Integrating astaxanthin production by using Haematococcus lacustris with the LDP phycoremediation provides multiple benefits. However, as a highly sensitive organism, the cultivation needs to be regulated, such as selecting the most favorable media, regulating light intensity, adjusting LDP conentration, and evaluating the effect of NaCl on astaxanthin biosynthesis. In this study, we developed a new cultivation method, three-stage continuous cultivation system, by regulating the light intensity at each stage of microalgae growth. Based on the evaluation, the ideal medium was MES-volvox. The favorable light intensity for the adaptation phase was 600 lux, with the best LDP concentration of 3.75% and NaCl 1 g/L. The desirable light intensity in the three-stage cultivation system was 600 lux (days 0-6), 1,200 lux (days 6-16), and 3,500 lux (days 16-30). This system produced density, biomass, and astaxanthin content up to 91.54 × 104 cells/mL, 1 g/L, 0.83 g/L, respectively. Remarkably, the enrichment of NaCl did not reduce the density, carbohydrate, protein, or pigment content. Therefore, it may serve as a safe astaxanthin-enhancing stressor in the cultivation of this microalgae.

Astaxanthin is an attractive carotenoid that contains beneficial properties as a powerful antioxidant capable of preventing and treating degenerative diseases, while Xanthophyllomyces dendrorhous is one of its main sources. Astaxanthin from X. dendrorhous is generated in response to oxidative or metabolic stress but, to increase the astaxanthin yields, it is necessary to stimulate the metabolic pathway in a way that allows the transcriptional reprogramming of involved genes. Research about the stimulation of carotenoids metabolic pathway posed an important question, could the expression of genes involved in the oxidative stress response be reprogrammed to enhance astaxanthin biosynthesis? Several investigations have demonstrated that astaxanthin biosynthesis is assisted and regulated by electron donor proteins similar to cytochrome P450 reductase (encoded by crtR). Here, we assessed some of these reprogrammed transcriptional responses in wild-type X. dendrorhous under two stimulating conditions: (i) abscisic acid (ABA), and (ii) 6-benzylaminopurine (6-BAP) combined with H2O2. mRNA levels of cytochrome P450 genes (CYP51, CYP61, and crtR), electron donor CBR.1, P450 regulator DAP1, and mitochondrial defense genes (CAT, SOD, GPX, and NDUFA), increased during astaxanthin biosynthesis stimulation. The highest stimulating responses were observed using 6-BAP in combination with H₂O₂ between 38 and 50h of fermentation. Moreover, the profiles of organic acids, glycerol, and volatile compounds were altered, thereby redirecting the carbon flux toward astaxanthin biosynthesis. Nonetheless, the X. dendrorhous wild-type strain required several stimulating agents additions. Astaxanthin overproduction may be achieved by reprogramming the transcriptional levels of genes involved in the biosynthetic pathway, as well as by enhancing cellular responses that mitigate oxidative stress, including those mediated by cytochrome P450 and mitochondrial damage-response proteins.

Harnessing machine learning for astaxanthin biosynthesis: Neural network-driven optimization of Haematococcus pluvialis

Astaxanthin, derived from Haematococcus pluvialis, is a potent antioxidant with significant therapeutic potential. However, its large-scale commercialization is hindered by the "thick-wall challenge", a phenomenon where the stress conditions required for astaxanthin accumulation also trigger the formation of resistant secondary cell walls. This challenge complicates extraction and reduces bioaccessibility, thereby increasing production costs. Recent advancements have focused on uncoupling astaxanthin biosynthesis from cell wall reinforcement, utilizing metabolic engineering and strain selection to reduce wall formation while maintaining high yields. Furthermore, green extraction techniques, such as electrotechnologies and ionic liquids, are being explored to improve efficiency and environmental sustainability. This review synthesizes these innovations, including biorefinery systems that maximize biomass valorization, and discusses emerging clinical applications. We highlight the challenges in bridging the gap between laboratory successes and clinical translation, and suggest future directions for resolving the thick-wall challenge, advancing astaxanthin production, and expanding its therapeutic uses in nutraceuticals and pharmaceuticals.

