This work explores astaxanthin (AXT), a valuable xanthophyll ketocarotenoid pigment with significant health benefits and diverse applications across various industries. It discusses the prevalence of synthetic AXT, and the development of natural-based alternatives derived from microorganisms such as microalgae, bacteria, and yeast. The chapter examines the potential of microbial AXT production, highlighting the advantages and challenges associated with natural AXT. Key microorganisms like Haematococcus pluvialis, Paracoccus carotinifaciens, and Phaffia rhodozyma are emphasized for their role in commercially producing this valuable ketocarotenoid. The narrative covers the complexities and opportunities in microbial AXT production, from cell structure implications to downstream processing strategies. Additionally, the chapter addresses current applications, commercialization trends, and market dynamics of natural microbial AXT, emphasizing the importance of cost-effective production, regulatory compliance, and technological advancements to reduce the market cost of the final product. As demand for natural microbial-based AXT rises, this chapter envisions a future where research, innovation, and collaboration drive sustainable and competitive microbial AXT production, fostering growth in this dynamic market.

Astaxanthin, a ketone carotenoid known for its high antioxidant activity, holds significant potential for application in nutraceuticals, aquaculture, and cosmetics. The increasing market demand necessitates a higher production of astaxanthin using Phaffia rhodozyma. Despite extensive research efforts focused on optimizing fermentation conditions, employing mutagenesis treatments, and utilizing genetic engineering technologies to enhance astaxanthin yield in P. rhodozyma, progress in this area remains limited. This review provides a comprehensive summary of the current understanding of rough metabolic pathways, regulatory mechanisms, and preliminary strategies for enhancing astaxanthin yield. However, further investigation is required to fully comprehend the intricate and essential metabolic regulation mechanism underlying astaxanthin synthesis. Specifically, the specific functions of key genes, such as crtYB, crtS, and crtI, need to be explored in detail. Additionally, a thorough understanding of the action mechanism of bifunctional enzymes and alternative splicing products is imperative. Lastly, the regulation of metabolic flux must be thoroughly investigated to reveal the complete pathway of astaxanthin synthesis. To obtain an in-depth mechanism and improve the yield of astaxanthin, this review proposes some frontier methods, including: omics, genome editing, protein structure-activity analysis, and synthetic biology. Moreover, it further elucidates the feasibility of new strategies using these advanced methods in various effectively combined ways to resolve these problems mentioned above. This review provides theory and method for studying the metabolic pathway of astaxanthin in P. rhodozyma and the industrial improvement of astaxanthin, and provides new insights into the flexible combined use of multiple modern advanced biotechnologies.

Effective metabolic regulators play an essential role in regulating astaxanthin biosynthesis in Phaffia rhodozyma. In this study, it was found that 5 mM glutamate increased the astaxanthin yield and biomass of P. rhodozyma D3 to 22.34 mg/L and 6.12 g/L, which were 1.22 and 1.33 times higher than the control group, respectively. Meanwhile, glucose uptake was increased and the level of reactive oxygen species (ROS) was reduced with 5 mM glutamate. To further explore the interrelationship between glutamate and astaxanthin synthesis, the energy metabolism of P. rhodozyma D3 with and without glutamate was analysed. Glutamate promoted the Embden-Meyerhof-Parnas pathway (EMP) metabolic flux, modulated the tricarboxylic acid (TCA) cycle and the pentose phosphate pathway (PPP), activated the ornithine cycle and purine metabolism, and provided more ATP and NADPH for astaxanthin accumulation. This study clarified the possible mechanism by which glutamate promoted astaxanthin accumulation in P. rhodozyma.

