How to Get Synthetic Astaxanthin?

Astaxanthin (C40H52O4) is a keto-type carotenoid with the chemical name 3,3'-dihydroxy-4,4'-dione-beta,beta'-carotene. Its chemical structure is shown in Figure 1: four isoprene units are connected by conjugated double bonds, with two isoprene units at each end to form a six-membered ring.

Astaxanthin has three optical isomers. The difference between the three optical isomers is that among the all-trans astaxanthin stereoisomers, the antioxidant activity of the racemic astaxanthin is the lowest, the dextrorotatory astaxanthin has the strongest ability to scavenge free radicals, and the levorotatory astaxanthin has a stronger inhibitory effect on lipid peroxidation and immune activity [1-2]. Astaxanthin has a variety of effects in addition to its antioxidant activity, including anticancer, anti-inflammatory, and anti-diabetic effects [3]. In addition, astaxanthin is the only carotenoid that can penetrate the blood-brain and blood-retina barriers and has a positive effect on the central nervous system and brain function. Therefore, astaxanthin is widely used in food, healthcare, cosmetics, and feed additives [4].

Natural astaxanthin is mainly found in the marine environment in the form of free and esterified astaxanthin. Free astaxanthin is unstable and easily oxidized. Due to the presence of hydroxyl groups in the terminal cyclic structure, astaxanthin is easily combined with fatty acids to form astaxanthin esters and exist stably. About 95% of the astaxanthin molecules in Haematococcus pluvialis are esterified with fatty acids and stored in cytoplasmic lipid bodies rich in triacylglycerols [5].

Esterified astaxanthin is divided into astaxanthin monoesters and astaxanthin diesters according to the fatty acids they are bound to. H. pluvialis can accumulate up to 4% astaxanthin (dry weight), and HOLTIN et al. [6] found that 95% of the astaxanthin accumulated under strong light stress was esterified with fatty acids. Although the mechanism of the interaction between astaxanthin and fatty acids in organisms is still unclear, the stoichiometry of astaxanthin and fatty acid biosynthesis has been observed in H. pluvialis. Chen et al. [7] analyzed the coordination mechanism between the two biosynthesis pathways of astaxanthin and fatty acids in H. pluvialis  the coordination mechanism between the two biosynthesis pathways of astaxanthin and fatty acids, and revealed that this interaction occurs at the metabolite level rather than the transcriptional level. In vivo and in vitro experiments have shown that the esterification of astaxanthin promotes its formation and accumulation.

At present, the methods for preparing astaxanthin at home and abroad can be divided into two main categories: chemical synthesis and biosynthesis. Chemically synthesized astaxanthin is a mixture of three structures [5,8] (l-: racemic: d-1:2:1), and is mainly used as an industrial dye. However, it is not allowed to be used in the food and pharmaceutical fields. Biosynthesized astaxanthin is allowed to be used in the food and pharmaceutical fields. Some microalgae, fungi, bacteria and specific plant species have the ability to synthesize astaxanthin in nature.

H. pluvialis is considered to be one of the most promising astaxanthin producers in nature. In recent years, it has been found that many strains of the genus Thraustochytrium also have the ability to synthesize astaxanthin [9], and the synthetic levo-astaxanthin accounts for more than 90% of the total astaxanthin. Previous researchers have reviewed the chemical synthesis methods and pathways of astaxanthin [10] and outlined the current production levels of natural astaxanthin producers [11]. This review will focus on astaxanthin biosynthesis and the anabolic pathways of astaxanthin in different organisms, based on a review of chemical synthesis routes, with a focus on chemical synthesis and biosynthesis. This article aims to provide readers with a macro-level overview of astaxanthin biosynthesis and help them quickly understand the research progress in astaxanthin synthesis methods.