With the continuous growth of the global population, todays food system is facing severe challenges. Researchers have begun to explore how to maximize possible yield while minimizing environmental pollution. During the last few years, there has been extraordinary research in biosynthesis of astaxanthin, a ketocarotenoid that offers numerous properties such as antioxidant, anticancer, and eye-protective function. This study focuses on discovery of the best possible pathway, gene editing strategies, host organism, and feedstock for biosynthesis of astaxanthin. Ultimately, we concluded the final biosynthetic pathway with specially focused gene modification, chosen the host organism as Saccharomyces cerevisiae, and sweet potato juice as the feed. Through genetic engineering methodology, this study demonstrates that astaxanthin can be efficiently produced by microorganisms, reducing the environmental impact of traditional extraction from aquatic organisms and addressing the safety concerns of chemical synthesis. These findings thereby have profound implications for the food system.

Light boosts cell proliferation and astaxanthin accumulation in nitrogen-starved Chromochloris zofingiensis via TOR signaling pathway.

Astaxanthin is a valuable carotenoid with potent antioxidant properties and has broad applications in the pharmaceutical, nutraceutical, and cosmetic industries. In this study, Rhodotorula toruloides CB6-10/1, isolated from Canna indica L. flowers, was evaluated for astaxanthin production. The orange-red pigment was confirmed as astaxanthin via thin-layer chromatography and high-performance liquid chromatography, with quantification performed by spectrophotometry. Comprehensive genome analysis and production optimization of R. toruloides CB6-10/1 confirmed the presence of key astaxanthin biosynthesis genes, such as CrtYB, CrtI, CrtW, CrtZ, and CrtR, which facilitate the conversion of β-carotene to astaxanthin through hydroxylation and ketolation. The key parameters, including carbon and nitrogen sources, their concentrations, trace elements, agitation speed, and pH, were systematically evaluated to optimize production. Although copper appeared beneficial in the Plackett-Burman screening, its effect and those of other metals were not significant. Optimization using Response Surface Methodology for cost-effective nitrogen sources determined that a combination of 1.10g/L yeast extract, 10.0g/L peptone, and 0.50g/L ammonium sulfate yielded the maximum astaxanthin production of 4.728mg/L, under cultivation conditions of pH 4.5, 200rpm, and 30g/L glucose, representing a fourfold increase compared with the basal medium. This optimization not only enhances pigment production efficiency but also reduces dependency on costly trace elements, improving process scalability and economic feasibility. Overall, these results demonstrate R. toruloides CB6-10/1 as a promising microbial source for sustainable astaxanthin production with potential further applications.

A comprehensive approach to optimized ultrasound stimulation for enhanced astaxanthin synthesis in Haematococcus pluvialis: The cultivation strategy.

Regulation of astaxanthin accumulation in Phaffia rhodozyma under titanium dioxide and rapeseed oil stress by amino acid metabolism.

Stress-induced astaxanthin biosynthesis in Phaffia rhodozyma: Bridging mechanistic understanding to industrial Feasibility.

Flow-through and cyclic fed-batch cultivation of the green microalga Haematococcus pluvialis in recirculating aquaculture effluent: Strategies for sustained biomass production and astaxanthin biosynthesis

Functional identification of carotene hydroxylases from the green alga Chromochloris zofingiensis.

