Polyamine-Mediated Growth Regulation in Microalgae: Integrating Redox Balance and Amino Acids Pathway into Metabolic Engineering

Abstract

Polyamines play a pivotal role in regulating the growth and metabolic adaptation of microalgae, yet their integrative regulatory roles remain underexplored. This review advances a comprehensive perspective of microalgae growth, integrating polyamine dynamics, amino acid metabolism, and redox balance. Polyamines (putrescine, spermidine, and spermine) biology in microalgae, particularly Chlamydomonas reinhardtii, is reviewed, exploring their critical function in modulating cell cycle progression, enzymatic activity, and stress responses through nucleic acid stabilization, protein synthesis regulation, and post-translational modifications. This review explores how the exogenous supplementation of polyamines modifies their intracellular dynamics, affecting growth phases and metabolic transitions, highlighting the complex regulation of internal pools of these molecules. Comparative analyses with Chlorella ohadii and Scenedesmus obliquus indicated species-specific responses to polyamine fluctuations, linking putrescine and spermine levels to important tunable metabolic shifts and fast growth phenotypes in phototrophic conditions. The integration of multi-omic approaches and computational modeling has already provided novel insights into polyamine-mediated growth regulation, highlighting their potential in optimizing microalgae biomass production for biotechnological applications. In addition, genomic-based modeling approaches have revealed target genes and cellular compartments as bottlenecks for the enhancement of microalgae growth, including mitochondria and transporters. System-based analyses have evidenced the overlap of the polyamines biosynthetic pathway with amino acids (especially arginine) metabolism and Nitric Oxide (NO) generation. Further association of the H₂O₂ production with polyamines metabolism reveals novel insights into microalgae growth, combining the role of the H₂O₂/NO rate regulation with the appropriate balance of the mitochondria and chloroplast functionality. System-level analysis of cell growth metabolism would, therefore, be beneficial to the understanding of the regulatory networks governing this phenotype, fostering metabolic engineering strategies to enhance growth, stress resilience, and lipid accumulation in microalgae. This review consolidates current knowledge and proposes future research directions to unravel the complex interplay of polyamines in microalgal physiology, opening new paths for the optimization of biomass production and biotechnological applications.

1. Introduction

The green microalgae Chlamydomonas reinhardtii (C. reinhardtii) has a markedly low growth rate characteristic of many microalgae species, which impairs large-scale biotechnological applications. Modifying such a natural phenotype to improve microalgae growth performance is a significant technological challenge. Once overcome, it could bring many novel biotechnological applications ranging from bioenergy to pharmaceuticals [1,2]. Understanding the fundamental principles of cell growth control is therefore essential for unraveling the mechanisms underlying the slow-growth phenotype and providing novel strategies for its modulation.

Microalgae biomass has received global attention as a versatile feedstock for industries spanning bioenergy, human nutrition, pharmaceuticals, and agriculture [1,2]. Microalgae can be cultivated associated with bioremediation processes, either in consortium or axenically, and possess favorable traits for use in sustainable and renewable biomass production systems [3]. Besides that, microalgae are also capable of growth in exclusive photoautotrophic conditions, which implies that they can be combined with decarbonizing strategies compatible with future low-emission industrial models [4]. However, to achieve broader industrial deployment, a deeper understanding of microalgal growth is imperative. Unfortunately, besides their enormous potential to generate molecules of broad interest to humankind, important limitations of their application at very large scales persist, including their slow growth phenotype, which increases cultivation costs [5], limiting the economic viability of large-scale cultivation systems [6].

Recent efforts have demonstrated that traditional process optimizations alone, such as controlling or improving process parameters, such as media composition, light conditions, stressors, and gas exchange, are still insufficient to overcome the intrinsic growth rate maximum. In this context, integrative omics analysis emerges as a powerful strategy to unravel regulatory networks and prioritize target pathways for metabolic engineering towards enhancing biomass productivity [7,8,9,10,11,12,13,14,15,16,17].

Previous metabolic models have indicated that current microalgae cultivation systems do not reach their theoretical maximum productivity, suggesting that total biomass production could be increased [17].