1 Chemical synthesis of astaxanthin

Chemical synthesis of astaxanthin can be divided into total synthesis and semi-synthesis. Total synthesis of astaxanthin uses chemical raw materials as the starting material to obtain astaxanthin through chemical synthesis reactions. Semi-synthesis uses carotenoids such as canthaxanthin, lutein and zeaxanthin as the starting material to prepare astaxanthin.

1.1 Chemical total synthesis of astaxanthin

Both at home and abroad, a series of studies have been carried out on the chemical total synthesis route of astaxanthin. The two major enterprises in the chemical synthesis of astaxanthin are Hoffmann-La Roche and BASF. The two companies use similar synthetic routes to produce astaxanthin by chemical synthesis, using the C9 + C6 → C15, 2C15 + C10 → C40 route. Hoffmann-La Roche uses 6-oxo-isophthalone as a raw material [12]. First, acetone and formaldehyde are used to generate α-β unsaturated butenone by hydroformylation condensation and dehydration under weak alkaline conditions. Then, 1, 2-nucleophilic addition to form a six-carbon tertiary alcohol, which is rearranged under the action of sulfuric acid. The hydroxyl group of the product is protected to react with 6-oxoisophthalone, and finally a bilateral Wittig reaction occurs under the action of a strong base to synthesize astaxanthin.

In the synthetic route of BASF [13-14], the intermediate 6-carbon-1-yne-3-ol is not first acidified and rearranged, but the hydroxyl group is protected and undergoes a series of transformations with 6-oxoisophthalone, and the rearrangement occurs during the transformation, and the final target product astaxanthin is obtained. The synthetic route for astaxanthin used by Chinese researcher Pi Qing, etc. [10] is different from the foreign synthetic route. It uses the synthetic route C13 + C2 → C15, 2C15 + C10 → C40 to prepare astaxanthin. He uses α-ionone as a raw material, which is treated with m-chloroperoxybenzoic acid, undergoes a series of intermediate transformations, is acidified and rearranged under the action of hydrobromic acid, and then reacts with triphenylphosphine to form a pentadecyl triphenylphosphonium salt, and finally undergoes a two-way Wittig reaction to form astaxanthin. The unique feature of the synthetic route of Pi Qingping et al. [10] is the use of a new method to synthesize the key intermediate C15 compound. The starting material of this method is easy to obtain, the reaction has high selectivity, and the overall yield is high.

The Witting reaction is a characteristic reaction of the total synthesis route of astaxanthin. This type of synthesis route has the advantages of simple technology and low cost. Although the processes of the two routes of Hoffmann-La Roche and BASF are very complex, the production process is long, the control of the intermediate process is difficult and strict, but the synthesis cost is low, the price is cheap, and industrial production has been realized. It is the main industrial source of astaxanthin supply on the market (Figure 2).

1.2 Chemical semi-synthesis of astaxanthin

Semi-synthesis is a method that uses carotenoids such as canthaxanthin, lutein and zeaxanthin as raw materials to prepare astaxanthin [15]. The classic method uses lutein as the starting material, and lutein is catalysed by an alkali to undergo an isomerisation reaction to produce zeaxanthin. Using 1,2-propanediol as a solvent and potassium hydroxide as a catalyst, the reaction was carried out at 110 °C for 168 h. Zeaxanthin was directly oxidized to astaxanthin under the action of iodine and sodium bromate.

When canthaxanthin is used as the raw material, astaxanthin is synthesized through four processes: alkalization, silylation, epoxidation, and hydrolysis. It is characterized by fast synthesis and high yield (approximately 60% yield). Due to the high cost of canthaxanthin and the certain dangers in the production process, it is difficult to achieve large-scale industrial production at present. Compared with the total synthesis method, the semi-synthesis method has high biological activity, but low yield, and it is difficult to achieve large-scale production (Figure 3).

2 Biosynthesis of astaxanthin

2.1 Astaxanthin metabolic pathway

Astaxanthin is the end product of C40 carotenoid metabolism. Carotenoid synthesis in living organisms can be divided into three stages: the first is central carbon metabolism, the second is the synthesis of carotenoid precursors isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DAMPP), and the third is carotenoid synthesis.

