The WT of Porphyridium cruentum LIMS-PS-1061 was obtained from the Library of Marine Samples of Korea. Cells were axenically maintained in an artificial seawater (ASW), according to the previous conditions (Jeon et al., 2021).

EMS (Sigma-Aldrich Co., MO, USA) was employed as a chemical mutagen to initiate random mutagenesis. Cells at the exponential stage were harvested and washed twice with distilled water (pH = 4) to eliminate excess exopolysaccharides (EPS) around the cells. Cells were resuspended in ASW supplemented with 0.5% (v/v) EMS, followed by continuous agitation for 1 h at 25°C in the dark. Then, cells were washed twice with ASW to remove excess EMS. To avert potential light-induced DNA repair, cells were incubated overnight under the same dark conditions as above. Subsequently, mutagenized cultures were spread onto 1% agar-ASW plates supplemented with 2 µg mL⁻¹ zeocin and the plates were further incubated at 25°C under constant illumination (approximately 60 μmol photons m⁻² s⁻¹). Zeocin served as the selection marker. After 15 d, surviving colonies were separated.

Cells at the exponential stage were inoculated at a concentration of 0.2 g L⁻¹. Then, cells were cultivated in ASW at 25°C under constant illumination (approximately 60 μmol photons m⁻² s⁻¹) on a rotary shaker at 120 rpm (Han et al., 2020). As a light source, white (400–700 nm) and blue (420–450 nm) light-emitting diodes (LEDs) were used (Han et al., 2021).

Each experiment was conducted with at least three independent specimens. One-way analysis of variance (ANOVA) with Tukey’s tests was performed for statistical analysis using SAS 9.4 software. The p-value of< 0.05 was considered to be statistically significant. The data are presented as mean values ± standard error (SE).

Ethyl methanesulfonate (EMS)-induced mutagenesis and zeocin selection were employed for the development and screening of P. cruentum mutants. EMS-induced mutagenesis is a simple and cost-effective method to generate extensive mutations. However, since EMS generates random mutations, considerable time and effort are required to isolate only specific mutants in which DNA is extensively altered. Zeocin is an antibiotic to which numerous species of microalgae present sensitivity (Osorio et al., 2019). Also, zeocin can act as a mutagen that induces mutations in microalgae (Lin et al., 2017). Therefore, zeocin was used as a subsequent mutagen and selection marker. The mutant was selected based on colony size on zeocin-containing agar medium and subsequent experiments were performed.

Differences in growth parameters between the WT and the mutant were compared under white light and blue light conditions. Under white light conditions, the cell dry weight of the mutant (0.854 g L⁻¹) increased by 33.9% compared to the WT (0.638 g L⁻¹) ( Figure 1A ). Under blue light conditions, however, there was no significant difference between the cell dry weights of the mutant (0.736 g L⁻¹) and the WT (0.756 g L⁻¹). Additionally, there was no significant difference in the yield of exopolysaccharides (EPS) between the WT and the mutant under both conditions ( Figure 1B ). Interestingly, the mutant exhibited prominent cell growth under white light conditions, while the WT exhibited prominent cell growth under blue light conditions. Various factors such as light source, temperature, pH, salinity, and the availability of nutrients influence the cellular growth of microalgae (Lee et al., 2015; Srinuanpan et al., 2018). Among these, the light source is considered the most critical factor influencing cell growth. Photosynthesis is a fundamental process through which microalgae produce energy, and this process is heavily reliant on light. The intensity, quality, and photoperiod of the light source can profoundly impact the photosynthetic efficiency in microalgae, with the energy generated being vital for cell growth and reproduction (Kwan et al., 2021). In addition, microalgae collect light through antenna pigments and convert it into energy. Microalgae possess a diversity of pigments, which are categorized into primary and accessory pigments, each absorbing light within a specific wavelength range and exerting a significant influence on cell growth (Sirisuk et al., 2018). Moreover, light is not just instrumental in energy production but also plays a critical role in the physiological regulation of microalgae. For instance, variations in light intensity and duration can have direct effects on the cell cycle as well as various metabolic pathways (Jaubert et al., 2017). In this experiment, all other environmental factors were kept constant except for the light source. Therefore, based on the observed cell growth results, it can be inferred that alterations in pigment occurred in the mutant.