Astaxanthin has multiple physiological functions and is applied widely. The yeast Phaffia rhodozyma is an ideal source of microbial astaxanthin. However, the stress conditions beneficial for astaxanthin synthesis often inhibit cell growth, leading to low productivity of astaxanthin in this yeast. In this study, 1 mg/L melatonin (MT) could increase the biomass, astaxanthin content, and yield in P. rhodozyma by 21.9, 93.9, and 139.1%, reaching 6.9 g/L, 0.3 mg/g DCW, and 2.2 mg/L, respectively. An RNA-seq-based transcriptomic analysis showed that MT could disturb the transcriptomic profile of P. rhodozyma cell. Furthermore, differentially expressed gene (DEG) analysis show that the genes induced or inhibited significantly by MT were mainly involved in astaxanthin synthesis, metabolite metabolism, substrate transportation, anti-stress, signal transduction, and transcription factor. A mechanism of MT regulating astaxanthin synthesis was proposed in this study. The mechanism is that MT entering the cell interacts with components of various signaling pathways or directly regulates their transcription levels. The altered signals are then transmitted to the transcription factors, which can regulate the expressions of a series of downstream genes as the DEGs. A zinc finger transcription factor gene (ZFTF), one of the most upregulated DEGs, induced by MT was selected to be overexpressed in P. rhodozyma. It was found that the biomass and astaxanthin synthesis of the transformant were further increased compared with those in MT-treatment condition. Combining MT-treatment and ZFTF overexpression in P. rhodozyma, the biomass, astaxanthin content, and yield were 8.6 g/L, 0.6 mg/g DCW, and 4.8 mg/L and increased by 52.1, 233.3, and 399.7% than those in the WT strain under MT-free condition. In this study, the synthesis and regulation theory of astaxanthin is deepened, and an efficient dual strategy for industrial production of microbial astaxanthin is proposed.

Microorganisms, such as yeasts, filamentous fungi, bacteria, and microalgae, have gained significant attention due to their potential in producing commercially valuable natural carotenoids. In recent years, Phaffia rhodozyma yeasts have emerged as intriguing non-conventional sources of carotenoids, particularly astaxanthin and β-carotene. However, the shift from academic exploration to effective industrial implementation has been challenging to achieve. This study aims to bridge this gap by assessing various scenarios for carotenoid production and recovery. It explores the use of ionic liquids (ILs) and bio-based solvents (ethanol) to ensure safe extraction. The evaluation includes a comprehensive analysis involving Life Cycle Assessment (LCA), biocompatibility assessment, and Techno-Economic Analysis (TEA) of two integrated technologies that utilize choline-based ILs and ethanol (EtOH) for astaxanthin (+β-carotene) recovery from P. rhodozyma cells. This work evaluates the potential sustainability of integrating these alternative solvents within a yeast-based bioeconomy.

Phaffia rhodozyma is a basidiomycetous yeast characterized by its production of the carotenoid pigment astaxanthin, which holds high commercial value for its significance in aquaculture, cosmetics and as nutraceutics, and the UV-B-absorbing compound mycosporine-glutaminol-glucoside (MGG), which is of great biotechnological relevance for its incorporation into natural sunscreens. However, the industrial exploitation has been limited to the production of astaxanthin in small quantities. On the other hand, the accumulation of MGG in P. rhodozyma was recently reported and could add value to the simultaneous production of both metabolites. In this work, we obtain a mutant strain that overproduces both compounds, furthermore we determined how the accumulation of each is affected by the carbon-to-nitrogen ratio and six biotic and abiotic factors. The mutant obtained produces 159% more astaxanthin (470.1 μg g-1) and 220% more MGG (57.9 mg g-1) than the parental strain (295.8 μg g-1 and 26.2 mg g-1 respectively). Furthermore, we establish that the carotenoids accumulate during the exponential growth phase while MGG accumulates during the stationary phase. The carbon-to-nitrogen ratio affects each metabolite differently, high ratios favoring carotenoid accumulation while low ratios favoring MGG accumulation. Finally, the accumulation of both metabolites is stimulated only by photosynthetically active radiation and low concentrations of hydrogen peroxide. The mutant strain obtained is the first hyper-productive mutant capable of accumulating high concentrations of MGG and astaxanthin described to date. The characterization of how both compounds accumulate during growth and the factors that stimulate their accumulation, are the first steps toward the future commercial exploitation of strains for the simultaneous production of two biotechnologically important metabolites.