The first stage is the central carbon metabolism cycle. Organisms use glucose, fructose and other carbon sources to synthesize substances such as glycerol-3-phosphate (G3P), pyruvate and acetyl-CoA via the glycolytic pathway. Glycerol-3-phosphate, pyruvate and acetyl-CoA flow into the next stage as precursors of IPP and DAMPP. At the same time, some acetyl-CoA enters the tricarboxylic acid cycle (TCA). The tricarboxylic acid cycle is the final metabolic pathway for the three major nutrients (sugars, lipids, and amino acids) and is also the hub that connects the metabolism of sugars, lipids, and amino acids. The tricarboxylic acid cycle synthesizes a variety of metabolites that flow in all directions of cellular metabolism. At the same time, the tricarboxylic acid cycle also produces a large amount of adenosine triphosphate (ATP) and reduced coenzyme II (NADPH), which provide energy and reducing power for the transformation of substances in the latter two stages.

The second stage is the synthesis of IPP and DAMPP, the precursors of carotenoids. There are two natural synthetic pathways for the synthesis of IPP and DAMPP: the methyl-D-erythritol-4-phosphate (MEP) pathway and the mevalonate (MVA) pathway. The MVA pathway is mainly found in eukaryotes and archaea, and is the only pathway for IPP formation in archaea, yeast and some gram-positive bacteria [16]. The MEP pathway is found in plants, algae and most bacteria. These plants and algae can produce IPP through the MVA pathway in the cytoplasm and the MEP pathway in the plastids [17-18]. Until the end of the 20th century, MVA was considered to be the only source of isopentenyl diphosphate precursors for the synthesis of terpenoids, including carotenoids. In the MVA pathway, acyl-CoA is converted to hydroxymethyl-trimethyl-pentanoyl-coenzyme coenzyme (HMG-CoA). HMG-CoA is converted to methyl-D-malonyl-D-glutaronate by HMG-CoA reductase, and methyl-D-malonyl-D-glutaronate is converted to IPP through a series of phosphorylation reactions.

In the MEP pathway, the molecules of glycerol triphosphate and pyruvate undergo condensation and isomerization reactions to form MEP. After MEP is coupled with cytidine triphosphate, a series of phosphorylation reactions are carried out to form IPP, which isomerizes to produce the isomer DAMPP isomerization to generate the isomer DAMPP. Early studies showed that the MVA pathway has been lost in many green algae and red algae, and the MEP pathway is the only pathway for IPP synthesis in Haematococcus pluvialis [19]. As research continues to deepen, multiple results indicate that the phenomenon that the MEP pathway is the only pathway for IPP synthesis may be common in green algae cells [20].

However, most of the MEP pathway genes were not found in the transcriptome data of Aurantiochytrium sp. SK4, and the mevalonate (MVA) pathway was involved in the formation of IPP in Aurantiochytrium sp. SK4 cells [21]. In addition, Henry et al. [22] discovered a third pathway in plants that is catalyzed by cytosolic isopentenyl phosphate kinase. This MVA pathway is the same as the other MVA pathway found in some archaea and the Chlorophyta phylum of bacteria in the process of forming MVAP. The difference is that MVAP in bacteria is converted by phosphomethylpentenate decarboxylase (MPD) into isopentenyl phosphate (IP), which is then phosphorylated to IPP by isopentenyl monophosphate kinase (IPK). Although both the MVA and MEP pathways are present in plants, the MEP pathway is the main source of carotenoid precursors in plants [23].

