Increasing lipid accumulation in Chlamydomonas by serial knocking out of DYRKP1 kinase and ADP-glucose pyrophosphorylase

Abstract

Microalgae are promising sustainable feedstocks for biodiesel production. Among the primary carbon reservoirs in microalgae, starch and lipids are the main targets for metabolic engineering aimed at enhancing productivity. Redirecting carbon flux from starch toward lipid biosynthesis has been considered an effective strategy to improve lipid yield, and manipulating upstream regulators may allow broader control over metabolic networks. DYRKP1, a plant-specific dual-specificity tyrosine-phosphorylation-regulated kinase conserved in photosynthetic eukaryotes, has been implicated in regulating intracellular carbon partitioning. In this study, we investigated the physiological and metabolic effects of DYRKP1 deficiency in a cell-wall-less strain of Chlamydomonas reinhardtii. To further enhance lipid accumulation, we additionally knocked out ADP-glucose pyrophosphorylase (AGP), a key enzyme involved in starch biosynthesis. The total fatty acid content of DYRKP1-AGP double knockout (dKO) mutants was higher than that of their parental strain (CC4349) under both nitrogen-replete and deplete conditions, and was even 1.2-fold higher than that of the AGP single knockout (agp) mutant under nitrogen-deplete conditions. The DYRKP1 single knockout mutants exhibited fatty acid composition similar to the parental strain, regardless of nitrogen depletion. The fatty acid composition of the dKO mutants resembled that of the agp mutant under nitrogen-replete conditions, but diverged upon nitrogen starvation, suggesting a conditional interaction between upstream regulation and metabolic flux. This finding implies that disrupting upstream regulators like DYRKP1 may offer limited additional benefit when key downstream bottlenecks, such as starch biosynthesis, are already removed. Overall, our study underscores the layered complexity of carbon partitioning in C. reinhardtii and the importance of context-dependent metabolic regulation in optimizing lipid production.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12934-025-02824-8.

Introduction

Microalgae have the remarkable ability to convert CO₂ or organic carbon in the culture medium into industrially valuable bioproducts such as lipids, starches, and proteins [1]. Owing to their photosynthetic capacity and carbon fixation potential, microalgae are considered a promising bio-feedstock for sustainable industrial applications [2]. In this context, extensive research has been dedicated to producing biofuels from microalgal biomass [2–4], with growing societal demand for sustainable aviation fuels further underscoring the importance of such efforts [5].

Recent advances in genome editing techniques, such as CRISPR-Cas9, have enabled targeted metabolic engineering to optimize carbon flux toward desired bioproducts [1]. A widely adopted strategy involves repressing metabolic pathways that compete with lipid biosynthesis for carbon allocation. For instance, suppression of ADP-glucose pyrophosphorylase (AGP), a key enzyme in starch metabolism, has been shown to increase lipid content by up to 81% in the model green microalga Chlamydomonas reinhardtii [6]. Meanwhile, direct manipulation of lipid metabolism has also been extensively explored through overexpression of lipid biosynthetic enzymes [7, 8] and inhibition of lipases responsible for lipid degradation [9, 10]. More recently, the focus has shifted to upstream regulators, such as transcription factors, which can be involved in broader metabolic changes [11, 12]. For instance, the overexpression of bZIP1 and MYB1 transcription factors enhanced lipid accumulation in microalgae [13, 14]. These studies suggest that modulating upstream regulatory elements may be an effective strategy for enhancing lipid accumulation in microalgae.

