Cyanobacteria, also known as blue-green algae, are the oldest and most ecologically pivotal photoautotrophic prokaryotes on Earth (Afreen et al., 2021). They play a key role in the global carbon/oxygen cycle (Sánchez-Baracaldo et al., 2022) and are harnessed by diverse industries as sustainable feedstock for the production of high-value compounds (Boutarfa et al., 2022; Santos-Merino et al., 2023). Moreover, they are considered ideal candidates for the development of next-generation biofuels and other biotechnologically relevant substances because of their potential for genetic manipulation and their ability to proliferate under diverse environmental conditions (Tan et al., 2015; Patel et al., 2019).

Among these, Arthrospira sp., colloquially known as ‘Spirulina’, is a helical filamentous cyanobacterium, and has attracted commercial interest because of its distinctive appearance and outstanding nutritional benefits (Selmi et al., 2011; Esquivel-Hernández et al., 2021; Tzachor et al., 2021). Arthrospira sp. contains an abundance of protein (60–70% of dry weight, DW), carbohydrates (15–25% of DW), lipids/PUFAs (3–9% of DW), and other nutrients, making it suitable as a functional food (Wu et al., 2021; AlFadhly et al., 2022). In addition, Arthrospira spp. contain abundant phycocyanin, a blue photosynthetic pigment utilized in the food, cosmetic, and pharmaceutical industries (Wu et al., 2021). These commercial advantages have led to the widespread cultivation of Arthrospira spp. Consequently, research aimed at improving the productivity of this organism is of significant value.

Numerous studies have been conducted to determine the optimal growth conditions that maximize the production of biomass and commercially valuable products from cultures of Arthrospira spp (Gómez et al., 2021; de Souza da Silva et al., 2022). An alternative approach involves developing mutants with increased biomass and metabolites via genetic engineering and random mutagenesis. Although genetic engineering focuses on gene expression and transcription (Wei et al., 2019), random mutagenesis begins with the phenotypes of strains (Cheng et al., 2017), thereby conferring an advantage in identifying beneficial phenotypic mutants (Cheng et al., 2019). Ultraviolet (UV) mutagenesis, a random mutagenesis method, can cause extensive DNA damage, can be applied on a large scale, and is relatively simple and cost-effective to implement (Sivaramakrishnan and Incharoensakdi, 2017). Thus, UV mutagenesis is an effective tool for developing phenotypic Arthrospira spp. mutants with increased biomass and metabolite production.

Arthrospira sp. possesses distinctive morphological characteristics that are essentially regarded as part of their identity (‘spira’ in Latin means spiral). Nonetheless, under diverse environmental conditions such as nutrient availability, salinity, temperature, and changes in the UV spectrum, Arthrospira sp. undergoes morphological change from a helical to a linear shape (Nübel et al., 2000; Mühling et al., 2003; Wu et al., 2005; Mühling et al., 2006; Gao et al., 2008). This morphological change is accompanied by enhanced cell flotation and biomass productivity (Yadav et al., 2020). In addition, unlike other spiral-shaped bacteria that are unicellular, Arthrospira spp. are multicellular and consist of cells connected to each other in a single row that elongates through cell division (Chaiyasitdhi et al., 2018). Therefore, the development of elongated linear Arthrospira sp. mutants could provide potential industrial advantages such as enhanced biomass productivity and simplified harvesting. However, to the best of our knowledge, there are no cases in which UV mutagenesis has been used to develop elongated linear Arthrospira spp. mutants.

In this study, we developed an elongated linear Arthrospira sp. mutant from an indigenous Korean Arthrospira sp. by UV mutagenesis ( Figure 1 ). The physiological characteristics and biomass productivity of the mutant strain were evaluated and compared with those of the most phylogenetically similar strain, A. platensis NIES-39. Furthermore, both the phenotypic and genotypic characteristics of the mutant strain were characterized. To the best of our knowledge, this is the first study to report an elongated linear Arthrospira spp. mutant development via UV mutagenesis. We anticipate that our research will pave the way for new commercial and ecological applications of Arthrospira spp.