Astaxanthin, a high-value keto-carotenoid with exceptional antioxidant capacity, has significant commercial potential for industrial applications. Microbial biosynthesis via engineered synthetic yeast presents an environmentally sustainable production platform. In this study, we developed a multistrategy optimization framework to enhance astaxanthin biosynthesis in synthetic yeast. Our systematic approach initiated with the construction of a de novo astaxanthin pathway in synthetic yeast strain 2369R, achieving a baseline production of 0.11 mg/L. Through rigorous screening of heterologous enzymes, we identified optimal variants of β-carotene hydroxylase (CrtZ) and ketolase (CrtW) that increased the titer to 0.65 mg/L. Subsequently, the combined enhancement of MVA pathway flux (via tHMG1 overexpression) and lipid metabolism regulation (through DGK1 overexpression) synergistically boosted astaxanthin production to 2.59 mg/L. Through combinatorial implementation of genome-scale diversification using the Synthetic Chromosome Rearrangement and Modification by LoxP-mediated Evolution (SCRaMbLE) system coupled with an absorption-based semi-high-throughput screening platform (A450/A600), we successfully isolated an elite mutant strain, YgM97, that achieved 6.85 mg/L astaxanthin production in shake-flask culture. This represents a remarkable 61.27-fold enhancement compared with the parental strain. Transcriptomic and genomic analyses subsequently revealed the potential molecular mechanisms underlying this significant yield improvement. Collectively, this study demonstrates the powerful synergy between rational metabolic engineering and randomized genome evolution, providing a novel paradigm for high-value compound biosynthesis in a microbial chassis.

Astaxanthin is a strong antioxidant and is widely applied in food industry. The yeast Phaffia rhodozyma is an ideal microbial astaxanthin resource. However, the nitrogen-deficiency stress, which is beneficial for astaxanthin synthesis, often impairs cell growth, leading to low productivity. In this study, an imbalance between cellular growth and astaxanthin synthesis in P. rhodozyma under nitrogen-deficient (H) and nitrogen-sufficient (L) conditions was identified. A comparative RNA-seq transcriptome analysis between the H and L groups revealed well-discriminated patterns. The differentially expressed genes (DEGs) indicated that the regulation of nitrogen deficiency does not occur directly in the astaxanthin biosynthesis pathway but rather operates at the global cellular level, involving processes such as central and energy metabolism, antioxidative stress responses, signal transduction, competitive metabolic pathways, and material transportation. Based on these findings, a regulatory mechanism is proposed, which involves cellular sensing of nitrogen sources in the medium, alterations in signaling pathways that direct effectors, and the regulation of multiple downstream target genes through post-translational modifications, protein interactions, gene transcription, and the protein and metabolite levels. Six DEGs were overexpressed in the wild strain (WT) of Phaffia rhodozyma, and the mutants M2 and M6, expressing the NHEJ gene for DNA repair and the ferric reductase gene, showed higher biomass and astaxanthin content compared with the WT strain under nitrogen-deficient conditions. However, the remaining mutants exhibited unchanged or even reduced biomass and astaxanthin productivity. Subsequently, a co-expression mutant (M7) carrying the two DEGs was constructed. This mutant exhibited further increases in both biomass and astaxanthin content, with 61.5 and 133.3% higher yields than the WT strain, respectively, and a 265.8% increase in final astaxanthin production.

Efficient production of astaxanthin in Yarrowia lipolytica through metabolic and enzyme engineering.

Population growth throughout the world has led to increased pollution and overconsumption of fossil resources. Microalgae are increasingly recognized as sustainable biofactories for producing lipids and astaxanthin, two commercially significant metabolites with wide-ranging applications in biofuel, pharmaceutical, cosmetic, and nutraceutical industries. Enhancing the yields of these compounds remains a major challenge due to growth-productivity trade-offs and limited understanding of regulatory mechanisms. This review aims to bridge that gap by providing a comprehensive and comparative analysis of traditional and modern strategies employed to enhance lipid and astaxanthin production in microalgae. We critically evaluate stress-based methods (e.g., salinity, light, nutrient limitation), phytohormone treatments, cultivation system optimization, and genome editing technologies, including CRISPR/Cas9. Special emphasis is given to gene-level responses and pathway-level regulation involved in these enhancements. This review article highlights the novel synchronization between astaxanthin and fatty acid biosynthesis under various stress conditions which emphasizes the role of diacylglycerol acyltransferase (DGAT) enzymes to enhance astaxanthin accumulation. Editing technologies with base suggest a novel strategy to reduce off-target effects and enhance metabolic efficiency related to lipid and astaxanthin biosynthesis.

Developing high-value-added cotton via rebuilding astaxanthin biosynthesis pathway.