The plant-specific dual-specificity tyrosine-phosphorylation-regulated kinase (DYRKP) has been implicated in the regulation of intracellular carbon metabolism [15, 16]. In C. reinhardtii, a knockout mutant of DYRKP1 (Cre07.g337300) in a cell-walled strain exhibited significant starch accumulation under nitrogen depletion when cells were grown in autotrophic conditions with a 2% CO₂ supply [15]. This phenotype was proposed to be linked to increased photosynthetic efficiency of photosystem II due to an elevated capacity for electron dissipation [15]. Similarly, in the unicellular microalga Euglena gracilis, knockdown of DYRKP homologs markedly delayed cell growth but led to substantial accumulation of wax esters under anaerobic conditions [16]. Under aerobic conditions, increased levels of paramylon (a storage polysaccharide) further supported the association between DYRKP homologs and intracellular carbon metabolism, particularly starch metabolism [16]. Phosphoproteomic analysis in C. reinhardtii revealed a 2.1-fold increase in phosphorylation at Ser76 of DYRKP1 from 24 to 72 h of nitrogen depletion [17], suggesting that nitrogen-deficient stress induces phosphorylation of DYRKP1, potentially leading to rapid shifts in carbon partitioning. These results collectively indicate that repression of DYRKP1 could be an effective strategy to enhance starch accumulation in microalgae.

Based on this, we hypothesized that a double knockout of DYRKP1 and AGP could maximize lipid accumulation by diverting excess starch, induced by DYRKP1 knockout, into lipid biosynthesis. However, DYRKP1 deficiency impairs degradation of cell wall glycoproteins, forming multicellular structures [18] that hinder accurate normalization of starch and lipid contents by cell number. To overcome this, we investigated nitrogen-deplete responses under the culture conditions tested in the previous work (autotrophic and mixotrophic conditions) using a dyrkp1 mutant generated in a cell-wall-less C. reinhardtii background, to assess their potential for improved lipid accumulation.

Methods and materials

Microalgae strains and culture conditions

Three independent dyrkp1 mutants (dyrkp1-1, dyrkp1-2, and dyrkp1-3), generated from Chlamydomonas reinhardtii CC4349 (cw15, mt⁺), were previously constructed and genotyped in our previous study [18]. In addition, the dyrkp1 mutant obtained from the cell-walled strain 137AH was kindly provided by Gilles Peltier and Yonghua Li-Beisson [15]. Cells were cultured under mixotrophic or autotrophic conditions using Tris-acetate phosphate (TAP) medium or MOPS-buffered minimal medium (MM-MOPS), respectively [19]. Cells were grown in 100 mL flasks containing 20 mL of medium, maintained at 25 ± 1 °C with continuous shaking (120 rpm) under constant light irradiation (80 ± 10 µmol photons m^− 2 s^− 1).

Generation of double knockout mutants

To generate the double knockout (dKO) mutant of DYRKP1 and AGP, the AGP small subunit gene (Cre03.g188250) was disrupted in the dyrkp1-1 mutant background using a CRISPR-Cas9 ribonucleoprotein (RNP) complex, following the protocol previously described [20]. The sgRNA target was designed using Cas-Designer (http://www.rgenome.net/cas-designer) and selected in the range of 5’UTR and 1st exon (5′-TAGCATGGCCCTGAAGA.

TGCGGG-3′). To help with mutant screening, we combined the insertion and expression of the paromomycin resistance (ParR) gene cassette. A total of 100 µg of purified Cas9 protein (Cas9 expression plasmid: pET-NLS-Cas9-6xHis (Plasmid #62934)) and 70 µg of sgRNA synthesized using the GeneArt™ Precision gRNA Synthesis Kit (ThermoFisher, MA, USA) were mixed to form the RNP complex and co-transformed with the ParR gene cassette. After transformation, cells were plated on a TAP medium containing 1.5% agar and 25 µg/mL of paromomycin (Sigma-Aldrich, MO, United States). To screen dKO mutants, colonies grown onto the selection plate were confirmed by genomic PCR using the target-specific primer sets (forward: 5′-TGGGCACGACTTGCATTGTGT-3,’ reverse: 5′- AATGGGCCAGCGCGAG.

CATA-3′). The PCR products were sequenced using Sanger sequencing (Macrogen Inc., Seoul, South Korea).

