How to Biosynthesis of Astaxanthin?

Astaxanthin is a fat-soluble carotenoid pigment originally isolated from lobsters [1]. It is found in a variety of algae, microorganisms, crustaceans and marine fish, but is rarely reported in higher plants [2]. Since the mid-1980s, with the discovery of the antioxidant and other biological activities of astaxanthin, it has gradually been used in products such as animal feed, health foods, cosmetics and medical preparations. The European Union has long approved astaxanthin for use as a dietary supplement; the US Food and Drug Administration has also approved astaxanthin as a food coloring agent for animal and fish feed; and China also allows the extensive use of astaxanthin in food and feed additives. Therefore, the market demand for astaxanthin is increasing.

In the current commercial supply of astaxanthin, astaxanthin is mainly synthesized by chemical methods or extracted from natural astaxanthin-containing Haematococcus pluvialis, Rhodopseudomonas palustris, and the shells of shrimp and crab, and then further processed to obtain products for different uses. Hydrophilic delivery vehicles are often used in health products and pharmaceutical preparations to ensure the effective absorption of active substances such as astaxanthin in the human body. At present, the methods used for large-scale production of astaxanthin all have certain limitations, resulting in a shortage of astaxanthin. In recent years, with the development of genetic engineering technology, transgenic crops rich in astaxanthin, such as rice, tomatoes, and corn, have been reported one after another, bringing new possibilities for the future industrial production of high-quality astaxanthin.

1 Properties of astaxanthin

Astaxanthin, also known as shrimp red pigment, is 3,3'-dihydroxy-4,4'-dione-beta,beta'-carotene with the molecular formula C40H52O4. The molecular structure of astaxanthin consists of a long conjugated polyene chain and two hexaene ketone rings at the ends, with C-3 and C-3' at the end rings as the chiral centers. Depending on the conformation of the chiral carbon atom at the end ring, astaxanthin has three stereoisomers: levogle (3S,3'S), dextrogyre (3R, 3'R) and meso (3R, 3'S) (Figure 1).

These different conformations of astaxanthin are all found in nature. For example, astaxanthin in Rhodotorula glutinis is a free form of the levo-conformation; Antarctic krill is dominated by astaxanthin esters of the dextro-conformation; wild salmon contains mainly astaxanthin in the free form of the levo-conformation; in Haematococcus pluvialis, it is the free form of astaxanthin with the left-handed conformation. Among these, monoesters account for about 80%, while diesters account for about 15%. The fatty acids that participate in esterification at the 3 or 3' hydroxyl groups mainly include oleic acid, trans-oleic acid, ricinoleic acid, and arachidic acid. In addition, depending on the spatial arrangement of the carbon-carbon double bond-linked groups in the astaxanthin structure, there are also cis-trans isomers. If the two groups are on the same side of the double bond, it is called the cis structure (Z), otherwise it is called the trans structure (E) (Figure 1). Among them, all-trans astaxanthin is the most stable and is found in large quantities in nature.

2  The biosynthetic pathway of astaxanthin

As a keto-carotenoid, astaxanthin only has a complete biosynthetic pathway in bacteria, fungi, algae and a few plants, including the well-known Rhodopseudomonas palustris[3] and Haematococcus pluvialis[4‑5]. Animals such as salmon and lobsters have poor ability to synthesize astaxanthin de novo and generally can only accumulate it in the body through the food chain[6‑7]. The metabolic pathways for carotenoid synthesis are similar. In flowering plants [8], geranylgeranyl pyrophosphate (GGPP) is produced from glyceraldehyde-3-phosphate and pyruvate through a series of reactions; then, under the action of phytoene synthase (PSY), two GGPP molecules to form phytoene, which is the rate-limiting step in carotenoid synthesis.