The third stage is the synthesis of carotenoid-like substances. DAMPP and IPP are synthesized in a ratio of 1:3 by the action of pyrophosphate synthase (CrtE) to form farnesyl diphosphate (FPP). FPP is then converted by pyrophosphate synthase to form geranylgeranyl pyrophosphate (GGPP).  GGPP is condensed by octahydro-lycopene synthase (CrtB) and phytoene desaturase (CrtI) to form lycopene, which is synthesized into β-carotene by lycopene cyclase (CrtY). The third stage, astaxanthin synthesis, differs in the synthesis pathway in different organisms, but is mainly produced by the hydroxylation and ketone formation of β-carotene.

In Phaffia rhodozyma, astaxanthin is synthesized from zeaxanthin by cytochrome P450 enzymes [24]. In bacteria and algae, it is mainly synthesized by β-carotene hydroxylase (CrtZ) and β-carotene ketolase (CrtW or BKT). Corn xanthophyll is converted from β-cryptoxanthin by the action of the specific enzyme β-carotene hydroxylase. The ketene and 4-ketone bodies of β-carotene are converted to canthaxanthin by β-carotene ketolase, and canthaxanthin is converted to astaxanthin via phycoerythrin (astaxanthinamide).

In different species, the order of action of β-carotene hydroxylase and β-carotene ketolase in the catalytic conversion of β-carotene to astaxanthin is different. LIU et al. [25] used heterologous expressed Haematococcus pluvialis β-carotene ketolase in Synechocystis sp. PCC 6803), Liu et al. found that astaxanthin was first synthesized in Synechocystis cells and reached a content of (4.81±0.06) mg/g dry cell weight (DCW). In vitro experiments [26-27] have also further confirmed that the optimal pathway for astaxanthin synthesis in Haematococcus pluvialis is the catalytic reaction of the ketolase enzyme, followed by the hydroxylation reaction of the hydroxylase enzyme (Figure 4).

2.2 Bacteria synthesize astaxanthin

Astaxanthin has been found in several types of bacteria, including the Gram-positive bacterium Brevundimonas sp. and the Gram-negative bacteria Sphingomonas sp., Par acoccus haeundaensis), Methylomonas sp. and Altererythrobacter ishigakiensis (Table 1).

The presence of precursors for astaxanthin biosynthesis in some bacteria and the identification of a number of key genes in the astaxanthin biosynthesis pathway have made it possible to construct high-yielding engineered astaxanthin-producing strains. It was found that by transferring the carotenoid genes crtW, crtZ, crtY, crtI, crtB and crtE from the marine bacterium Pseudoalteromonas luteoviolacea into E. coli, an engineered E. coli strain that produces astaxanthin was successfully constructed, and the yield was as high as 400 μg/g DCW [36]. In E. coli, the overexpression of the two major rate-limiting enzymes, DXP synthase (DXP) and IPP isomerase (IDI), increases the supply of IPP and DMAPP.

By increasing the metabolic flux of isopentenyl diphosphate precursors, the production of carotenoids such as lycopene or β-carotene can be significantly increased. However, for the heterologous biosynthesis of astaxanthin in E. coli, the conversion of β-carotene to astaxanthin is the most critical step for achieving efficient astaxanthin biosynthesis. Using λ-Red recombination technology, a plasmid-free E. coli was constructed, and the xanthophyll biosynthesis genes of Pantoea ananatis and Phaffia were integrated into the chromosome of E. coli BW-CARO to obtain the engineered strain E. coli BW-ASTA. This strain produced 1.4 mg/g DCW of astaxanthin after heterologous expression. In Corynebacterium glutamicum Corynebacterium glutamicum successfully synthesized astaxanthin after expressing the coding genes of the lycopene cyclase CrtY, β-carotene ketolase CrtW and β-carotene hydroxylase CrtZ from Fulvimarina pelagi, and the yield could reach 0.4 mg/L/h [32].

Although the level of astaxanthin synthesis by the bacteria itself is quite different from that of algae, the synthesis of astaxanthin in bacteria is of great significance and provides the corresponding gene sequences for the construction of subsequent engineered strains.