Growth analysis

All the cell lines were adapted to autotrophic (MM-MOPS, 2% CO₂) or mixotrophic (TAP, air) conditions for one week before starting the growth measurement. The cell was inoculated to make an initial cell number of 0.2 million cells. The cells were manually counted using Neubauer Counting chambers (Marienfeld, Lauda-Königshofen, Germany) on the light microscope (ECLIPSE Ni; Nikon, Tokyo, Japan) during the experiment.

Starch analysis

For starch extraction, 1 mL of culture was collected in a 2 mL screw cap tube and centrifuged at 15,000×g for 2 min. The pellet was completely dried after pigment extraction using 1 mL of methanol. The sample tube was filled with 0.4 mL of distilled water and autoclaved for 15 min at 120 °C to solubilize starch. Then, starch was digested to glucose by 0.2 mL of amyloglucosidase solution (Starch Assay Reagent; Sigma-Aldrich), and glucose concentration (µg mL^− 1) was assayed using an automated sugar analyzer (model 2700 select; YSI, Yellow Springs, OH, USA). The measured glucose concentration was normalized by cell number (10⁶ cells mL^− 1) of particle volume (mm³ mL^− 1) in each sample.

Chlorophyll fluorescence analysis

Chlorophyll fluorescence was measured using a Dual Pam-100 (Heinz Walz, Germany). Cells were placed into a cuvette under constant stirring at room temperature and were dark-adapted for 15 min before measurements. Light curves were recorded by increasing stepwise (3 min per step) the light intensity (0, 25, 100, 200, 400, 600, and 950 µmol photons m^− 2 s^− 1). Saturating flashes (10,000 µmol photons m^− 2 s^− 1, 200 ms duration) were applied to determine PSII yield [21].

Lipid analysis

For lipid extraction and esterification, 20 million cells were harvested in a 2 mL screw cap tube by centrifugation (15,000×g, 5 min, 20 °C). The sample tube was filled with 0.9 mL of acetyl chloride: methanol solution (5:100 (v/v)), 0.1 mL of methyl heptadecanoate (C17:0, internal standard; 3 mg mL^− 1 dissolved in hexane), and glass micro-beads (Sigma-Aldrich, USA). Then, the sample tube was vortexed vigorously for 2 min, and incubated for 1 h at 80 °C with gentle mixing (200 rpm). After chilling for 5 min on the ice, 1 mL of n-hexane was added, and the separated supernatant was transferred to a GC glass vial.

Total fatty acid methyl esters (FAMEs) were analyzed using a gas chromatograph GC2400 (PerkinElmer, MA, USA). Instrumental parameters for GC-FID analysis of FAMEs were set following the protocol described in the reference paper [22]. FAME species were identified based on the peak retention times of the reference standard (Supelco 37-component FAME mix; Sigma-Aldrich, USA) and were quantified based on the peak area compared to the internal standard.

Reactive oxygen species analysis

Intracellular reactive oxygen species (ROS) levels in C. reinhardtii were measured using the DCFH-DA (2′,7′-dichlorofluorescin diacetate) fluorescence assay [19]. To exclude interference from extracellular substances, approximately 2 mL of algal culture was harvested by centrifugation (15,000×g, 5 min, 20 °C), and the cell pellet was resuspended in fresh TAP medium containing 5 µM DCFH-DA (Invitrogen, MA, USA). The cells were incubated in the dark at 25 °C for 1 h. Following incubation, the fluorescence intensity of DCFH-DA was measured using the Guava HT Flow Cytometer (Millipore, MA, USA). For analysis, approximately 200,000 cells were acquired per sample. A fluorescence window was established by comparing it with a negative control to isolate the DCFH-DA-positive population, and the mean fluorescence intensity was determined. The excitation and emission wavelengths for DCFH-DA detection were 488 nm and 525/30 nm, respectively.