Growth rate limitations have been attributed to both light and CO₂ uptake under photoautotrophic conditions, while nutrient limitation shifts growth constraints towards light usage efficiency [11]. Further models indicated phenylalanine, tyrosine, and tryptophan biosynthesis, porphyrin, and chlorophyll metabolism, and purine metabolism as essential ones in mixotrophic growth [12]. Previous flux balance analysis implicates ATP production in mitochondria as a critical bottleneck, reinforcing the idea that energy balance and mitochondrial activity are central to growth regulation [13,17]. Thus, despite robust metabolic reconstructions, the precise regulatory genes and control nodes that modulate microalgae growth rates remain elusive and urgently require functional validation [18,19,20,21,22,23].

Among the molecules linked to growth modulation, polyamines have long attracted attention for their association with cell proliferation across a wide range of organisms, including bacteria, yeast, plants, microalgae, and animal cells [24,25,26,27]. These molecules participate in diverse molecular functions and stress adaptation responses, reflecting a complex systemic role in growth control [8]. Studies in various microalgae species have demonstrated that fluctuations in polyamine concentrations correlate with growth phase transitions and may function as signaling elements that influence cell division and metabolic activities [25,28,29,30].

Here, we review and summarize the current understanding of polyamine biosynthesis, function, and regulatory potential in green microalgae, focusing on their dynamics across metabolic phases and their roles as systemic regulators.

By integrating and interpreting omics-derived regulatory models and metabolic engineering strategies, we propose an updated conceptual framework for using polyamines as tools to modulate growth performance and adaptation in microalgae, with potential implications for their biotechnological applications and microalgae stress resilience.

Furthermore, we explore how synthetic biology approaches can leverage the functional diversity of polyamine biosynthetic pathways to design genetically tunable growth which also consider redox balance and amino acids pathways as an integrative strategy towards growth enhancement devices. This could enable translational applications in biotechnology, offering a roadmap for creating resilient and productive microalgal strains suitable for deployment in carbon-efficient bioprocesses.

2. Polyamines’ Biology in Microalgae

2.1. Biosynthesis Routes

In microalgae, the key substrates for polyamines’ production are the amino acids arginine and ornithine. In separate pathways, two key enzymes, Ornithine decarboxylase (ODC, EC 4.1.1.17) and Arginine decarboxylase (ADC, EC 4.1.1.19), participate in the metabolic biosynthesis of putrescine, which may be further transformed into spermidine and spermine [14]. In the microalgal species expressing the enzyme arginase, the amino acid arginine is the main substrate for both the ODC and ADC pathways. Arginine is decarboxylated into agmatine, a putrescine precursor. In some conditions, arginine is converted into ornithine, which is the main substrate of ODC to produce putrescine in a direct decarboxylation reaction [15].

Of note, in the model microalgae C. reinhardtii, no arginase gene has been annotated so far, which suggests that the cells have separated functional ODC and ADC pathways, with ornithine provided only by the metabolism of glutamine. The ADC pathway in Chlamydomonas starts with the arginine being converted to agmatine, which leads to the production of putrescine [14].

On the ODC pathway, ornithine is a product of the nitrogen assimilation mechanism and is decarboxylated into putrescine, which is converted into spermidine through the spermidine synthase enzyme (spd1 gene) [16] by the addition of an aminopropyl group derived from decarboxylated S-adenosylmethionine (dcSAM) [9,10]. The spermidine synthesized can be metabolized into spermine or thermospermine by the transferring of a second aminopropyl group to the N8 (aminobutyl) or N1(aminopropyl) ends of spermidine by spermine synthase (sps1 gene) or thermospermine synthase (acl5 gene) [31], respectively, even though the concentration of spermine compound is undetectable experimentally in some reports, including the microalgae C. reinhardtii [15,32] (Figure 1).

In Chlamydomonas, putrescine is mainly synthesized by the activity of the ODC, being a rate-limiting step for polyamine biosynthesis [16], with the ADC pathway considered inactive in some conditions [15]. The concentration of both subunits of ODC (ODC1: gene ID 5723008; ODC2: gene ID 5724425) [14] is regulated by their translational rate and by non-ubiquitinated proteolysis of ODC by 26S proteasome. The proteasome degradation of ODC involves its interaction with an inhibitor protein named “antizyme” as it occurs in mammalian cells [18,33,34], a mechanism that is still not completely elucidated in microalgae [32]. Several authors reported an increase in ODC activity before cell division [10,19,20,32], pointing out the ODC enzyme and its degradation as an important regulatory process in the mechanism of cell proliferation. In addition, the increase in polyamine concentration decreases ODC activity. Ten times less spermidine and spermine than putrescine concentration is enough to decrease the ODC activity [32], by affecting its enzymatic activity and the ODC mRNA translation.