Background: Natural astaxanthin, a commercially valuable carotenoid, is primarily sourced from Haematococcus pluvialis, a microalga known for its remarkable resilience to environmental stress. Methods: In this study, the physiological and transcriptomic responses of H. pluvialis to ND were investigated at various time points under high light conditions. Results: Under high light conditions, nitrogen deprivation (ND) enhances astaxanthin content (33.23 mg g−1) while inhibiting the formation of the secondary cell wall (SCW), increasing astaxanthin content by 29% compared to the nitrogen-replete group (25.64 mg g−1); however, the underlying mechanisms remain unclear. ND reduced chlorophyll fluorescence parameters, elevated reactive oxygen species (ROS) levels, and increased starch and total sugar accumulation while decreasing protein and lipid content. Fatty acid content increased on the first day but had declined by the fifth day. A transcriptomic analysis revealed substantial alterations in gene expression in response to ND. Genes associated with the TCA cycle, glycolysis, astaxanthin biosynthesis, and cell motility were upregulated, while those involved in photosynthesis, lipid synthesis, ribosome biogenesis, amino acid synthesis, and SCW synthesis were downregulated. Additionally, ND modulated the expression of genes involved in ROS scavenging. Conclusions: These findings provide critical insights into the adaptive mechanisms of H. pluvialis in response to ND under high light, contributing to the development of strategies for enhanced production of astaxanthin-rich motile cells.

Objective: This study evaluates recent advances in deep eutectic solvent (DES) systems for astaxanthin extraction from Haematococcus pluvialis, comparing their efficiency and limitations. Theoretical Framework: The review synthesizes knowledge on H. pluvialis biology and astaxanthin biosynthesis, limitations of physical/chemical extraction methods, and DES design principles for algal biorefining. Method: A systematic literature review was conducted in Web of Science databases using search terms aligned with the research objectives. Selected articles were analyzed according to inclusion/exclusion criteria to compose the discussion. Results and Discussion: Recent DES innovations have transformed astaxanthin extraction from H. pluvialis, achieving 83-99.6% yields under mild conditions (25-50°C). Hydrophobic DES (thymol-oleic acid) demonstrated dual extraction-stabilization functions, while IL-DES hybrids enabled selective fractionation. Aqueous DES-ATPS systems reached 99.6% efficiency through targeted cell wall disruption, and acidic DES (menthol-acetic acid) combined 91.3% recovery with extended extract stability. Research Implications: The reviewed advances position DES as sustainable alternatives for industrial astaxanthin production, offering integrated extraction-stabilization processes and biorefinery potential through biomass valorization. GRAS-compliant formulations address safety concerns, though future work should optimize solvent viscosity. Originality/Value: This is the first comparative analysis of DES systems for H. pluvialis biorefining, providing design guidelines for industrial implementation.

Microalgae have evolved a diverse carotenoid profile, enabling efficient light harvesting and photoprotection. Previous studies have demonstrated the feasibility of genome editing in the green algal model species Chlamydomonas reinhardtii, leading to significant modifications in carotenoid accumulation. By overexpressing a fully redesigned β-carotene ketolase (bkt), the metabolic pathway of C. reinhardtii was successfully redirected toward astaxanthin biosynthesis, a high-value ketocarotenoid with exceptional antioxidant properties, naturally found in only a few microalgal species. In this study, a tailor-made double knockout targeting lycopene ε-cyclase (LCYE) and zeaxanthin epoxidase (ZEP) was introduced as a background for bkt expression to ensure higher substrate availability for bkt enzyme. The increased zeaxanthin availability resulted in a 2-fold increase in ketocarotenoid accumulation compared to the previously engineered bkt1 or bkt5 strain in the UVM4 background. Specifically, the best Δzl-bkt-expressing lines reached 2.84 mg/L under low light and 2.58 mg/L under high light, compared to 1.74 mg/L and 1.26 mg/L, respectively, in UVM4-bkt strains. These findings highlight the potential of rationally designed microalgal host strains, developed through genome editing, for biotechnological applications and high-value compound production.