Study on Synthesis of Astaxanthin by Microbial Fermentation Method

Astaxanthin is an orange-red keto-type carotenoid with the molecular formula C40H52O4 and the chemical name 3,3'-dihydroxy-4,4'-dione-beta,beta'-carotene [1-2]. Astaxanthin is insoluble in water, fat-soluble, soluble in organic solvents such as benzene, chloroform, acetone and carbon disulfide, and slightly soluble in polar organic solvents such as methanol and ethanol [3]. Astaxanthin is a tetraterpenoid composed of eight isoprene units linked by conjugated double bonds, with unsaturated hydroxyl and ketone groups at the ends of the conjugated double bonds [4]. Therefore, astaxanthin exists in different conformations (Figure 1), including: levorotary (3S, 3'S), dextrorotary (3R, 3'R) and racemic (3S, 3'R) [5]. Among them, the (3S, 3'S) isomer is the most common astaxanthin conformation in nature and also the one with the highest antioxidant activity, followed by the (3R, 3'R) and (3S, 3'R) conformations [6].

Astaxanthin has very high application value. Its long conjugated polyene chain can quench singlet oxygen, scavenge free radicals, enhance cell activity, and protect human body lipids, thus improving immunity and anti-aging to a certain extent. Some studies have shown that astaxanthin's antioxidant capacity is 6,000 times that of vitamin C, making it the strongest antioxidant reported in nature [7-9]. At the same time, it can prevent most oxidative stress and related inflammation, including hypertension, cancer, obesity, cardiovascular disease, inflammatory diseases, bone diseases, skin diseases, etc., so it can be used as a multi-target drug preparation [10]. For example, Lignell et al. [11] showed that orally administered astaxanthin-containing drugs can significantly improve muscle strength and exercise endurance.

Astaxanthin is also a natural coloring agent that exists in different species in different configurations, giving the body a unique color. The red color of salmon meat is often a visual treat that makes it easy to judge the freshness and flavor of the food. In addition, astaxanthin can also be used as an additive in poultry feed. Studies have shown that adding 10 mg/kg of natural astaxanthin to the feed can effectively deposit it in the meat of ducks, giving the beaks and feet of live ducks a healthy golden color. It can also improve the oxidative stability of muscle lipids and make them more nutritious [12].

Astaxanthin also plays a protective role in environmental plants. For example, astaxanthin sprays can increase the photosynthesis of grape leaves, improve their stress resistance, and change their color. As a result, astaxanthin is currently being used in an increasing number of applications in the fields of medicine, cosmetics, food, feed additives, health products, agriculture, etc. (Figure 2) [13-14]. The global carotenoid market was valued at 1.5 billion US dollars in 2017 and is expected to reach 2 billion US dollars by 2022 [15]. The global market value of astaxanthin, the second largest carotenoid, is expected to grow to nearly 3.4 billion U.S. dollars by 2027 [16].

At present, the main methods of producing astaxanthin include natural extraction, chemical synthesis and microbial fermentation. Natural extraction involves extracting astaxanthin from lobster, crab and other crustacean waste, but the yield is extremely low, the process is complex and costly, and the extraction process is susceptible to contamination, making it economically unviable [17-18]. The chemical synthesis method has a long production cycle and a complex process [19]. The product of synthesis is a mixture of astaxanthin in various configurations and the accumulation of various by-products [20]. The absorption and utilization rate in living organisms is lower than that of naturally extracted astaxanthin, so it is not approved for human use [19].

With the continuous development of synthetic biology technology, the use of microbial fermentation to produce natural products has shown great potential [21-23]. Astaxanthin produced using microorganisms has the advantages of a clear configuration, being environmentally friendly and having few by-products [16]. Therefore, this is a very promising method of astaxanthin production [18, 24].