Phaffia rhodozyma represents an excellent microbial resource for astaxanthin production. However, the yeast\'s low astaxanthin productivity poses challenges in scaling up industrial production. Although P. rhodozyma originates from plant material, and phytohormones have demonstrated their effectiveness in stimulating microbial production, there has been limited research on the effects and mechanisms of phytohormones on astaxanthin biosynthesis in P. rhodozyma. In this study, the addition of exogenous salicylic acid (SA) at a concentration as low as 0.5 mg/L significantly enhanced biomass, astaxanthin content, and yield by 20.8%, 95.8% and 135.3% in P. rhodozyma, respectively. Moreover, transcriptomic analysis showed that SA had discernible impact on the gene expression profile of P. rhodozyma cells. Differentially expressed genes (DEGs) in P. rhodozyma cells between the SA-treated and SA-free groups were identified. These genes played crucial roles in various aspects of astaxanthin and its competitive metabolites synthesis, material supply, biomolecule metabolite and transportation, anti-stress response, and global signal transductions. This study proposes a regulatory mechanism for astaxanthin synthesis induced by SA, encompassing the perception and transduction of SA signal, transcription factor-mediated gene expression regulation, and cellular stress responses to SA. Notably, the polyamine transporter gene (PT), identified as an upregulated DEG, was overexpressed in P. rhodozyma to obtain the transformant Prh-PT-006. The biomass, astaxanthin content and yield in this engineered strain could reach 6.6 g/L, 0.35 mg/g DCW and 2.3 mg/L, 24.5%, 143.1% and 199.0% higher than the wild strain at the SA-free condition, respectively. These findings provide valuable insights into potential targets for genetic engineering aimed at achieving high astaxanthin yields, and such advancements hold promise for expediting the industrialization of microbial astaxanthin production.

This study aimed to produce carotenoids by Phaffia rhodozyma in a stirred-tank bioreactor under the influence of magnetic fields (MF) and to evaluate a sustainable approach to recover them from the yeast biomass. MF application proved to be effective in increasing 8.6 and 22.9 % of β-carotene and astaxanthin production, respectively. Regarding solid-liquid extraction (SLE), the ability of aqueous and ethanolic solutions of protic ionic liquids (PILs) was determined. β-carotene and astaxanthin recovery yields increased with the anion alkyl chain length hydrophobicity. [Pro][Oct]:EtOH (50 % v v-1) was selected as the effective solvent. Moreover, it led to improvement in carotenoid stability at different storage temperatures over time in comparison with the control. This study is one of the first to describe an effective and sustainable approach to move carotenoid production from shake flasks to a bioreactor under the influence of MF and recover carotenoids from P. rhodozyma biomass.

Astaxanthin is widely used in food, aquaculture, cosmetics, and pharmaceuticals due to its strong antioxidant activity and coloring ability, but its production from Phaffia rhodozyma remains the main challenge due to the high fermentation cost and low content of carotenoid. In this study, the production of carotenoids from food waste (FW) by a P. rhodozyma mutant was investigated. P. rhodozyma mutant screened by UV mutagenesis and flow cytometry could stably produce high carotenoids at 25°C, with carotenoid production (32.9 mg/L) and content (6.7 mg/g), respectively, increasing by 31.6% and 32.3% compared with 25 mg/L and 5.1 mg/g of wild strain. Interestingly, the carotenoid production reached 192.6 mg/L by feeding wet FW, which was 21% higher than batch culture. The 373 g vacuum freeze-dried products were obtained from the fermentation of 1 kg FW by P. rhodozyma, which contained 784 mg carotenoids and 111 mg astaxanthin. The protein, total amino acids, and essential amino acids content of the fermentation products were 36.6%, 40.5%, and 18.2% (w/w), respectively, and lysine-added fermentation products had the potential of high-quality protein feed source. This study provides insights for the high-throughput screening of mutants, astaxanthin production, and the development of the feed potential of FW.

The attractive biological properties and health benefits of natural astaxanthin (AXT), including its antioxidant and anti-carcinogenic properties, have garnered significant attention from academia and industry seeking natural alternatives to synthetic products. AXT, a red ketocarotenoid, is mainly produced by yeast, microalgae, wild or genetically engineered bacteria. Unfortunately, the large fraction of AXT available in the global market is still obtained using non-environmentally friendly petrochemical-based products. Due to the consumers concerns about synthetic AXT, the market of microbial-AXT is expected to grow exponentially in succeeding years. This review provides a detailed discussion of AXT\'s bioprocessing technologies and applications as a natural alternative to synthetic counterparts. Additionally, we present, for the first time, a very comprehensive segmentation of the global AXT market and suggest research directions to improve microbial production using sustainable and environmentally friendly practices. KEY POINTS: • Unlock the power of microorganisms for high value AXT production. • Discover the secrets to cost-effective microbial AXT processing. • Uncover the future opportunities in the AXT market.