Phytoene is then oxidized to form lycopene, which undergoes further branching. One branch synthesizes lutein under the action of lycopene epsilon cyclase (LCY-e), while the other branch proceeds in the direction of β-carotene, finally forming abscisic acid. Beta-carotene is the precursor for the synthesis of astaxanthin in bacteria, fungi, algae and a few plants. The structural difference between astaxanthin and beta-carotene lies in the hydroxyl groups on the C3 and C3' rings and the carbonyl groups on the C4 and C4' rings at the ends of the carbon chain. Therefore, extending the β-carotene biosynthesis pathway to the astaxanthin process is actually a process of adding hydroxyl and carbonyl groups to the corresponding sites on the β-rings at both ends of the β-carotene molecule. However, the methods and pathways of hydroxylation and carbonylation are slightly different in different species, and can be divided into three pathways in general, as shown in Figure 2.

Tagetes erecta is the only higher plant reported to date to be able to synthesize astaxanthin. Cunningham et al. [9] screened a cDNA library of Tagetes erecta petals for cDNAs similar to β-carotene 3-hydroxylase genes (cbfd1 and cbfd2) and transferred these two cDNAs into Escherichia coli for gene function verification. The results showed that CBFD1/CBFD2 has substrate specificity and can hydroxylate the C4 of unmodified carotenoid β-rings and the C3 of carotenoid 4-keto-β-rings, but cannot hydroxylate the C3 of unmodified β-rings or 4-hydroxy-β-rings. The team further verified this result in 2011 and identified two other genes, hbfd1 and hbfd2, which encode HBFD[2] and can dehydrogenate the hydroxyl group on the 4-hydroxy-β-ring to form a 4-carbonyl-β-ring. In marigold, when β-carotene is used as a substrate for the synthesis of astaxanthin, CBFD first hydroxylates the C4 of the β-carotene β-ring; then the hydroxyl group at this site is dehydrogenated by HBFD to form a carbonyl group; finally, CBFD adds a hydroxyl group to the C3 of the 4-carbonyl-β-ring to form astaxanthin.

The astaxanthin metabolic pathway of marine bacteria seems to be more concise. There is neither interference between β-carotene ketolase CrtW and β-carotene hydroxylase CrtZ, nor is there a strict catalytic reaction sequence between the ketolase and hydroxylase in the marigold.

In algae, the ketolase BKT and the CrtW enzyme from Haematococcus pluvialis have similar amino acid sequences [10], but the cytochrome P450 reductase [11] acts as a hydroxylase in conjunction with BKT. In yeast, the functional gene that converts β-carotene to astaxanthin is still controversial. This is because there are two different reactions in the conversion of β-carotene to astaxanthin: hydroxylation and ketolation. Only one relevant gene, CrtS, has been cloned for the first half of this process. Ojima et al. [12] introduced CrtS into E. coli that could produce β-carotene and detected the intermediate product astaxanthin, thereby proposing the hypothesis that CrtS has two functions: hydroxylation and ketolation. However, Álvarez et al. [13] only detected the hydroxylated products of β-carotene, β-cryptoxanthin and zeaxanthin, when they introduced CrtS into the β-carotene-producing Streptomyces. Therefore, they believed that β-carotene hydroxylase only has a hydroxylation function. Alcaíno et al. [14] cloned another gene, CrtR, which encodes cytochrome P450 reductase, and research has shown that CrtR is necessary for CrtS to convert β-carotene into astaxanthin.

3 Applications of astaxanthin

3.1 Application of astaxanthin in aquaculture and livestock feeds

Astaxanthin is a natural coloring agent that occurs in different species in different conformations, giving the organism its characteristic color. A typical example is the red color of salmon flesh. This red color is visually pleasing and people are accustomed to seeing this bright color as a sign of freshness and flavor. Astaxanthin can accumulate in fish lipoproteins[15], myosin[16] and α-actinin[17]. For this reason, in order to make farmed salmon more vividly colored, a moderate amount of astaxanthin is added to conventional feed.

It is estimated that the market demand for animal feed and nutritional products was 300 million and 30 million US dollars respectively in 2009, but will reach 800 million and 300 million US dollars respectively in 2020, with the annual demand for astaxanthin as salmon feed being 200 million US dollars (2,500 US dollars·kg-1) [18].