Therefore, as ornithine biosynthesis is dependent primarily on the glutamate content from nitrogen assimilation, the reduction in the polyamines’ biosynthesis in conditions such as nitrogen deprivation may trigger a cell quiescent state, a well-known phenotype that is associated with lipid accumulation in many microalgae species [21]. In this condition, the de novo production of ornithine, which is the main precursor for putrescine in Chlamydomonas reinhardtii, is heavily reduced, as well as the expression of S-adenosylmethionine synthetase (SAS1). Therefore, the reduced production of putrescine through the ODC pathway due to the lack of available substrate and the possible inhibitory effect of antizyme over ODC activity would be accompanied by the reduced generation of conjugated forms of polyamines [21,34].

Our previous findings indicated the existence of a regulatory sub-network coordinating arginine biosynthesis and degradation pathways with the triggering of lipid accumulation and cell quiescence in the microalga Chlamydomonas reinhardtii. The gene promoter motif analysis of co-expressed genes also suggested that cell growth might be controlled by a regulatory network that is partially independent of the ones leading to lipids accumulation [22]. Thus, the understanding of the functional principles of cell quiescence and polyamines’ metabolism may reveal novel possible designs of genetic devices for cell growth control.

2.2. Functional Roles of Polyamines

Polyamines are linear or branched aliphatic metabolites that are positively charged at physiological pH, containing several amines, mostly functioning in the coordination of the metabolism and growth rate in eukaryotic organisms, acting in surveillance pathways or as regulatory molecules [23]. The most common forms of these compounds in plants are putrescine, spermidine, and spermine, with some consideration also for cadaverine and diaminopropane. Since they are associated with cell division, the plants’ meristematic tissues are the richest in polyamines, as is reported by their accumulation in cells under high division rates or exponential growth phases [24,25,26,27,35]. Besides these forms, there are reports of uncommon polyamines, like nor-spermine, nor-spermidine, homospermine, homospermidine, and thermospermine. Polyamines exist in three major states, namely free state, perchloric acid-soluble conjugated state, and perchloric acid-insoluble conjugated state [28], which can influence complex regulatory processes of plant adaptation to several metabolic states [31,36].

In microalgae, these polyamines, especially putrescine and spermidine, can accumulate in high concentrations, leading to processes associated with cell proliferation, growth, and resistance to environmental stress by regulating the cell cycle and antioxidant responses [29]. Of note, in C. reinhardtii (green algae), spermine has not consistently been identified so far, and its cellular uptake from the environment is not consistently demonstrated [15,30].

Moreover, the exposure of the green microalgae Chlorella vulgaris to spermidine stimulates antioxidant activities of the superoxide dismutase, ascorbate peroxidase, and catalase enzymes. Concomitant accumulation of ascorbate and glutathione increased the antioxidant capacity in cells growing under heavy metal abiotic stress [37]. In plants, the catabolism of polyamines by flavin-containing polyamine oxidases (PAO) generates endogenous hydrogen peroxide (H₂O₂), which has been related to the induction of signaling events triggering mild antioxidative defense responses related to abiotic stress [38,39]. The oxidative deamination of polyamines in plants by copper-containing amine oxidases (CuAO) and the PAO has been associated with modifications in the cell wall structure, wound closure, and resistance to biotic and abiotic stress [40]. In animal cells, spermidine binds to mitochondrial trifunctional proteins, enhancing longevity and maintaining mitochondrial functionality [36]. In Chlamydomonas reinhardtii, at least five genes coding for polyamine oxidases (CuAOs and PAOs) have been annotated and predicted to be involved in the metabolism of spermine, spermidine, and putrescine, likely generating H₂O₂ in the process [15].

In diatoms, a major group of eukaryotic microalgae, the regulation of the deposition of cell wall structure is performed in unique siliceous cell walls [31] by an organic template consisting of proteins and aliphatic long-chain polyamines (LCPAs) [41]. LCPAs are crucial in controlling the shape and morphology of the frustules, acting as both structural and catalytic agents in silica condensation.

Polyamines also participate in stabilizing nucleic acids and stimulating their replication [42], impacting protein synthesis and modulating enzyme activities. Therefore, the ability to bind to DNA and RNA, especially mRNA, can be highlighted as one of the main characteristics that promote cell division and growth regulation [29], making it possible to pinpoint some causality across these events.