2.3 Astaxanthin synthesis by yeast

Currently, Rhodotorula glutinis is the main yeast source of natural astaxanthin and has been applied in the aquafeed industry. Research on the synthesis of astaxanthin by Rhodotorula glutinis has focused on the isolation of strains, mutagenesis, and genetic engineering to obtain high-yield astaxanthin-producing strains. Red yeast is a basidiomycete fungus that is facultatively cold-loving and a low-temperature yeast.

The astaxanthin it synthesizes has a dextrorotatory structure and is the main carotenoid synthesized by red yeast as a secondary metabolite. The astaxanthin synthesis concentration of the wild-type red yeast is about 200–400 μg/g DCW, and strain mutation can obtain mutant strains with high astaxanthin production. The wild-type Phaffia rhodozyma strain was mutagenized using chemical reagents such as antimycin, nitroguanylin (NTG), and methylnitro-nitroguanidine, as well as ultraviolet light and low-energy ion beam technology. A high-yield astaxanthin strain was obtained through screening (For a summary of the mutant strains, see Table 2). The yield of the E5042 strain, which was induced by low-energy ion beam implantation in the Phaffia rhodozyma ZJB00010 mutant strain, can reach 2512 μg/g [37]. The advantages of red yeast rice, such as its ability to utilize a variety of carbon sources, short fermentation cycle, high-density cultivation in fermenters, and fast production speed, have made it an excellent strain for industrial production of astaxanthin.

In addition, yeast engineered strains have good application prospects in astaxanthin production (Table 2). Yarrowia lipolytica has high IPP and DMAPP production. Studies have found that CrtZ is the key enzyme that catalyzes the conversion of β-carotene to astaxanthin. The β-carotene hydroxylase CrtZ coding gene from Pantoea ananatis and the β-carotene ketolase CrtW coding gene from Paracoccus sp. N81106 were introduced into the Yarrowia lipolytica genome. enzyme CrtZ coding gene and the β-carotene ketolase CrtW coding gene from Paracoccus sp. N81106. The engineered strain ST7403 obtained a high astaxanthin yield of 3.5 mg/g DCW (54.6 mg/L) [40]. The introduction of the crtZ and bkt genes from Haematococcus pluvialis into Saccharomyces cerevisiae through genetic engineering can increase the conversion efficiency of β-carotene to astaxanthin and achieve the accumulation of astaxanthin in cells. In a positive mutant of GGPP synthase, tHMG1 was overexpressed, and the encoding genes of the three rate-limiting enzymes CrtI CrtY and CrtB were overexpressed. In an optimized diploid strain, the encoding genes of CrtZ and BKT were overexpressed, and the astaxanthin accumulation reached 8.10 mg/g DCW [42]. It is worth noting that the synthesized astaxanthin is of the levorotary structure.

2.4 Microalgae synthesis of astaxanthin

Microalgae generally refers to the collective term for microorganisms that contain chlorophyll a and can photosynthesize. Most microalgae can not only synthesize various bioactive ingredients such as polyunsaturated fatty acids and microalgal polysaccharides, but can also accumulate a large amount of carotenoids. Some microalgae have their own complete astaxanthin synthesis pathway. Among them, freshwater unicellular microalgae such as Haematococcus pluvialis and Chlorella vulgaris are the main sources of astaxanthin biosynthesis. In addition, euglena (Halamidomonas), euglena (Euglena), and Aceta- bularin also contain astaxanthin.

When exposed to environmental stress, microalgae cells can switch from the green, photosynthetic form to the red, cystic form. This is because the microalgae cells have synthesised large amounts of astaxanthin to counteract the unfavourable environment for their growth. The biosynthesis of astaxanthin in the Chlorella pyrenoidosa microalgae begins early in the exponential growth phase. The cells usually grow under optimal conditions in the green, photosynthetic form. Stressful conditions induce astaxanthin accumulation and the cells take on a red, cystic form. Unlike primary carotenoids, which form the structural and functional components of photosynthesis (e.g. β-carotene, zeaxanthin and lutein), astaxanthin can accumulate in large quantities under stress conditions such as high light, high salinity and nutrient deficiency. Under environmental stress conditions such as low nutrition and high light, the formation of capsules begins and a large amount of astaxanthin is accumulated. Light, temperature, salinity and chemical reagents all affect astaxanthin synthesis at the molecular level.