Fascinating changes in pigment were observed between the WT and mutant. There was no statistically significant difference in the total amount of major pigments (chlorophyll and phycobiliproteins, referred to as ‘PBs’) between the WT and mutant under white light conditions ( Figure 2A ). Under white light, the total amounts of major pigments in the WT and mutant were 7.80 mg g⁻¹ and 8.06 mg g⁻¹, respectively. Under blue light conditions, the total amount of major pigments in the WT and mutant significantly increased to 12.7 mg g⁻¹ and 11.4 mg g⁻¹, respectively, but there was no statistical difference between them. However, despite the total amount of pigment being similar, the composition of pigments exhibited a dramatic difference. Under white light, the chlorophyll content in the mutant was 1.74 mg g⁻¹, which was 2.20 times lower than that of the WT (3.83 mg g⁻¹) ( Figure 2A ). In contrast, the phycobiliproteins (PBs) content in the mutant was 6.33 mg g⁻¹, which was 1.59 times higher than that of the WT (3.97 mg g⁻¹). Similarly, under blue light, the chlorophyll content in the mutant was 1.25 mg g⁻¹, which was 3.61 times lower than that of the WT (4.50 mg g⁻¹), while the PBs content was 10.1 mg g⁻¹, which was 1.23 times higher than that of the WT (8.22 mg g⁻¹). Based on these remarkable differences in pigment content, the mutant was designated as a phycobiliprotein production-enhancing mutant (PPE).

To investigate the pigment composition profiles in greater detail, the contents of chlorophyll, phycoerythrin (PE), phycocyanin (PC), and allophycocyanin (APC) in the WT and PPE were analyzed ( Figures 2A, B ). The significant increase in PB content in PPE was primarily attributed to increases in APC and PC. Under white light, the contents of APC and PC in PPE increased by 2.23- and 1.63-fold, respectively, compared to the WT, while PE increased only by 1.07-fold. The weight percentages of PBs in the total amount of major pigment in WT and PPE were 50.9% and 78.5%, respectively. Similarly, under blue light, the contents of APC, PC, and PE in PPE increased by 1.28-, 1.20-, and 1.18-fold, respectively, compared to the WT. The weight percentages of PBs in the total amount of major pigment in WT and PPE were 64.6% and 89.0%, respectively. These alterations in pigment composition between WT and PPE are presumed to be a consequence of chlorophyll deficiency. In P. cruentum, chlorophyll and PBs have a complementary relationship. It is known that a deficiency in chlorophyll promotes the production of PBs as a compensatory mechanism to maintain the photosynthetic capacity of cells (Jeon et al., 2021). In addition, the increase in PBs observed in both WT and PPE under blue light is inferred as a defense mechanism against stress induced by blue light. Blue light has a short wavelength (typically 380–500 nm) and has higher energy photons than are required for photosynthesis (Schulze et al., 2014). Excess photon energy can cause photooxidative damage to cells due to the generation of reactive oxygen species (ROS) (Coward et al., 2016). In P. cruentum, PBs play a role in scavenging ROS (Peña-Medina et al., 2023). It has been demonstrated that PE acts through the primary pathway by directly sequestering ROS and possesses high reducing power. In contrast, PC and APC function through both primary and secondary pathways, notably serving as chelators of metal ions that are involved in ROS synthesis (Sonani et al., 2015). Indeed, it has been reported that PB production is enhanced for stress reduction in Porphyridium sp. under blue light (Coward et al., 2016; Huang et al., 2021).