Transcriptome and proteome analysis

Data related to ROS scavenging were collected from RNA-seq and pellet proteome data of the cell-walled strains: the 137AH strain and the dyrkp1 mutant (137AH background). The transcriptome data (BioProject: PRJNA1042620) and pellet proteome data (PRIDE: PXD047255) used in this study were produced using the cells cultured under autotrophic conditions with a 2% CO₂ supply, and the analysis parameters of the data were described in our previous study [18].

Statistical analysis

All the experiments were performed with at least three biological replicates. Data were statistically analyzed using the Student’s t-test, and are presented as mean ± standard deviation (SD). Significant differences with a significance level of 95% (p < 0.05) are clearly indicated in the figure legends for each figure.

Results

DYRKP1 deficiency promotes starch accumulation under mixotrophic conditions

To enable normalization by cell number, we selected three independent dyrkp1 mutants (dyrkp1-1, dyrkp1-2, and dyrkp1-3) generated from the cell-wall-less C. reinhardtii background, CC4349 (parental strain). These correspond to mutants previously designated as dyrkp1-7, dyrkp1-8, and dyrkp1-9 [18]. In the previous study, the dyrkp1 mutant generated from the cell-walled background (137AH strain), exhibited elevated starch accumulation and enhanced photosynthetic performance following nitrogen depletion [15]. These phenotypes may vary depending on the presence or absence of the cell wall. To determine whether DYRKP1 plays a role in starch accumulation regardless of the presence of the cell wall, we examined starch accumulation and the effective quantum yield of Photosystem II under autotrophic conditions with 2% CO₂ and mixotrophic conditions supplemented with acetate.

Cells were initially cultured in a nitrogen sufficient medium for two days and subsequently exposed to nitrogen depletion (Fig. 1A). Under autotrophic conditions, the starch content of dyrkp1 mutants was significantly lower than that of their parental strain after nitrogen depletion (Fig. 1B). Meanwhile, in mixotrophic conditions, the starch content of the dyrkp1 mutants was significantly higher than those of the parental strain before and after nitrogen depletion, respectively (Fig. 1B). Nevertheless, the photosynthetic parameter of photosystem II (Y(II)) did not differ significantly before and after nitrogen depletion (Fig. 1C).

Fig. 1.

Starch accumulation and photosystem II efficiency under different nutritional conditions. A, Schematic diagram of the experimental design under autotrophic and mixotrophic conditions. Cells were cultured autotrophically in MM-MOPS medium supplied with 2% CO₂, or mixotrophically in TAP medium supplied with ambient CO₂. Two days after inoculation, cells were harvested and resuspended in nitrogen-free medium for inducing nitrogen depletion (ND); B, Starch content under autotrophic and mixotrophic conditions with and without nitrogen. Starch content was normalized to cell number; C, Effective quantum yield of photosystem II under autotrophic and mixotrophic conditions with and without nitrogen. The experiments were performed in biological replicates (n = 6). Statistical analysis was performed using Student’s t-test (^* p < 0.05, ^** p < 0.005, and ^*** p < 0.0005). The ‘ns’ indicates no significant difference

dyrkp1 mutants exhibit high TFA content under mixotrophic conditions

To assess lipid accumulation, we conducted FAME analysis under mixotrophic conditions. All dyrkp1 mutants showed elevated total fatty acid (TFA) content even before the onset of nitrogen depletion (Fig. 2A), and further increased TFA content (25–40% higher than CC4349) was observed after nitrogen depletion (Fig. 2B). Before nitrogen depletion, the fatty acid composition was largely comparable across strains (Fig. 2C); however, after nitrogen depletion, several fatty acid species, particularly C16:0 and C18:2 cis (n6), showed significantly higher accumulation in the dyrkp1 mutants relative to the CC4349 strain (Fig. 2D). Despite these increases, the overall fatty acid composition did not differ markedly between CC4349 strain and the dyrkp1 mutants across the nitrogen depletion (Fig. S1).