Free polyamines have an impactful role in metabolic modulation due to concentration shift towards intracellular accumulation or depletion in ordinary and stress responses associated with growth control. On the other hand, the conjugated polyamines, or the binding of polyamines into cell components, have been associated with morphogenic processes and the formation of secondary metabolites, as the long-chain polyamines can be covalently conjugated to proteins as a post-translational modification. Thus, the dynamic contribution of the conjugated and unconjugated forms of polyamines affects several metabolic regulation events. The unconjugated forms have been associated with regulatory events occurring at the level of metabolic flux or substrate-level regulation, serving as precursors of conjugated forms by interacting with decarboxylated S-adenosylmethionine molecules and as substrates to aminopropyltransferase enzymes in polyamines elongation [43,44].

Protein post-translational modifications (PTMs) may occur by the covalent interaction with conjugated forms of polyamines, leading to functional alterations of target proteins due to allosteric inhibition or protein’s sub-cellular translocation and localized distribution inside the organelles, such as the distribution of LHCII proteins inside the chloroplasts in Scenedesmus obliquus [45].

In mammalian cells, polyamine-like PTM effectors preferentially activate protein kinases, triggering a cascade of signaling events, such as the mitogen-activated protein kinase (MAPK) cascade. Among them, putrescine can stimulate transcription factor regulation, and spermidine enhances the phosphorylation of threonine and tyrosine residues in ERK1 and ERK2, with both putrescine and spermidine stimulating the phosphorylation of proteins by tyrosine kinases [42].

The metabolic intermediates of the polyamine’s biosynthesis pathway may also exert an important regulatory function in polyamine-based signaling, associating the role of the arginine biochemical pathway with polyamine biosynthesis. This has already drawn some attention during the investigation of a fast-growing green microalgae, highly resistant to high illumination and isolated from the Negev desert crust in Israel [25,46]. The fast-growing Chlorella ohadii (C. ohadii) has demonstrated an optimal capacity to resist photodamage, generating carotenoid molecules to protect its photosystems under high light. Most interestingly, they show increased metabolite fluxes towards the tricarboxylic acid cycle, amino acid synthesis, and lipid synthesis compared to land plants [47]. Besides that, putrescine has been identified as markedly more abundant in C. ohadii in a growth stage, preceding an increase in the cell growth rate suggesting its role as a potent regulator of cell growth in this fast-growing microalgae species.

The intricate roles of polyamines as metabolic intermediates, cell structure compounds, and signaling mechanisms urge a deeper investigation of their function in microalgae and how their interplay can influence growth performance in different microalgae species.

2.3. Species-Specific Variation and Structural Roles

Long-chain polyamines (LCPAs) play an important structural function by composing the cell structure of diatoms. The LCPAs found in diatoms are predominantly N-methylated derivatives of oligo-propyleneimine chains attached to polyamine building blocks such as putrescine, ornithine, spermine, spermidine, and 1,3-diaminopropane. These compounds exhibit species-specific variations in chain length, degree of methylation, and the positioning of secondary, tertiary, and quaternary ammonium groups. The molecular diversity of LCPAs has been documented across various diatom species, including Chaetoceros, Coscinodiscus, Cylindrotheca, Navicula, Stephanopyxis, and Thalassiosira representatives. Such diversity is indicative of their essential role in the biosilica morphogenesis process [48,49,50].

Studies using high-resolution mass spectrometry and other spectroscopic techniques have revealed that LCPAs from different marine environments exhibit unique molecular distributions, likely reflecting variations in the local diatom community. For instance, putrescine-based LCPAs are consistently found across different regions, whereas 1,3-diaminopropane-based LCPAs were identified with certain exclusivity by Bridoux et al. (2012) in samples from the northeastern Pacific coast [44].

The biosynthetic pathways leading to the production and methylation of LCPAs in diatoms remain largely unknown but are believed to originate from the spermidine/spermine route with ornithine as the primary precursor. Inhibition of ornithine decarboxylase in Thalassiosira pseudonana significantly altered frustule morphology, supporting this hypothesis [44,51]. It is suggested that up to 20 successive aminopropyltransferase enzymes may be required for LCPA chain elongation to consider the same metabolic pathway from green algae to diatoms, along with a substantial supply of decarboxylated S-adenosylmethionine, which reinforces the differences between these organisms mainly by different genome incorporation of bacterial genes [31,43].