Excessive low-activity oxygen produced in cells under high-temperature conditions weakens carotenoid metabolism. High light[44] and acetate[45], methyl jasmonate[46] and gibberellin[46] all have the function of promoting the expression of key genes related to the carotenoid biosynthesis pathway. Acetate, methyl jasmonate and gibberellic acid further promote astaxanthin biosynthesis by enhancing the expression of the crtZ gene and inhibiting the expression of the lcyE gene. Compared with the induction conditions such as acetate, high light intensity affects the expression of the pds, crtISO, lcyB, lut1, lut5 and zep genes, which promotes carotenoid biosynthesis to a greater extent and is the main driving force behind the changes in the expression of genes related to carotenoid synthesis. Studies have shown that under high light conditions, the Calvin cycle and the tricarboxylic acid cycle provide more precursors for other metabolism. The β-carotene hydroxylase, hexahydro-lycopene synthase, and octahydro-lycopene desaturase are all upregulated, thereby increasing the intracellular astaxanthin accumulation.

The industrial production of astaxanthin extracted from Haematococcus pluvialis began on a large scale in the late 1990s. As a type of single-celled photosynthetic organism, the wild-type Haematococcus pluvialis cell can contain up to 4% astaxanthin by dry weight. It also has the characteristics of high light energy utilization and fast growth, and has been recognized as a safe production strain in China.

However, the industrialization of Haematococcus pluvialis requires the use of a photoreactor to ensure photosynthesis, which significantly increases production costs. Therefore, the development of new resources and technologies to reduce production costs has become the focus of current research.

2.5 Marine eukaryotic microorganisms synthesize astaxanthin

Thraustochytrium is a type of eukaryotic microorganism similar to a microalga but lacking chloroplasts and therefore not photosynthesizing. The cells can accumulate a large amount of active substances beneficial to the human body, such as lipids, pigments, and squalene. In addition, Thraustochytrium, Schizochytrium, and Aurantiochytrium can also accumulate carotenoids such as β-carotene and astaxanthin. Studies have found that the metabolites of Thraustochytrium, Schizochytrium, and Aurantiochytrium differ under different carbon source conditions. Related metabolic studies are currently underway (Table 3). During the fermentation of glycerol as a carbon source by Schizochytrium, glycerol mainly promotes the biosynthesis of secondary metabolites in Schizochytrium by enhancing glycolytic activity and producing NADPH. Using brewing by-products and waste molasses as a carbon source for Thraustochy- triidae sp. and Aurantiochytrium sp., the astaxanthin production was successfully increased while reducing production costs, further enhancing the possibility of commercializing astaxanthin biosynthesis in Thraustochy- triidae sp.

In addition, the astaxanthin production in cells can be further enhanced through environmental stress, mutagenesis, genetic engineering and other means. Astaxanthin has strong antioxidant capacity. When cells are under stress, the metabolism of carotenoids in cells is enhanced, which greatly increases the production of astaxanthin and helps cells resist adverse environments. Studies have found that butanol and methanol at certain concentrations have the effect of inducing astaxanthin synthesis in Schizochytrium limacinum B4D1. When 5.6% methanol was added to the culture medium, the total astaxanthin content increased to about 3300 μg/g, and the astaxanthin synthesized was mainly 3S-3'S structure [47].