Microorganisms currently used for fermentative synthesis of astaxanthin include algae, bacteria, yeast, etc. [25]. This paper discusses the latest progress in the production of astaxanthin by Haematococcus pluvialis, Escherichia coli, Xanthophyllomyces dendrorhous, Yarrowia lipolytica and other microorganisms, and summarizes the strategies for screening and metabolic engineering of astaxanthin-producing strains to increase astaxanthin production and reduce costs. Yarrowia lipolytica and other microorganisms to produce astaxanthin, the latest developments are discussed, and strategies for the selection and metabolic engineering of astaxanthin-producing strains to increase astaxanthin production and reduce costs are summarized.

1 The biosynthesis pathway of astaxanthin

The biosynthesis of astaxanthin can generally be divided into three stages [10]:

The first stage is central carbon metabolism, in which organisms use glucose and other carbon sources to generate pyruvate and acyl-CoA through the Embden-Meyerhof-Parnas (EMP) pathway. These are used as precursors for the synthesis of terpenes substances are transported to the mevalonate pathway (MVA) and the methylerythritol phosphate (MEP) pathway as precursors for terpene synthesis.

MV A and MEP are the second stage of the astaxanthin synthesis pathway (Figure 3). The MVA pathway not only provides the precursors required for terpene synthesis, but also the precursors for substances essential for cell growth. It starts from acetyl coenzyme A, isopentenyl pyrophosphate (IPP) is produced in a six-step enzymatic reaction, and isopentenyl diphosphate isomerase (I DI) isomerizes IPP to dimethylallylpyrophosphate (DMAPP), and finally uses IPP and DMAPP as precursors to synthesize the precursors of terpenoids. The M EP pathway is another supply route for synthesizing the precursors of natural terpenoids, and is widely found in bacteria, fungi, plants and algae. This pathway starts with pyruvate, and DMAPP is produced in seven enzymatic reactions. DMAPP is then isomerized into IPP by IDI, and finally IPP and DMAPP are used as precursors to synthesize terpenoid precursors.

The third stage is the astaxanthin synthesis stage, in which IPP and DMAPP are converted to geranylgeranyl pyrophosphate (GPP) by the action of the enzyme farnesyl diphosphate synthase (ispA). GP P continues to be produced by ispA. Farnesyl pyrophosphate (FPP) is produced by farnesyl pyrophosphate synthase (CrtE), and geranylgeranyl diphosphate (GGPP) is produced by geranylgeranyl diphosphate synthase (CrtE). ). GGPP is converted into lycopene by the action of the octahydro-lycopene synthase/cyclase (phytoene synthase, lycopene cyclase) CrtYB and the octahydro-lycopene desaturase (phytoene desaturase) CrtI. Lycopene is converted into β-carotene by the action of CrtYB.

The structural difference between β- The structural difference between β-carotene and astaxanthin lies in the hydroxyl and carbonyl groups on the rings at the ends of the carbon chains. Therefore, the process of converting β-carotene to astaxanthin is a process of adding hydroxyl and carbonyl groups to the ends of the β-carotene molecule ring. However, the synthetic pathways in different organisms may differ. For example, in Pantoea, astaxanthin is synthesized mainly through β-carotenoid ketolase (CrtW) and β-carotene hydroxylase (CrtZ); in in Haematococcus pluvialis, astaxanthin is synthesized mainly by β-carotenoid ketolase (BKT) and β-carotene hydroxylase (CrtR); in the yeast Red Faction, astaxanthin is synthesized mainly by Cr tR and CrtS. In some other engineered yeasts, astaxanthin is also synthesized by expressing β-carotene ketolase and β-carotene hydroxylase. Tagetes erecta is currently the only plant that can produce astaxanthin, which is synthesized by expressing the carotenoid 4-hydroxy-β

2 Astaxanthin synthesis chassis cells

At present, regulating the fermentation process and metabolic engineering to modify the strain are still commonly used strategies to improve microbial astaxanthin synthesis. For example: ① improving microbial astaxanthin production by optimizing fermentation conditions; ② enhancing the supply of precursor substances by strengthening the MVA and MEP metabolic pathways; ③ screening for the expression of key genes from different sources;

④ modular engineering to connect expression genes and increase their copy number;

⑤ localize different subcellular organelles, etc.