Polyamine metabolism in diatoms is closely associated with silicon availability. Research on Skeletonema dohrnii (diatom) has shown that low silicate concentrations result in reduced growth rates, lower polyamine content, and downregulation of PAO gene expression [52]. Also, higher silicate concentrations increase polyamine content and upregulate PAO gene expression. This response suggests that polyamine metabolism is part of the diatom’s adaptive mechanism to environmental stresses and cell cycle metabolism.

LCPAs extracted from fossilized diatom frustules in marine sediments have demonstrated the ability to induce silica precipitation in vitro, even after several years [44]. It also highlights a biotechnological application to modulate the polyamine metabolism in diatoms, to provide silica precipitation ex vivo for industrial purposes, or to modulate the cell architecture to achieve better culture performance under limiting conditions. Then, long-chain polyamines play a fundamental role in diatom silica morphogenesis, polyamine metabolism, and environmental responses. Understanding these processes provides novel insights into the molecular mechanisms underlying diatom adaptability and their contribution to marine biogeochemical cycles.

3. Polyamine Dynamics and Cell Growth Progression

3.1. Intracellular Concentration Shifts and Growth Phase Transitions

The regulation of cell growth by the polyamine’s concentration shifts involves multiple cellular processes, including biosynthesis, uptake, oxidation, and acetylation, since the intracellular polyamine levels are primarily controlled by these mechanisms.

Biosynthesis is a crucial step in polyamine regulation, with the rate-limiting step being the conversion of ornithine into putrescine, catalyzed by ODC. Uptake mechanisms, extensively studied in other organisms, involve energy-dependent transporters whose activity is influenced by external pH and amine concentration. Oxidation and excretion contribute to reducing cellular polyamine levels, primarily mediated by specific polyamine or diamine oxidases, with some oxidation products exhibiting biological activity. Acetylation, catalyzed by acetyltransferases, decreases the net positive charge of polyamines, reducing their interactions with negatively charged molecules like nucleic acids and phospholipids. This modification promotes excretion, further regulating intracellular polyamine concentrations [42].

In microalgae, including C. reinhardtii and Scenedesmus obliquus, polyamine levels rise before the transition to the cell division phase, emphasizing their essential role in growth regulation [26,32]. In C. ohadii, polyamine concentration shift predicts growth transitions, in light-independent conditions (high or low illumination—3000 or 1000 µmol·m⁻²·s⁻¹), while a higher photosynthetic rate occurs during the transition from phase I to II, coinciding with metabolite fluctuations, such as peaks in glutamine, alanine, and serine content. Notably, putrescine and spermine exhibit opposite metabolic shifts during different growth phases [25].

In a previous report, it was inquired whether there is a specific function for each polyamine, or if their global concentration oscillation is the responsible factor to modulate growth [7]. Investigation across different microalgal species following alterations of the concentration of putrescine, spermidine, and spermine suggests that specific types of shifts in their intracellular concentration occur at target metabolic moments (e.g., specific phases of the cell growth curve). Some shifts seem to be characteristic of a broad range of organisms, and some of them are species-specific, as reported in the literature [53].

Considering the association between the growth phase and polyamine accumulation, the increased putrescine concentration during cell proliferation and its decline at the mid-exponential phase seem to be controlling and stabilizing the C. ohadii growth in phase II. Furthermore, the inverse relationship between putrescine and lipid content, including a-linoleic and a-linolenic acids profiles, suggests metabolic interactions between growth and lipid biosynthesis [25]. In synchronized Euglena cultures (Euglenophyceae), growth arrest blocks polyamine utilization, pointing to this mechanism as a negative feedback regulator of polyamines’ production [54]. The concentration of polyamines increases during exponential growth in various algal species, closely linking them to DNA replication and cell division [24,25,26]. High spermidine strongly correlates with the growth of Chattonella antigua (Raphidophyte) and Heterosigma akashiwo (Raphidophyte), while putrescine plays a more predictive metabolic role in growth shifts [29,30,55]. In Alexandrium minutum (Dinoflagellate), putrescine and cadaverine rise significantly in the exponential phase, while Thalassiosira pseudonana (diatom) exhibits a similar pattern with nor-spermidine [24].

The reviewed topics indicate that different polyamines may be required at distinct growth stages (Figure 2). Free polyamine levels are higher in the lag phase, whereas conjugated polyamines dominate during exponential growth, increasing sevenfold compared to free forms. In A. minutum T1 cells, free polyamine levels peak in the lag phase before declining in the exponential phase, except for cadaverine and nor-spermidine, which increase slightly before decreasing mid-exponential phase. Putrescine and cadaverine are the primary conjugated polyamines, and spermine levels rise significantly during the death phase [24]. Similarly, in Thalassiosira pseudonana, polyamine concentrations increase significantly during exponential growth [56]. In Nicotiana tabacum L. (land plant), corolla spermine also appears correlated with cell death promotion [57].