In addition to aquatic products, astaxanthin can also be used in poultry feed. Adding 10 mg·kg-1 of natural astaxanthin to the feed of meat ducks can effectively deposit it in the ducks, causing the beaks and shins of live ducks to take on a natural and healthy golden yellow color. It can also effectively inhibit lipid peroxidation in the muscles and improve nutritional value [19]. Using high-astaxanthin corn to completely replace corn in traditional feed to feed laying hens (Figure 3) can produce eggs with astaxanthin levels of 12.10–14.15 mg·kg-1 in the yolk, with each egg containing about 540 µg of astaxanthin, which can meet the daily antioxidant health needs of the human body [20].

3.2 Astaxanthin in health foods and cosmetics

The long conjugated polyene chain in the astaxanthin molecule can quench singlet oxygen and scavenge free radicals, so astaxanthin has extremely strong antioxidant capacity [21]. It has been reported that the antioxidant activity of astaxanthin is 10 times higher than that of zeaxanthin, lutein, canthaxanthin and β-carotene, and 100 times higher than that of tocopherol [22]. Therefore, it is believed that adding astaxanthin to food and skin care products can use its antioxidant activity to achieve whitening and skin care, immune enhancement and anti-aging effects. As of November 2022, there were 2,371,474 registered products in China labeled as containing astaxanthin, of which 70,765 were skin care and beauty products; 45,156 were food products; however, astaxanthin health products are basically all imported from abroad[23‑24].

imported products[23‑24].

3.3 Application of astaxanthin in medical drugs

Because astaxanthin has a strong antioxidant effect, it can be used as a multi-target pharmacological agent. Astaxanthin can prevent and improve non-alcoholic fatty liver disease and liver fibrosis by regulating liver immune response, liver inflammation and oxidative stress[25]. In addition, Fakhri et al. [26] believe that astaxanthin can prevent most diseases related to oxidative stress and inflammation, including inflammatory diseases, cancer, obesity, hypertriglyceridemia, hypercholesterolemia, cardiovascular, gastrointestinal , liver, neurodegenerative ophthalmic, skeletal, reproductive system diseases and skin diseases. Lignell et al. [27] also showed that oral administration of astaxanthin-containing drugs can significantly enhance human muscle strength and exercise tolerance.

4 Main sources of commercial astaxanthin

4.1 Chemically synthesized astaxanthin

The chemical synthesis of astaxanthin is the main source of commercial astaxanthin. In China, Pi et al. [28] reported a chemical synthesis method for astaxanthin, which has the advantages of easy access to raw materials, high reaction selectivity, and high yield. Abroad, the main sources of commercially available synthetic astaxanthin are BASF in Germany and Roche in Switzerland. The synthetic methods used by these two companies are similar, and the process is complex and stringent [29], but the cost is relatively low. In addition, there is also a method for synthesizing astaxanthin using canthaxanthin. Although the astaxanthin synthesized by this method has higher biological activity, it has high cost, low yield, and the synthesis process is dangerous [30]. At present, most industrially synthesized astaxanthin is used as a feed additive in the aquaculture of salmon and other aquatic products.

4.2 Natural extraction method

In addition to chemical synthesis, astaxanthin can also be extracted from organisms that naturally contain astaxanthin. Existing extraction methods mainly extract astaxanthin from organisms such as Haematococcus pluvialis, Chromatococcus purpureus, Rhodopseudomonas palustris, and crustacean shells. The form of astaxanthin in different species is different, and the production generally requires the extraction of the more stable all-trans astaxanthin. At present, in industrial production, natural astaxanthin with all-trans configuration can only be extracted from Haematococcus pluvialis, but algae have a long growth cycle, low biomass, and the induction of astaxanthin accumulation by adverse stresses conflicts with the accumulation of cell biomass, resulting in an astaxanthin content of 1% to 5% in Haematococcus pluvialis [31]. Although the red yeast Rhodotorula glutinis has a fast growth rate and high biomass, its astaxanthin content is only 0.4% [31]. The method of extracting astaxanthin from discarded crustaceans is costly, has a low yield and low purity, and is therefore rarely used. In general, the existing industrial production methods have more or less the problems of being technically difficult, costly to produce and yielding low levels of astaxanthin. However, the demand for natural astaxanthin with higher safety and biological activity in the fields of health products and cosmetics has led to the high price of such goods.