In general, the harvest stage accounts for 20–30% of the total production cost of microalgae (Grima et al., 2003; Mata et al., 2010; Verma et al., 2010), and various technologies are being researched to improve harvesting methods (Singh and Patidar, 2018). Our research focused on strain improvement and the induction of random mutations through UV irradiation. Strain NCB002, newly obtained through UV irradiation, is dramatically longer than previously known Arthrospira spp. strains. Based on these characteristics, strain NCB002 has a significant advantage in the harvesting process. When harvested through a thin sieve, almost 80% of strain NCB002 was recovered, whereas NIES-39, which was the reference strain, was not recovered at all through this sieving process ( Figures 4F, G ). Strain NCB002 can be washed with a sieve because of its length, which makes sieving effective for removing impurities from the culture. The cell culture-related characteristics of this strain include a high growth rate and high phycocyanin and chlorophyll contents ( Figures 4C–E ). The increase in the content of the main pigments, phycocyanin and chlorophyll, which are major high-value-added substances, appears to be due to the increase in biomass.

Here, we conducted genomic, transcriptomic, microfluidic technology, and TEM analysis to obtain a comprehensive understanding of this strain and sought to identify any differences compared to existing Arthrospira spp. strains. Strain NCB002 has a long linear form, which is different from the helical form of A. platensis ( Figure 4B ). These morphological characteristics significantly increased the harvest efficiency ( Figure 4F ). However, it was difficult to measure the exact length of this strain in the single-cell state, although its elongated appearance could be observed with the naked eye. In General, the length of a single cell of Arhrospira spp. is 50–500 μm in diameter and 3–4 μm in width and can only be measured using a microscope (Vo et al., 2015). However, strain NCB002 was so long that it was difficult to measure it even under a microscope. A microfluidic device was used to measure the length of strain NCB002 more accurately ( Figure 5 ). This device could not measure cells that were excessively long because they broke or agglomerated during passage. Therefore, the current length was measured based on the cells that passed through this device, showing an average length of 11.69 ± 1.35 mm and a maximum length of 15.15 mm ( Figures 6A–C ). Transmission electron microscopy (TEM) was performed to confirm the differences within the cells; however, no notable differences were identified ( Supplementary Figure 3 ).

Whole genome sequencing (WGS) confirmed that this strain belonged to Arthrospira platensis phylogenetically at 99.86%, with a total genome size of 6,864,973 bp and a G + C content of 44.3 mol% ( Figures 2B , 3 ). Compared with existing reference strains, it was confirmed that it had 3,821 core genes and 58 unique genes ( Supplementary Figure 2 ).

To determine why strain NCB002 was longer than the existing strains, a transcriptomics analysis was conducted. GO enrichment analysis was conducted to categorize differentially expressed genes (DEGs) ( Figure 7A ). Genes within the catalytic activity functional group are primarily associated with proteins that play catalytic roles in diverse chemical reactions, particularly enzymes that are pivotal to cellular metabolic pathways. Therefore, the differential expression of genes related to catalytic activity indicates potential metabolic or enzymatic process modifications in the NCB002 strain. In strain NCB002, 72 genes linked to catalytic function were overexpressed, suggesting a possible enhancement or reduction in metabolic activity. This can influence nutrient uptake, growth kinetics, and the production of specific metabolites in cells. The cytoplasm contains proteins and cellular components that are integral to the cytoplasm, which is the primary internal cellular matrix. It is associated with fundamental cellular functions and elements involved in a range of biological processes, including metabolism, signaling pathways, and cell division. Therefore, differential expression of cytoplasm-linked genes suggests potential alterations in cell organization, intracellular transport, and cellular homeostasis in NCB002 cells. Given that Arthrospira sp. exhibits a filamentous structure connecting its multicellular organization, alterations in intercellular signaling, transport mechanisms, cell division machinery, and cell-to-cell adhesion may have contributed to the enhancement of the chain length.