Fig. 2.

FAME analysis of DYRKP1 single knockout mutants under mixotrophic conditions with and without nitrogen. A, Total fatty acid (TFA) contents before nitrogen depletion (ND 0d; nitrogen-sufficient condition); B, TFA contents after 3 days of nitrogen depletion (ND 3d); C, Quantification of fatty acid species in the cells before nitrogen depletion; D, Quantification of fatty acid species in the cells after nitrogen depletion. The experiments were performed in biological replicates (n = 6). Statistical analysis was performed using Student’s t-test (^* p < 0.05, ^** p < 0.005, and ^*** p < 0.0005). The ‘ns’ indicates no significant difference

Mutant generation of DYRKP1-AGP double knockout

Given that the dyrkp1 mutants exhibit enhanced starch and lipid accumulation, we hypothesized that the combined knockout of DYRKP1 and AGP might further improve lipid accumulation by redirecting carbon flux from starch to the lipid pathway. To test this, we knocked out the AGP small subunit gene in the dyrkp1-1 mutant background, which exhibited the highest starch accumulation under mixotrophic conditions. Transformation was performed using an RNP complex consisting of sgRNA and Cas9 protein targeting the first exon of AGP (Fig. 3A). Colonies were selected on the TAP agar plate containing paromomycin, and successful insertions of the resistance cassette were identified through colony PCR. Three AGP-knockout mutants were confirmed by gDNA PCR (Fig. 3B), and Sanger sequencing (Fig. 3C), and designated as dKO #1, dKO #2, and dKO #3.

Fig. 3.

Genotyping of DYRKP1-AGP double knockout mutants. A, Schematic diagram of AGP small subunit gene (Cre03.g188250). The underlined sequence is an sgRNA target region. The ATG highlighted in bold indicates a start codon, and the GGG highlighted in bold indicates PAM sequence; B, The genomic DNA PCR targeted to AGP small subunit gene; C, Sanger sequencing analysis. Three dKO mutants indicated different insertion and deletion patterns. ParR indicates a paromomycin resistance gene cassette. Light gray and a dashed line indicate the gene deletion

Under mixotrophic conditions, the CC4349 strain and the agp mutant exhibited similar growth. In contrast, both the dyrkp1-1 mutant and dKO mutants showed significantly reduced growth (Fig. 4A). To assess starch accumulation, cell cultures were stained with Lugol’s iodine solution after nitrogen depletion. The cell cultures of the CC4349 strain and the dyrkp1-1 mutant were stained, but the agp mutant and dKO mutants were not stained (Fig. 4B). This indicates that starch accumulates in the CC4349 strain and the dyrkp1-1 mutant, but not in the agp mutant and dKO mutants, which is consistent with the quantitative analysis results of starch (Fig. 4C).

Fig. 4.

Growth and starch accumulation of DYRKP1 or AGP single knockout mutants, and DYRKP1-AGP double knockout mutants. A, Growth patterns under mixotrophic conditions; B, Starch staining of the cell cultures using a Lugol’s solution. Cell cultures were exposed to nitrogen-deplete conditions for three days; C, Starch content under mixotrophic conditions before nitrogen depletion (ND 0d; nitrogen-sufficient condition) and three days after nitrogen depletion (ND 3d). Starch content was normalized to cell number. The experiments were performed in biological replicates (n = 3). Statistical analysis was performed using Student’s t-test (^*** p < 0.0005). The ‘ns’ indicates no significant difference

Double knockout of DYRKP1 and AGP exhibits higher TFA content than the parental strain under mixotrophic conditions