5 Genetic engineering for the commercial production of astaxanthin

Genetic engineering research on astaxanthin biosynthesis was first carried out in algae and microorganisms, with the astaxanthin content in Rhodopseudomonas palustris reaching about 0.5% of the cell dry weight; the astaxanthin content in Haematococcus pluvialis can reach about 4% to 5% of the cell dry weight (Table 1). However, the biomass of these receptor organisms is low, and the mechanism by which they store astaxanthin is not yet clear, so astaxanthin production is generally low.

In recent years, many researchers have used environmentally friendly crops as bioreactors to achieve astaxanthin biosynthesis through genetic engineering (Table 1). This method has many advantages, such as low cost, strong operability, high yield, high biomass, and storage resistance.

The first study on astaxanthin gene transfer was in the model plant tobacco. The CrtO gene from Haematococcus pluvialis was transferred to tobacco, and astaxanthin was synthesized in its flowers for the first time [35]; while in transgenic tobacco with transferred cyanobacterial CrtO, ketocarotenoids were detected in the leaves, with a content of 165.00 µg·g-1 DW [44]; at the same time, the transgenic tobacco and tomatoes with fused expression of CrtW and CrtZ the amount of astaxanthin accumulated was increased but still very low [45]. 

The CrtW and CrtZ from the marine bacterium Brevundimonas sp. strain SD212 were transformed into tobacco by chloroplast transformation, and the astaxanthin content in the leaves of the transgenic tobacco was as high as 5.44 mg·g-1 DW [37 ]; after the ketolase gene BKT and CrtB were transferred into potatoes, the transgenic plants accumulated 13.90 µg ·g-1 DW of astaxanthin [36]; the transfer of the BKT gene to carrots resulted in astaxanthin accumulation of 91.60 µg ·g-1 FW [38]. Chen Feng's team at Peking University found that after transferring different ketolase genes from algae to Arabidopsis and tobacco, the CrBKT from Chlamydomonas reinhardtii resulted in the highest astaxanthin accumulation in transgenic plants, reaching 2.07 and 1 . 60 mg ·g-1 DW[39‑40]; further, the simultaneous expression of CrBKT and the hydroxyase gene HpBHY from Haematococcus pluvialis in tomatoes resulted in astaxanthin accumulation of 16.10 mg ·g-1 DW in transgenic tomato fruits[41].

Subsequently, the biosynthesis of astaxanthin was focused on food crops. Liu Yaoguang's team [43] successfully reconstructed the astaxanthin metabolic pathway in rice endosperm. Farré et al. [42] obtained transgenic maize expressing both the Brevundimonas sp. strain SD212 Crbkt and BrcrtZ genes by gene gun co-transformation, and the accumulation of astaxanthin in its seeds reached 1 6.77 µg·g-1 DW. Liu Xiaoqing et al. [20] introduced key enzyme genes in the astaxanthin synthesis pathway into corn to create high astaxanthin corn germplasm, with astaxanthin levels as high as 47.76–111.82 mg·kg-1 DW, which is 6 times that of previous transgenic astaxanthin grains (Fig. 3).

6 Astaxanthin active delivery system

Astaxanthin is a highly unsaturated molecule that is extremely sensitive to conditions such as high temperatures, light exposure and oxidation. Therefore, astaxanthin is highly prone to degradation, which in turn reduces the biological activity of astaxanthin products. Only by simultaneously improving the bioavailability and stability of astaxanthin in the application system can the industrial production and commercial application of astaxanthin be promoted, and effective protection of human health be achieved.

Delivery systems are currently available strategies that are highly protective and practical, including traditional delivery systems such as emulsions, nanoparticles, and liposomes. Khalid et al. [46] used high-pressure homogenization and modified lecithin and sodium caseinate (SC) raw materials to prepare an “oil-in-water” nanoemulsion of astaxanthin. sodium caseinate (SC) to prepare an oil-in-water nanoemulsion of astaxanthin. Ribeiro et al. [47] used a premixed film to protect astaxanthin, but some degradation still occurred. At present, liposomes mostly use materials such as lecithin, dimyristoyl phosphatidylcholine [48], and soy phosphatidylcholine [49] to encapsulate astaxanthin. These materials have higher oral safety and can be digested and absorbed by the body.