With the metabolic pathway clarified, high-yield astaxanthin strains have also been obtained in Aurantiochytrium through genetic engineering techniques. In the Aurantiochytrium sp. SK4 strain, the gene encoding the hemoglobin of the diatom Vitreoscilla stercoraria (vhb) was overexpressed, and the astaxanthin production increased 9-fold to 131.09 μg/g [21]. In addition, high-yield astaxanthin strains can be obtained by mutagenesis of wild strains using γ-rays, NTG chemicals, and other methods. The astaxanthin yield of the high-yielding strain Schizochytrium SH104 obtained using γ-rays was 3 times that of the original strain, reaching 3.689 mg/L [51]. Schizochytrium also has the characteristic of not requiring light, in addition to being a safe strain for DHA production, making it a potential strain for industrial astaxanthin production.

2.6 Plants synthesize astaxanthin

A few species of marigold are the only terrestrial plants that can produce astaxanthin [56]. The petals of Adonis aestivalis and Adonis annua in the genus Adonis show a bright blood-red color due to the accumulation of astaxanthin. However, due to the small size of the marigold flowers, it is limited in the industrial production of astaxanthin. However, it is a good carrier of the astaxanthin synthesis pathway in higher plants and provides a reference for the development of astaxanthin bioreactors. Astaxanthin is the final product of carotenoid metabolism. Although many plants do not have the ability to accumulate astaxanthin, they contain high levels of carotenoids.

The relevant genes in the metabolic pathway from β-carotene to astaxanthin are missing in these plant cells, which causes the metabolism to break down at the β-carotene synthesis stage. Researchers have obtained high-yield astaxanthin-producing engineered plant strains through genetic engineering. In tomatoes, the co-expression of Chlamydomonas reinhardtii β-carotene ketolase and Haematococcus pluvialis β-carotene hydroxylase resulted in the upregulation of most of the original carotenoid genes in tomatoes, effectively directing the carbon flux into carotenoids and accumulating large amounts of free astaxanthin in the leaves. The expression of Brevundimonas sp. SD212 genes encoding crtW and crtZ in tobacco produced astaxanthin in tobacco leaves at 0.5% DCW (more than 70% of total carotenoids) [57].

3 Conclusion and outlook

Astaxanthin has strong antioxidant properties. As market interest in astaxanthin increases and demand rises, astaxanthin has great application value and development potential in food nutrition enhancers, healthcare, feed and other fields. Chemically and biologically synthesized astaxanthin has different application spaces in different fields. Chemically synthesized astaxanthin is low in cost and inexpensive, has been industrialized, and is the main industrial source of astaxanthin supply on the market. With the rise of biosynthetic astaxanthin, countries have become increasingly strict in their management of chemically synthesized astaxanthin. The US Food and Drug Administration (FDA) has banned chemically synthesized astaxanthin from entering the food, health product and other markets.

Biosynthetic natural astaxanthin has higher biological activity and a safer source, meeting market needs, especially for natural pigments for human consumption, which has become a research hotspot. This market demand has also led to increasing attention on biosynthetic astaxanthin. However, the current low production of natural astaxanthin leads to high prices and cannot meet the general market demand. In response to the market's growing demand for astaxanthin, precise regulation of astaxanthin biosynthesis in plants or microorganisms through synthetic biology, metabolic engineering, fermentation engineering, and other means is an effective way to achieve large-scale industrial production of natural astaxanthin. The known organisms with the ability to synthesize astaxanthin from scratch are limited to several types of bacteria, yeast, microalgae and plants [19], so obtaining astaxanthin-producing microbial strains with high yields is an important research direction for the large-scale production of astaxanthin.

In addition, there are also significant challenges in the downstream processing of biosynthesis, especially in the efficient extraction and purification of astaxanthin. The production potential of biosynthesized astaxanthin is huge, and the main challenges that remain to be overcome still require better engineering and innovation to make the process more cost competitive. In short, the biosynthesis of astaxanthin is an attractive field and may develop rapidly. It is expected that biotechnology will open up new possibilities for the industrial production of bio-derived astaxanthin.

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