KEGG pathway enrichment analysis revealed alterations in metabolic pathways, metabolite production, intracellular transport, and cytoplasmic machinery responsible for cell division and intercellular adhesion ( Figure 7B ). Notably, pathways for the biosynthesis of secondary metabolites and cofactors were predominant. These pathways may facilitate the formation or maintenance of long chains by amplifying the production of specific molecules that are instrumental in stress responses, signaling, or cell-to-cell interactions. The observed changes in pathways related to amino acid biosynthesis, purine metabolism, nucleotide sugar biosynthesis, and carbon metabolism indicate the occurrence of a shift in the metabolic landscape of NCB002. These changes may have been accompanied by changes in nutrient assimilation, energy utilization, and growth in NCB002. Additionally, the ABC transporter pathway suggests changes in nutrient uptake and transport in NCB002. Nutrient uptake mechanisms and nutrient or molecular transport may have been enhanced or diversified in NCB002. The peptidoglycan biosynthesis pathway is likely linked to modifications in the cytoplasmic machinery that governs cell division and intercellular adhesion. Furthermore, pathways such as aminoacyl-tRNA biosynthesis, ribosome biosynthesis, and arginine biosynthesis implied potential changes in the protein expression landscape of NCB002. The photosynthetic pathway suggests potential modifications in light capture or energy conversion, which may affect both energy efficiency and growth rate in NCB002.

Integrating the phenotypic features of NCB002 with GO and KEGG pathway enrichment analyses, alterations in photosynthesis, nutrient uptake, nutrient assimilation, metabolic pathways, and metabolite production appear to be involved in cell division. In addition, modifications in intercellular adhesion seem to be instrumental in elongating chain formation. Furthermore, it can be inferred that changes in stress response, signal transduction, and intercellular interactions are involved in the maintenance of extended chains. In other words, it was inferred that changes in NCB002 are multifaceted, encompassing metabolic, morphological, and ecological changes. However, a deeper exploration of these pathways is necessary to elucidate the precise mechanisms driving these phenotypic features and comprehend their potential advantages or implications.

The bacterial cell shape is predominantly defined by the peptidoglycan that constitutes the cell wall (Rohs and Bernhardt, 2021). Therefore, the expansion and division of this structure play an integral role in bacterial growth and division. Peptidoglycan synthesis is regulated by the Rod complex, which includes mreB, mreC, mreD, rodA, rodZ, and PBP2 (Egan et al., 2020). Specifically, PBP2 functions as a transpeptidase essential for bacterial cell elongation (Sauvage et al., 2008) and rodA encodes a glycosyltransferase integral to the polymerization process of peptidoglycan synthesis (Sjodt et al., 2020). The genes mreC and mreD, along with mreB, form an operon central to maintaining the structural integrity of the cell wall (Ago et al., 2023). In addition, mreC interacts with PBP2 (Contreras-Martel et al., 2017), causing structural changes in PBP2 and stimulating peptidoglycan polymerization and cross-linking (Rohs et al., 2018). mreD negatively regulates the interaction between mreC and PBP2; consequently, the balance between mreC and mreD determines the activity of PBP2 (Liu et al., 2020). In NCB002, the observed upregulation of PBP2, mreC, and rodA coupled with the downregulation of mreD ( Figure 8 ) may be reflected in the promotion of peptidoglycan insertion into the cell surface and cell elongation by the Rod complex. Moreover, the upregulation of the pbpC gene, which encodes a transglycosylase involved in peptidoglycan synthesis, can lead to the production of long-linked sugar chains. This strengthens intercellular bonds, facilitating the development of elongated chain structures.