As a complementary tool, genetic modification of Chlamydomonas’ genome is an upcoming field that could bring a better understanding to this topic. Since the protein’s heterologous expression in algae is not a well-established field as it is in bacteria, considering some difficulties in cell transformation, gene codon bias, high CG content in DNA, and lack of strong promoters’ diversity [58], basic resources still need to be developed to make this alternative straightforward.

Previous reports about putrescine pathway genetic engineering in Chlamydomonas demonstrate that it cannot be controlled by a single gene and may need cloning of other components to start promoting substantial alterations in this process [15]. Then, due to the signaling nature of polyamines’ concentration shift, specific modifications like interruptions in the natural concentration oscillation process may expose the cells to severe stress from unpredictable metabolic interventions. These findings underscore the complexity of polyamine regulation in microalgae and their implications for growth and stress responses. At this point, neither a specific gene function nor an isolated biological network function can define a phenotype [59]. The complex network structure converging these biological entities, and the observed dynamic of the metabolic phase-specific responses, suggest that a deeper understanding of the biological systems is necessary, considering the non-linear nature of polyamines’ responses [60].

3.2. Exogenous Supplementation and Implications in Growth Phenotype

The microalgae C. reinhardtii lack high-affinity polyamine membrane transporters, which decreases their uptake rate. Nevertheless, exogenous supplementation of polyamines impacts cellular polyamine metabolism, influencing growth and gene expression [32].

Previous results suggested that under homeostasis, cells accumulate putrescine up to a maximum concentration, after which a metabolic homeostasis status is disrupted by putrescine “saturation” moment, after which the dynamics of the interconversion of the several polyamines, mainly modulating the synthesis rate of spermidine and spermine, is affected [7]. If the internal concentration of putrescine decreases, as it was reported for microalgae cells under nitrogen starvation and mammalian cells under serum starvation, this conversion rate remains minimal [42], while microalgae cells remained with unmodified growth yields in AMX1 and AMX2 knockout mutants where putrescine was not degraded and its levels were similar to a reference type strain. This analysis indicates that a minimum concentration of putrescine is necessary to provide growth yield in microalgae, while the overexpression of ODC reduces the cell concentration and increases the cellular putrescine during cell growth [15]. Further analysis of the fast-growing Chlorella ohadii (green algae) revealed its peculiar growth profile: the cells population grows exponentially (phase I), suffers retardation in growth rate (phase II), and then grows faster again, achieving its maximum density (phase III) [25]. However, when 0.1 mM of putrescine was introduced into C. ohadii culture, growth was stimulated, and the culture entered phase II (retardation) at a higher cell density.

This suggests that a certain level of putrescine depletion is necessary for the transition into growth retardation. Conversely, when putrescine was added during the retardation phase, the delay was shorter, further confirming that lower putrescine levels lead to slow growth. Spermine at 0.1 mM in phase I slowed growth and extended the retardation phase [25], which may be connected to the ODC inhibition. Moderate growth inhibition and an increase in 30% of cell diameter were reported at high concentrations of putrescine, indicating its positive impact on cell proliferation [15].

Polyamine uptake experiments in other microalgae species reinforce these observations. In Scenedesmus obliquus (green algae), low spermine doses promoted chlorophyll synthesis, whereas high concentrations altered thylakoid structures and reduced chlorophyll [45,61]. In Chlorella vulgaris, exogenous spermidine (100–300 µM) increased biomass and accelerated cell division [37,62].

Putrescine has also modulated cell responses in C. reinhardtii under low-temperature acclimation. The supplementation with 100 µM putrescine before cold stress (10 °C) increased growth compared to the control, though still lower than unstressed cultures. The treated samples showed lower lipid peroxidation, indicating reduced membrane damage, reinforcing putrescine’s role as a membrane stabilizer and a possible target of oxidation. Moreover, exogenous putrescine alleviated the cold-induced downregulation of odc2 and spd1 genes, crucial for polyamine biosynthesis [16]. This suggests that putrescine supplementation mitigates stress impacts on growth by sustaining polyamine-related gene expression.