These results support our hypothesis that the difference in cell growth between WT and PPE was due to variations in pigment composition. In P. cruentum, chlorophyll absorbs light at 400–500 nm and 650–720 nm (Dagnino-Leone et al., 2022), while PE absorbs at 490–570 nm (Sepúlveda-Ugarte et al., 2011), PC at 550–650 nm (Ma et al., 2008), and APC at 600–680 nm (Dagnino-Leone et al., 2020). The wavelengths of the white and blue LEDs used in the experiment were 400–700 nm and 420–450 nm, respectively. In terms of photosynthetic pigments, PBs have the capacity to absorb light across a broad spectrum (490–680 nm), allowing them to support photosynthesis by absorbing light in ranges where chlorophyll absorption is less efficient (500–650 nm) (Watanabe and Ikeuchi, 2013). Furthermore, in P. cruentum, light energy is sequentially transmitted along the pathways of PE, PC, APC, and chlorophyll a, with electron flow correlating with the quantity of PBs (Yokono et al., 2011). Consequently, cell growth in PPE was more pronounced than that of WT under white light conditions. On the other hand, interestingly, there was no significant difference in cell growth between WT and PPE under blue light, despite chlorophyll being more adept at absorbing blue light compared to PBs. As previously mentioned, it is inferred that this is due to the high energy levels of blue light and the stress-reducing effects of PBs. Blue light is absorbed by chlorophyll and utilized for cellular energy metabolism through photosynthesis, but it also acts as a stress-inducing factor that can inhibit cell growth. Under blue light, WT possesses a higher light energy conversion efficiency than PPE, owing to its higher chlorophyll content. Conversely, PPE exhibits higher stress resistance due to its higher PB content compared to WT. In other words, under blue light, WT has high light absorption efficiency but low stress tolerance, whereas PPE has high stress tolerance but low light absorption efficiency. Consequently, no significant difference in cell growth was observed between PPE and WT under blue light conditions. These results imply that the relationship between pigment and cell growth in microalgae involves the collective action of several pigments rather than the isolated function of individual pigments, and both light absorption efficiency and light inhibition should be taken into account.

A subsequent analysis was conducted on the EPS produced from PPE to ascertain its industrial potential. The EPS originated from P. cruentum is primarily composed of sulfated polysaccharides (SPs) and exhibits biological activities such as antioxidant, anti-inflammatory, anti-cancer, and UV protective properties, making it a valuable biological material in the cosmetic and medical industries (Li et al., 2019). Also, the biological activity of EPS is generally positively correlated with its sulfur content (Liberman et al., 2020). Elemental analysis revealed no significant differences in the weight percentages of carbon (C), hydrogen (H), and sulfur (S) between the EPS produced from WT and PPE ( Figure 3 ). The weight percentages of C, H, and S in the EPS derived from WT were 0.243 ± 0.028%, 0.848 ± 0.047%, and 14.3 ± 0.7%, respectively. The weight percentages of C, H, and S in the EPS derived from PPE were 0.485 ± 0.100%, 0.817 ± 0.003%, and 12.6 ± 0.4%, respectively. On the other hand, nitrogen (N) was not detected in both EPS, indicating that cell debris (especially lipids and proteins) and nucleic acids were absent from the extracted EPS. Despite the slight difference in weight percentage, an analysis of variance (ANOVA) revealed that these differences in the elemental compositions of both EPS were not statistically significant. These results suggest that the EPS produced from PPE likely has biological activity comparable to that of EPS from WT. Thus, the potential for skin improvement of EPS produced from PPE was evaluated in human skin cells.

Since AQP3, FLG, IVL, and LOR are all expressed in keratinocytes and play roles in water transport, hydration, and skin barrier formation, changes in the expression of these genes were investigated in epidermal cells (HaCaT). In contrast, ELN and FBN, which are fibrous proteins involved in maintaining skin elasticity, are produced in dermal fibroblasts; therefore, changes in the expression of these genes were evaluated in dermal cells (Hs68). The results of the real-time RT-PCR analysis showed that EPS could potentially be used for skin improvement ( Figure 4 ). Expression levels of aquaporin 3 (AQP3) in the EPS-treated groups were 1.66- and 2.80-fold higher at concentrations of 4 ppm and 40 ppm respectively, compared to the control ( Figure 4A ). In addition, expression levels of filaggrin (FLG) in the EPS-treated groups were 1.72- and 3.23-fold higher at concentrations of 4 ppm and 40 ppm respectively, compared to the control ( Figure 4B ). Retinoic acid, used as a positive control, exhibited AQP3 and FLG expression levels that were 2.30- and 1.72-fold higher, respectively, compared to the control. AQP3 and FLG are two pivotal proteins that play crucial roles in skin moisturization. AQP3, a water channel protein located in the cell membranes of skin cells, facilitates the transport of moisture, helping to maintain the moisture content of the skin (Bollag et al., 2020). Also, AQP3 has a glycerol transport capability, so it plays an important role in strengthening the moisturizing and barrier functions of the skin. FLG, which is present in the epithelial layer of the skin, enhances the adhesion between skin cells, minimizing water loss and protecting the skin against external stimuli (Kim et al., 2012). Also, FLG is involved in the production of natural moisturizing factors within the skin. Thus, an increase in the gene expression levels of AQP3 and FLG suggests an enhancement of skin moisturization.