2.1 Algae

Many algae in nature can produce astaxanthin, such as Haematococcus pluvialis, Chlamydomonas, Acetabularia, Euglena, etc. [26]. Haematococcus pluvialis is a freshwater single-celled green alga that can reach 5% astaxanthin content of the cell dry weight. It is the main alga for astaxanthin production, and the astaxanthin produced by Haematococcus pluvialis is the most antioxidant-rich levo (3S, 3'S) configuration [27]. However, Haematococcus pluvialis has a long growth cycle, high cultivation requirements, needs light, and astaxanthin is found in thick-walled spores, which have a low extraction rate, high cost, and poor continuity [19, 28].

The high cost of Haematococcus pluvialis production of astaxanthin limits its large-scale application. Therefore, there is an urgent need to develop new processes to achieve commercial application by reducing production costs and increasing the astaxanthin content in Haematococcus pluvialis. The growth of Haematococcus pluvialis requires light, but due to the uneven light intensity distribution and mixing inside the photobioreactor, the algae will be affected by the light and dark cycle, which will affect the biomass and the production of secondary metabolites. Ranjbar et al. [29] designed an air-lift photobioreactor, which, compared to a traditional bioreactor, has a more regular flow pattern for liquid circulation, resulting in a more stable light-dark cycle, more uniform liquid mixing, increased production of secondary metabolites, and a significant increase in astaxanthin production in Haematococcus pluvialis.

In addition to the above strategies, adding some exogenous substances is also a feasible way to increase astaxanthin production. Wang et al. [30] found that the addition of rac-GR24 (a synthetic plant hormone analogue) can effectively increase the biomass produced by Haematococcus pluvialis and the accumulation of astaxanthin. Rac-GR24 can increase photosynthesis in plants and increase the utilization rate of CO2 in carbohydrate synthesis, thereby increasing biomass accumulation. It also promotes the overproduction of NADPH and peroxidase, thereby reducing damage caused by reactive oxygen species. In addition, rac-GR24 treatment of Haematococcus pluvialis also alters the activity of fatty acid biosynthesis and the astaxanthin esterification pathway, thereby increasing the accumulation of astaxanthin.

Extracting astaxanthin from algae is the biggest challenge because the hard and thick cell walls of algae increase the mechanical and chemical resistance of the cells. Traditional extraction methods are not suitable for extracting astaxanthin from algae. Therefore, Huang Wencan et al. [31] proposed a new method for extracting astaxanthin from Haematococcus pluvialis using a switchable hydrophilic solvent. Dimethylamino cyclohexane (DMCHA) is a switchable hydrophilic solvent with low volatility and solubility. Using it, without the need for distillation, the extraction rate of astaxanthin from Haematococcus pluvialis can reach 87.2% by simply adding water and CO2 at the same time.

Haematococcus pluvialis is rich in natural astaxanthin, unsaturated fatty acids, etc., and has high research and utilization value [32]. At the same time, the demand for natural astaxanthin in domestic and foreign markets is increasing, and its development potential is huge. However, there are still several problems that need to be explored and solved: ① The conversion of the intermediate metabolites and the expression regulation of key enzymes involved in the synthesis of astaxanthin by Haematococcus pluvialis need to be further explored; ② Due to the complex cell wall structure of Haematococcus pluvialis, the extraction yield is low, and new extraction processes still need to be developed in the future to reduce production costs.