Exogenous polyamine uptake in Chlamydomonas does not seem to be intense, as the concentration of polyamines in the culture medium decreases slightly across cell proliferation phases. The addition of 1 mM putrescine in the culture medium slightly increased intracellular putrescine and spermidine levels, likely due to its uptake and putrescine-to-spermidine conversion, supporting previous reports that interconversions occur predominantly at intracellular polyamines saturation. Spermidine supplementation elevated intracellular spermidine and putrescine, possibly through enhanced spermidine degradation or reduced putrescine-to-spermidine conversion. Interestingly, no conversion into spermine was detected in putrescine- or spermidine-fed conditions [32].

Spermine supplementation led to a progressive increase in intracellular spermine while reducing spermidine and putrescine levels, suggesting that spermine may negatively regulate their accumulation. Also, the addition of 0.1 mM of spermine promoted a 50% reduction in growth in C. reinhardtii, while the supplementation with either putrescine or spermidine did not promote the same effect [14]. Furthermore, considering that Chlamydomonas may lack high-affinity polyamine transporters, labeled putrescine experiments showed no progressive depletion from the medium, and its uptake rate diminished when spermidine or spermine was also present. This suggests the presence of a shared transport system with varying affinities for different polyamines. Spermine supplementation induced a stronger decrease in putrescine uptake compared to spermidine. However, pre-incubation with spermidine or spermine for 1 to 3 h did not alter putrescine uptake kinetics, indicating that the competitive interactions between polyamines in uptake mechanisms are immediate rather than cumulative over time [32]. Thus, the supplementation studies can help us understand the natural impact and oscillation of polyamine concentrations, giving hints to investigate the role of polyamines and their role in regulating microalgae growth. A summary of the main effects of exogenous supplemented polyamines in C. reinhardtii cells is provided (Figure 3).

The scheme indicates the main cellular processes that are influenced by the supplementation with exogenous polyamines (Putrescine, Spermidine, Spermine, and Cadaverine).

The results of the polyamines supplementation studies point to a major role of polyamines as signaling molecules under non-stress growth conditions. The correlation of spermidine supplementation and the enhancement of mitochondrial functions [36], its essentiality in cell growth and survival [63], and the association of their catabolism with the H₂O₂ production reinforce the evidence of a connection between cell growth and necessary adjustments in mitochondrial activity, as predicted by growth and genomic-based modeling approaches [12,17].

4. Complex Overlap Between Polyamines and Arginine Synthesis Pathways: The Balance of Quiescence and Growth

The distinct characteristics and effects of polyamines’ uptake and metabolism in C. reinhardtii and other microalgae species reveal significant differences from mammalian systems, particularly in the absence of high-affinity membrane transporters and the concentration-dependent nature of polyamine interconversions. Polyamines’ dynamics play a crucial role in microalgal cell growth and proliferation, with putrescine, spermidine, and spermine each contributing differently to these processes (Figure 4).

Putrescine is essential for growth, with its levels increasing during cell proliferation and peaking in the early exponential phase before declining at the mid-exponential phase. It is the major component of conjugated polyamines and plays a key role in regulating polyamine balance [64]. An increase in putrescine concentration leads to a corresponding increase in spermidine levels, reinforcing its role in cell metabolism, while the AMX2 enzyme is the main copper-containing amine oxidase in C. reinhardtii [15]. However, putrescine depletion or over-accumulation results in decreased growth, affirming its significance in sustaining cellular proliferation. Exogenous supplementation of putrescine also reduces ODC activity affecting directly internal putrescine synthesis [64].

Spermidine actively promotes biomass accumulation and cell division. Its levels are higher during exponential growth, contributing to the overall increase in conjugated polyamines observed in this phase. Additionally, an increase in spermidine may be associated with an increase in putrescine, emphasizing their interconnected regulation. However, mutant strains of SPD1 have shown high accumulation of putrescine compared to reference strains [63], indicating that putrescine levels are influenced by the activity of AMX and PAO enzymes. Spermidine also inhibits ODC activity, a key enzyme in polyamine synthesis, further shaping cellular polyamine homeostasis [15,65].

In contrast, spermine acts as a growth inhibitor. It slows down cell growth and prolongs the time of growth retardation in fast-growing microalgae species, demonstrating its negative regulatory effect, likely due to reducing spermidine biosynthesis and abolishing cell division. Furthermore, spermine inhibits growth entirely when present at high concentrations [64]. Its influence extends to influencing polyamine balance, as an increase in spermine concentration leads to a decrease in both putrescine and spermidine levels, while supplementation of spermidine or putrescine reverts the spermine-induced cell cycle arrest [64]. Like spermidine, spermine also inhibits ODC activity, reinforcing its role as a negative regulatory checkpoint in polyamine metabolism. Nevertheless, it is positively correlated with enhanced lipids production, opening opportunities to better understand this interaction.