In addition to FLG, the expression levels of involucrin (IVL) and loricrin (LOR), which are related to skin barrier enhancement, were also analyzed. The expression levels of IVL were 1.12- and 2.60-fold higher in the EPS-treated groups at concentrations of 4 ppm and 40 ppm, respectively, compared to the control ( Figure 4C ). In addition, the expression levels of LOR were 1.86- and 3.60-fold higher in the EPS-treated groups at concentrations of 4 ppm and 40 ppm, respectively, compared to the control ( Figure 4D ). AdipoRon was used as a positive control and exhibited 1.30-fold higher IVL and 2.35-fold higher LOR expression levels than the control. IVL is a protein present in the epithelial cells of the skin and is one of the principal components involved in forming the keratinized epidermis, which serves as a protective barrier in the skin (Furue et al., 2015). IVL functions as a scaffold for the formation of the keratinized epidermis by cross-linking with other proteins through the catalytic action of an enzyme known as transglutaminase. LOR is another essential protein of the keratinized epidermis, which constitutes a significant part of the keratinized epidermis and is involved in the aggregation of keratin fibers (Furue et al., 2015). This aggregation and subsequent cross-linking result in a densely packed protective layer that is highly resistant to mechanical stress, thereby contributing to skin barrier enhancement. Thus, an increase in the gene expression levels of IVL and LOR is intimately associated with the strengthening of the skin barrier.

Subsequently, the expression levels of elastin (ELN) and fibrillin-1 (FBN1), which are associated with the improvement of skin elasticity, were analyzed. When treated with 40 ppm of EPS, the expression levels of ELN and FBN1 were 1.45- and 1.47-fold higher, respectively, compared to the control ( Figures 4E, F ). No statistical significance was observed when both genes were treated with 4 ppm of EPS. These results suggest that an improvement in skin elasticity can be expected when treated with EPS of a certain concentration or higher. Epigallocatechin gallate (EGCG) was used as a positive control and exhibited 1.91-fold and 1.63-fold higher expression levels of ELN and FBN1, respectively, compared to the control. ELN encodes elastin, which is a key protein in the skin that provides elasticity. Elastin allows the skin to stretch and then return to its original shape (Romana-Souza et al., 2020). As elastin levels decrease with age, the skin loses its ability to regain its shape, leading to sagging and wrinkles. In addition, FBN1 encodes fibrillin-1, a component of microfibrils that plays a crucial role in maintaining skin elasticity and structure (Romana-Souza et al., 2020). Moreover, fibrillin-1 interacts with elastin, aiding in the support of the skin’s microstructure, and helps the skin withstand physical stress, such as stretching. Thus, an increase in the expression levels of the ELN and FBN1 genes can contribute to improved skin elasticity and structural reinforcement, which can help slow down the aging of the skin and reduce wrinkles.

Finally, a scratch assay was conducted on Hs68 cells to test the ability of EPS to promote cell migration, which is related to wound healing ( Figure 5 ). In the absence of any treatment, the cells spontaneously migrated to induce re-epithelialization. Interestingly, when the cells were treated with EPS at a concentration of 40 ppm, a significant enhancement in wound closure was observed ( Figure 5A ). Indeed, the wound recovery rate after 16 hours was 54.7% with EPS treatment, which was significantly higher than that of EGCG (46.3%) used as a positive control, or untreated cells (control; 40.3%) ( Figure 5B ). Moreover, it was also confirmed that EPS has the potential to enhance the skin absorption rate of other skin active ingredients ( Supplementary Figure S1 ). These results demonstrate the potential of EPS derived from PPE for skin improvement and suggest that it can be applied in the cosmetic or medical industry.

Integrating the aforementioned results, we estimated the mechanism through which EPS derived from P. cruentum improves the condition of human skin cells ( Figure 6 ). The EPS was found to enhance skin moisturization, elasticity, barrier enhancement, and wound healing through the upregulation of six genes.