We then compared the lipid contents of the double knockout mutants with those of the single knockout mutants under mixotrophic conditions, where the dyrkp1 mutants showed higher starch contents than their parental strain (Fig. 1). In nitrogen-replete conditions, the TFA content in dKO mutants was more than 3-fold higher than that of the dyrkp1 mutant and similar to that of the agp mutant (Fig. 5A). This trend remained the same after nitrogen depletion, and the dKO mutants even showed a 1.2-fold higher TFA content than the agp mutant; only the dKO #1 mutant showed significantly higher TFA contents than the agp mutant (Fig. 5B). The FAME species analysis of the agp and dKO mutants before nitrogen depletion showed that both of them had higher contents of C16:0 and C18:0, C18:1 cis(n9), C18:2 cis(n6), and C18:3n3 than CC4349 strain (Fig. 5C). The agp and dKO mutants showed high proportions of C16:0 and C18:0 in the fatty acid composition, whereas the proportions of C18:2 cis (n6), C18:3n6, and C18:3n3 were reduced (Fig. S2). Under nitrogen-replete conditions, the agp and dKO mutants had similar proportions of their overall fatty acid composition, but this similarity was not confirmed under nitrogen-deplete conditions (Fig. S2). The dKO #1 and dKO #2 mutants had significantly higher C16:0 than the agp mutant, while dKO #3 had significantly lower C16:0. And C18:1 trans (n9) was higher in all dKO mutants than in the agp mutant, while C18:2 cis (n6) and C18:3n3 were lower in all dKO mutants than in the agp mutant. Consistent with this trend, in the per cell quantitative results of FAME species, C16:0 and C18:0, C18:1 trans (n9) of dKO #1 and dKO #2 were significantly higher than those of the agp mutant (Fig. 5D).

Fig. 5.

FAME analysis of DYRKP1 or AGP single knockout mutants, and DYRKP1-AGP double knockout mutants under mixotrophic conditions with and without nitrogen. A, Total fatty acid (TFA) contents before nitrogen depletion (ND 0d; nitrogen-sufficient condition); B, TFA contents after 3 days of nitrogen depletion (ND 3d); C, Quantification of fatty acid species in the cells in nitrogen-replete condition; D, Quantification of fatty acid species in the cells after nitrogen depletion. The experiments were performed in biological replicates (n = 6). Statistical analysis was performed using Student’s t-test (^* p < 0.05, ^** p < 0.005, and ^*** p < 0.0005). The ‘ns’ indicates no significant difference

Discussion

In this study, we analyzed the physiological characteristics and metabolic changes associated with DYRKP1 deficiency in the cell-wall-less strain, after minimizing the physical effects of palmelloid structures that were previously observed in the dyrkp1 mutants [15, 18]. The dyrkp1 knockout mutants accumulated more lipid under mixotrophic conditions, consistent with previous observations linking DYRKP1 deficiency to altered carbon partitioning [15]. To further enhance lipid accumulation, we additionally knocked out AGP, a key enzyme involved in starch biosynthesis [23]. Under nitrogen-depletion, the DYRKP1-AGP double knockout (dKO) mutants exhibited 2.4-fold and 1.2-fold higher TFA content compared to their parental strain (CC4349) and the agp mutant, respectively. However, considering the dramatic starch accumulation in the dyrkp1 mutant observed in a previous study [15], the consequences of reallocation of carbon flux toward lipid metabolism through the double knockout of DYRKP1 and AGP were less dramatic. These results underscore the complexity of metabolic regulation, where upstream genetic modifications do not always have synergetic effects due to the multilayered controls in carbon flux and feedback-regulated networks [3, 24].

The absence of dramatic increases in lipid accumulation may be related to the unexpectedly low increase in starch accumulation due to DYRKP1 knockout. In the case of the cell-walled strain 137AH, the dyrkp1 mutant accumulated 1.8–2.5 times more starch after nitrogen depletion under autotrophic conditions and approximately 4 times more starch even before nitrogen depletion (Fig. S3A). In contrast, the dyrkp1 mutants generated from the cell-wall-less strain (CC4349) showed lower starch content per cell than the parental strain under the same conditions (Fig. 1B), and no significant difference in starch content per particle volume (Fig. S3A). While these results cannot be directly compared due to impaired cell wall degradation in 137AH [18], the undigested parental cell wall may have affected intracellular metabolism.