The polyamines exhibit a complex interplay influencing microalgal cell growth with specific outcomes at different metabolic stages. Their regulation and interaction with several biological processes affect cellular proliferation, with genetic modifications and supplementation studies offering valuable insights into their natural fluctuations and impact on growth dynamics [66,67].

5. Insights into Growth Regulation from Multi-Omics and Metabolic Modeling

A careful observation of the broad information retrieved by several omics analyses investigating the cell quiescence in microalgae, especially under nutritional deprivation, brings critical analysis of the difference between cell quiescence and the low-growth phenotype typical of many microalgal species [21]. The genomic reconstructed models of microalgae in several light regimes [12] and CO₂ concentrations [17] have indicated that mitochondrial functions, membrane transporters and changes in amino acids pools are correlated to the phenotypes of cells with the highest growth performance, either in higher CO₂ availability [17] or optimal light conditions [12].

Investigation of events governing the cell cycle progression and diurnal behavior of microalgae cells has also identified similar general biological processes being affected during the cell cycle progression. Alterations in protein synthesis, amino acid metabolism, and transport biological function preceded the S/M phases of cell division, and characteristic alterations in the expression of cell cycle-related proteins (e.g., Cyclins and CDKs). These results suggest that cell proliferation regulation may take place in moments that anticipate the chloroplast division initiation, likely during the prophase. Gene expression alterations of mitochondrial ribosomal genes occur massively before S/M transitions, indicating an important metabolic adjustment towards cell division [68]. These processes are also affected by the ratio between H₂O₂/NO [69]. Furthermore, a deeper understanding of how these processes relate to each other would likely bring more information on how to control cell growth in microalgae.

6. Tunable Control by Synthetic Devices

Considering that the present most active and regulated pathway from the polyamines’ biosynthesis is the one involving ODC enzymes, it is also possible to hypothesize that the growth rate is dependent on the established homeostasis of polyamines, which could be tuned for each microalgae species, as it is for C. reinhardtii (Figure 5A). However, it is important to consider that there is an overlap between the metabolic pathways of nitrogen assimilation and carbon accumulation under normal and abiotic stress conditions, serving as the controlling point for cells to decide whether to grow and divide or to keep carbon backbones stored, by co-activation of the quiescence state.

The overlap between the arginine and polyamine pathways may significantly affect the balance between the H₂O₂/NO ratio during the cell response to environmental stress (e.g., nitrogen deprivation). Nitric oxide is enhanced under nutritional deprivation stress in microalgae, while enzymes related to the catabolism of polyamines are more expressed, indicating that H₂O₂ would be more abundant in those situations [21]. Therefore, a specific range of H₂O₂/NO ratio may be necessary for cells to keep their growth progression without activating cell quiescence, and a coordinated balance between both species may be definitive for the progression of cell growth or the quiescence activation.

The metabolic engineering of microalgae strains, modifying their main pathway of polyamines’ synthesis to overexpress the ADC enzyme in ODC1 mutants or inhibited forms, may create a linear supply of ornithine from arginine towards feeding the TCA cycle and deactivating a concurrence pathway (Figure 5B). That synthetic device could be developed with genes already described in different microalgae species or plants, which currently can provide arginase and ADC genes adapted to microalgae physiology. In this way, the preservation of putrescine synthesis is possible, warranting the supply of arginine and ornithine without the strong side regulations observed in promiscuous inhibition of ODC enzymes by several natural compounds. The constitutive expression of ADC would provide putrescine without the overproduction of NO, and the inhibition of ODC1 under selected gene promoters can modulate the amount of glutamate/glutamine towards equilibrating the nitrogen flow, avoiding the excess of NADH.

7. Prospects

The combination of our learning from modeling approaches and natural bio-prospected species indicates that the analysis of the mechanisms of microalgae growth regulation will likely help us better understand growth control and could provide novel insights and metabolic designs towards enhancing microalgae growth performance. Dedicated experimentation on the energy (ATP) pools, H₂O₂/NO ratio control, and amino acids metabolism may open new paths to explore novel applications to boost microalgae growth.