Compared to the previous two methods, nanoparticles provide better protection and higher utilization. Astaxanthin is embedded in a glutaraldehyde-crosslinked chitosan matrix using the multiple emulsification/solvent evaporation method to form a powdered astaxanthin microcapsule product with a diameter of 5–50 µm. This embedding can protect astaxanthin from isomerization or chemical degradation [50]. Using DNA/chitosan co-assemblies as nanocarriers, astaxanthin-loaded DNA/chitosan (ADC) colloidal systems can be obtained [51], with astaxanthin content is as high as 65 µg·mL-1 .

ADC nanoparticles can be absorbed by endocytosis of intestinal epithelial cells in a short period of time, and their reactive oxygen species scavenging efficiency is as high as 54.3%, which is twice that of free astaxanthin. Biopolymer nanoparticles prepared using stearic acid-chitosan conjugates and sodium tyrosine (NaCas) by the ionogel method can be used to encapsulate astaxanthin at a concentration of up to 140 µmol·L-1 [52]. Astaxanthin can also be effectively protected by solid lipid-polymer hybrid nanoparticles (SLPN) prepared by in situ coupling of oxidized dextran and bovine serum albumin [53].

Astaxanthin can interact with various proteins, and amphiphilic proteins are suitable as hydrophilic delivery vehicles for lipophilic substances. When fatty acids are used as protein ligands, the bovine serum albumin (BSA)-astaxanthin system [54] can effectively ensure the storage stability of astaxanthin. In addition, in vitro simulations have also shown that hydrophilic delivery vehicles significantly improve the bioavailability of astaxanthin.

If unsaturated fatty acids other than DHA or long-chain fatty acids are used instead, the stability of encapsulated astaxanthin decreases, but the bioavailability increases. Therefore, the fatty acids used to complex the protein should be selected according to the specific conditions of the actual application. Potato proteins (PP) extracted from potato starch processing by-products [55] can also form nanoparticles with astaxanthin molecules. Although only 80% retention is achieved after simulated gastrointestinal digestion, the low cost of the raw material can significantly reduce the cost of astaxanthin as a dietary supplement. Other delivery vehicles for hydrophobic substances, such as soy β-conglycinin (β-CG) [56], are also expected to be used for astaxanthin delivery in the future.

7 Prospects for future applications of astaxanthin

Astaxanthin has a wide range of biological functions and therefore has a huge market demand. However, there are certain limitations in the current industrial production and commercial applications. At present, the problem of the first production source can be solved by genetic modification technology. In particular, crops represented by corn and rice and fruits and vegetables represented by tomatoes and kale can be used as bioreactors to accumulate astaxanthin with strong biological activity in large quantities. In particular, fruits and vegetables containing astaxanthin can be directly supplied to the market, increasing the astaxanthin content of people's daily diet. Corn, as a crop that can be used for both food and feed, can meet people's daily dietary needs and the needs of related industrial uses.

However, due to problems such as the fragmentation of exogenous genes and gene segregation caused by the transformation method, as well as the lack of evaluation standards for the astaxanthin characteristics of related germplasm, the research results in China cannot meet market demand. The second step of effective utilization relies on a reasonable application delivery system, and the corresponding delivery system with different characteristics is selected according to the actual application scenario. With the increasing variety of hydrophobic active molecular delivery systems and relatively mature technology, especially nanoparticle technology, astaxanthin can be effectively protected and delivered using a variety of materials.

If proteins that interact with astaxanthin in a naturally specific manner are introduced into existing astaxanthin transgenic crops, it is hoped that astaxanthin molecules can be simultaneously accumulated and encapsulated in a bioreactor, and astaxanthin material extraction and delivery system assembly can be achieved in one step. As market demand continues to increase, research on astaxanthin will also deepen, and in the future, the low-cost and high-efficiency industrial application of astaxanthin resources will surely be better realized.

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