Aquaporin-3 (AQP3), a membrane transporter of water and glycerol, is expressed in the plasma membrane of basal keratinocytes in normal skin epidermis. This transporter plays a pivotal role in the stratum corneum’s (SC) water retention, elasticity, and biosynthesis. Notably, glycerol transport by AQP3 is more critical than water transport for skin physiology and phenotype alterations (Hara-Chikuma and Verkman, 2005). AQP3 enhances skin moisturization and elasticity by increasing glycerol content, a key determinant of skin hydration. Additionally, AQP3 facilitates re-epithelialization, an essential stage in wound healing, by promoting keratinocyte migration and proliferation from the surrounding epidermis and appendages such as hair follicles and sweat glands. The water and glycerol transport facilitated by AQP3 enhances keratinocyte migration and proliferation, respectively (Hara-Chikuma and Verkman, 2008). Increased glycerol contents due to AQP3 not only aid in skin barrier recovery but also contribute to the enhancement of epidermal barrier functions (Hara and Verkman, 2003). In summary, AQP3-facilitated water transport is involved in accelerating wound healing, also its glycerol transport contributes to skin hydration and elasticity, as well as cell proliferation.

Natural moisturizing faction (NMF) is a key factor in maintaining skin moisture, is present in keratinocytes, and contributes to both moisture retention and epidermal barrier functions. NMF, synthesized from filaggrin (FLG) in the SC, typically comprises amino acids (40%), pyrrolidone carboxylic acid (PCA, 12%), lactate (12%), and urea (7%). Its synthesis follows a sequence of processes (Tsukui et al., 2022): FLG is initially produced by the precursor protein profilaggrin (proFLG), which is metabolized to FLG through dephosphorylation and other mechanisms, subsequently binding with keratin in keratinocytes. In the upper SC, proteolytic enzymes degrade FLG from keratin. The arginine in FLG is citrullinated (Cit) by the protein arginine deiminase, and Cit residues are released and then fragmented into amino acids by bleomycin hydrolase. These amino acids are further transformed by degradative enzymes, with Glu and Gln converting into PCA, His into urocanic acid, and Arg into urea (Tang et al., 2016). Additionally, mammalian epidermal cells feature a cornified cell envelope (CE), a 15 nm thick protein layer cross-linked by isodipeptide and disulfide bonds. FLG, along with involucrin (IVL) and loricrin (LOR), forms this complex structure. The human epidermal CE comprises 65–70% LOR, about 10% FLG and cysteine-rich protein (CRP), and 2–5% IVL, small proline-rich proteins (SPRRs), and cystatin A (Tharakan et al., 2010). The formation of CE in vivo is a multistage process, starting with the initial attachment of IVL, SPRR, CRP, and cystatin A to the cell membrane, followed by a heavy deposition of LOR with some FLG. In summary, an increase in FLG expression enhances NMF biosynthesis, thereby strengthening the SC’s capacity for moisture retention. FLG also collaborates with IVL and LOR in forming the CE, further strengthening epidermal barrier functionality and moisture retention.

Elastic fibers are a crucial component of the extracellular matrix, imparting stretchability, resilience, and cellular interaction to a wide range of elastic tissues. Elastin (ELN) constitutes the majority of elastic fibers and is formed by the hierarchical assembly of its monomer, tropoelastin (Ozsvar et al., 2021). Fibrillin microfibrils, extensible polymers present in both elastic and non-elastic tissues, provide long-range elasticity to connective tissue. These microfibrils also act as a scaffold for elastin deposition during elastic fiber synthesis, playing a vital role in maintaining tissue integrity (Ramirez and Sakai, 2010). In elastic fiber assembly, elastin globules are directly deposited onto microfibril templates, forming elastic fiber composed of an amorphous elastin central core surrounded by a fibrillin microfibril sheath (Thomson et al., 2019). An increase in the expression of both ELN and FBN1, therefore, leads to an enhanced biosynthesis of elastic fibers, significantly contributing to the improvement of skin elasticity.

In summary, EPS derived from P. cruentum enhances skin moisturization, elasticity, barrier enhancement, and wound healing. This improvement is mediated through the individual or synergistic effects of six key genes implicated in skin health: (1) AQP3 enhances skin hydration, elasticity, wound healing, and barrier function. (2) FLG contributes to skin hydration. (3) FLG, IVL, and LOR jointly improve skin hydration and barrier function. (4) ELN and FBN1 play a significant role in enhancing skin elasticity.

This research introduces a mutant strain of P. cruentum, developed through EMS-induced mutagenesis, which exhibits superior industrial traits. This study further elucidates the relationship between pigment and cell growth in P. cruentum and the mechanisms by which EPS derived from P. cruentum ameliorates skin conditions. Given its skin care and pharmacological properties, EPS from P. cruentum is poised for varied industrial applications. Consequently, we anticipate that our findings will have broad implications and utility across industries utilizing P. cruentum.