ROS-related measurements further support these speculations. DCFH-DA fluorescence analysis revealed that the 137AH strain exhibited stronger ROS signals than the CC4349 strain, with the dyrkp1 mutant of the 137AH strain showing a markedly higher signal than its parental line. In contrast, the dyrkp1 mutants from the CC4349 strain did not differ significantly from their parent strain (Fig. S3B). Although the loss of intracellular ROS during medium replacement in the cell-wall-less background cannot be excluded, the high ROS levels in the dyrkp1 mutant of the 137AH strain suggest that DYRKP1 deficiency may cause oxidative stress. Indeed, transcriptome analysis detected increased expression of ROS scavenging enzymes (glutathione peroxidase, ascorbate peroxidase, and Mn superoxide dismutase) in the dyrkp1 mutant compared to the 137AH strain, as well as an increase in protein content (catalase and Mn superoxide dismutase) (Figures S3C and S3D). Given that cells have been reported to increase the expression of ROS-scavenging enzymes in response to increased ROS [25–28], the dyrkp1 mutant (137AH background) appears to have already been under high levels of ROS stress. ROS is known to induce starch and lipid accumulation in cells [29, 30], which explains the relationship between high ROS levels and starch accumulation in the dyrkp1 mutant (137AH background). Another possible explanation is that daughter cells trapped within an undigested parental cell wall in 137AH experience spatial restriction, impairing nutrient uptake and carbon utilization. Consequently, excess carbon may be stored as starch. Therefore, the starch accumulation observed in the dyrkp1 mutant (137AH background) could be attributed to oxidative stress or unutilized carbon surplus, although further research is needed to confirm the precise mechanisms.

Knockout of DYRKP1 and AGP did not significantly increase lipid production compared to a single AGP knockout, a limitation often reported in algal biotechnology. Comprehensive metabolic engineering strategies, along with manipulation of nutrient signaling, can be a promising solution. For example, in the oleaginous yeast Yarrowia lipolytica, coordinated rewiring of central carbon metabolism, enhancement of acetyl-CoA supply, and disruption of competing pathways have led to lipid contents exceeding 60% of dry cell weight [31–33]. Similarly, in microalgae such as C. reinhardtii, Nannochloropsis species, and Phaeodactylum tricornutum, overexpression of key enzymes in lipid biosynthesis combined with manipulation of nutrient signals has significantly enhanced triacylglycerol production [1, 34–36]. Even in cyanobacteria like Synechocystis species, similar approaches have enabled redirection of carbon flux toward fatty acid synthesis [37]. These successes highlight that substantial lipid enhancement often requires a systems-level strategy involving multi-gene regulation, pathway balancing, and environmental tuning. Therefore, to achieve higher lipid yields, future work should focus on integrated approaches that combine transcriptomic, proteomics, metabolomic, and carbon flux data to identify key nodes of regulation. Additionally, implementing CRISPR-based multiplex editing or inducible gene circuits targeting upstream regulators such as DYRKP1 could offer more precise and tunable control over carbon partitioning.

Author contributions

M.K. designed and moderated this study. M.K., J.Y.K., and K.H.H. performed the experiments. M.K. analyzed and interpreted the results. M.K. wrote a drafted the manuscript. M.K., H.H.S., and E.J. revised a draft version of the manuscript. All authors reviewed the final version of the manuscript.

Funding

M.K.: National Research Foundation (NRF) funded by the Korean government (MSIT) (No. RS-2024-00458958). E.J.: National Research Foundation (NRF) funded by the Korean government (MSIT) (No. RS-2024-00458958).

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

This declaration is not applicable.

Competing interests

The authors declare no competing interests.