Filamentous cyanobacteria grow through intercalary cell division (Corrales-Guerrero et al., 2018). The ftsZ gene is essential for bacterial cell division (Springstein et al., 2020) and drives peptidoglycan biosynthesis together with the divisome, a multiprotein complex (Wagstaff and Löwe, 2018). Previous research on Escherichia coli demonstrated that reduced ftsZ expression leads to elongated cells, whereas increased expression causes frequent cell division and formation of minicells (Ward Jr and Lutkenhaus, 1985). In contrast, the elongated mutant NCB002 exhibited overexpression of ftsZ ( Figure 8 ), suggesting a fundamental difference in cell division mechanics between E. coli, a unicellular rod-shaped bacterium, and filamentous cyanobacteria of the genus Arthrospira. In Arthrospira sp., as long as their peptidoglycan walls are maintained, the promotion of intercellular cell division implies an increase in cell length. Moreover, the FtsZ-driven divisome plays a role beyond cell division, maintaining multicellularity and intercellular communication in filamentous cyanobacteria (Ramos-León et al., 2015). Nevertheless, for the augmented expression of the ftsZ gene to result in extended cell lengths, precise Z ring assembly is essential. The minC, minD, and minE genes, which are part of the mitotic timer pathway, help ftsZ to move to the appropriate location and form a Z ring. The upregulation of minC and downregulation of minE observed in NCB002 appear to control the formation time and location of the Z ring, and are inferred to have influenced the chain length extension of NCB002.

As previously mentioned, maintenance of the peptidoglycan wall is a prerequisite for the promotion of cell division, leading to the elongation of cells in Arthrospira sp. Generally, as the cell length increases, a greater quantity of peptidoglycan is required; therefore, genes related to peptidoglycan biosynthesis must be upregulated (Zhao et al., 2022). However, in the elongated mutant NCB002, the key genes involved in peptidoglycan synthesis, such as mltG, murA, murC, and murG, were downregulated ( Figure 8 ). This downregulation is linked to a reduction in cell wall rigidity, as established in studies on E. coli (Zhang et al., 2018). Peptidoglycan layers undergo continuous turnover throughout the bacterial life cycle, with new monomers incorporated as old bonds are cleaved (Jacobs et al., 1994). Thus, when a new monomer is inserted into the existing peptidoglycan layer and cross-linked with the original layer, structural changes may occur in the peptidoglycan layer. Therefore, we propose that decreased cell wall rigidity in NCB002 cells may enhance the frequency of new monomer insertions, leading to cellular elongation. Indeed, the upregulation of genes associated with the Rod complex, which is responsible for the polymerization and cross-linking of peptidoglycans, the insertion of monomers, and the production of long glycan chains, supports this hypothesis in NCB002. However, the mechanisms underlying the downregulation of peptidoglycan biosynthesis genes are not yet fully understood.

Collectively, the long-chain phenotype of NCB002 was characterized by significant alterations in cell morphology, cell wall assembly, and cellular division dynamics. The distinctive gene expression pattern of NCB002, characterized by the upregulation of genes related to rod shape, PBP, and cell division, along with the downregulation of genes involved in peptidoglycan synthesis, suggests a complex regulatory mechanism. This mechanism orchestrates cell-to-cell adhesion, peptidoglycan synthesis, filament elongation, and cellular division, thereby contributing to the NCB002’s unique structural and physiological attributes. However, current information may be fragmentary. The roles of individual genes can vary by interactions with other genes, which can be very complex and controlled by multiple pathways. To fully elucidate the comprehensive characteristics NCB002, which has unprecedented cell length, may require the development of a novel device that can measure cell lengths non-invasively. This endeavor may also need acquiring genetic information that exceeds the current level of knowledge about Arthrospira sp. Thus, to gain additional insight into these changes, further in-depth studies are required.

In conclusion, strain NCB002 of NCell Co., Ltd. obtained through UV irradiation is an attractive strain, both academically and industrially, and we hope that it will be used more industrially in the future.

All relevant data supporting the key findings of this study are available within the article and its Supplementary Information files, or from the corresponding author upon reasonable request. The source data are provided in this study. The draft genome sequences were deposited in the DNA databank of Japan/European Molecular Biology Laboratory/GenBank under the accession numbers JARHUM010000001-JARHUM010000005.

CL: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Supervision, Validation, Writing – original draft, Writing – review & editing. SH: Data curation, Writing – original draft, Writing – review & editing. HN: Methodology, Writing – original draft. ZK: Methodology, Writing – original draft. JA: Data curation, Writing – original draft. BO: Methodology, Writing – original draft. HK: Data curation, Writing – original draft.