynthetic processes, serving a pivotal function in plant resistance to abiotic stress. To date, little is known about the CYP450s of Chlamydomonas reinhardtii. In our study, a total of 37 crP450 genes were identified from C. reinhardtii based on domain and sequence alignment, unevenly distributed on 12 chromosomes with 4 pairs of tandem replications shared among family members. Most of these genes contained 10 or more introns and encoded CYP450 proteins with an average of 593 amino acids and 3-9 conserved motifs. CYP450 enzymes were mainly distributed in the chloroplasts, cytoplasms, mitochondria, and cytoplasmic membranes. There were numerous light, jasmonic acid, abscisic acid, and salicylic acid response elements located in the upstream of gene coding sequences, suggesting that these genes could be modulated by plant hormones. Transcriptome analysis uncovered distinct expression patterns of crP450 genes under various stress conditions, with the 37 crP450 genes grouped into 9 clusters. In summary, this study presented a genome-wide characterization of CYP450 genes in C. reinhardtii, providing a strong foundation for further exploration into their biological functions.