2.2 Yeast

The main yeasts in nature that naturally produce astaxanthin are Rhodotorula rubra, Rhodotorula glutinis, R. benthica and others. With the development of synthetic biology, engineered yeast constructed based on genetic engineering can also produce astaxanthin, such as Yarrowia lipolytica, Saccharomyces cerevisiae, and Kluyveromyces marxianus. Compared to algae and other microorganisms, yeast has a wide range of substrate sources for astaxanthin production, fast growth, a short fermentation cycle, and relatively mature genetic modification tools. Therefore, yeast is currently one of the most promising chassis cells for the industrial production of astaxanthin.

2.2.1 Red fife yeast

Red yeast is considered, along with Haematococcus pluvialis, to be the most suitable microorganism in nature for the industrial production of astaxanthin [33-34]. It can ferment and synthesize astaxanthin using a variety of sugars as carbon sources [35], and its cells grow rapidly, with a short growth cycle, enabling high-density cultivation and significantly reducing production costs [34, 36]. The astaxanthin produced at the same time is in the (3R, 3'R) configuration and is easily absorbed by the human body. Therefore, Rhodotorula became one of the ideal chassis cells for astaxanthin synthesis. Table 1 shows the latest progress in astaxanthin production by Rhodotorula.

Optimization of fermentation conditions is the easiest and most direct way to increase astaxanthin production. Among these conditions, pH has an effect on both the growth of Red Faction yeast cells and astaxanthin accumulation. Some studies have shown that the optimum initial pH for Red Faction yeast cell growth is 6.0, the optimum pH for astaxanthin formation is 4.0, and the optimum pH for astaxanthin accumulation is 5.0 [37]. Therefore, by using the pH-modulation strategy, the astaxanthin production of Rhodotorula glutinis was increased by 24.1% compared with fermentation at a constant pH. The astaxanthin synthesis pathway is complex and requires the participation of a variety of substrates and precursors. Therefore, the addition of certain substances during the fermentation process can also promote astaxanthin biosynthesis.

For example, Yang Haoyi et al. [42] used red yeast as the chassis cell and found that the downregulation of the purine, pyrimidine, amino acid synthesis and glycolysis pathways all contributed to astaxanthin biosynthesis, and the upregulation of the lipid metabolism pathway helped astaxanthin accumulation. The addition of sodium protoporphyrin can inhibit the amino acid metabolic pathway and increase astaxanthin production by 19.2%; the addition of melatonin can promote lipid metabolism and increase astaxanthin production by 30.3%.

Through metabolic flux analysis, Ru Yi et al. [41] found that ethanol can increase the content of pyruvate and acetyl coenzyme A in the metabolism of Rhodaffia yeasts, which increases the flux to the astaxanthin synthesis pathway by 2.3 times, thereby promoting astaxanthin synthesis. At the same time, the metabolic nodes of α-ketoglutaric acid and 5-phosphoribosyl pyrophosphate in the astaxanthin synthesis pathway were regulated. It was found that the addition of 0.5 g/L α-ketoglutaric acid to the culture medium could increase the growth of red fife yeast cells by 0.4 g/L. The addition of 3 g/L glutamic acid to the medium increased the production of astaxanthin to 67.9 mg/L, which is 1.7 times the flux of the control group.

It is well known that yeast has strong metabolic capabilities and can not only utilize small molecules such as monosaccharides, disaccharides, polysaccharides, and organic acids, but also simple nitrogen sources and complex organic mixtures. The use of inexpensive substrates from industrial waste can effectively reduce the cost of astaxanthin production, such as bagasse and sweet sorghum bagasse (SSB). Zhuang Yuan et al. [38] conducted studies on the red fife yeast strain at room temperature and UV mutagenesis. The mutant strain obtained after breeding was fermented at 22 °C and 220 r/min for 96 h in a sugarcane bagasse hydrolysate, and the carotenoid concentration reached 88.57 mg/L. After further cell wall disruption using ultrasound and cellulase, the astaxanthin yield reached 96.01%. Stoklosa et al. [39] used sweet sorghum bagasse as a carbon source to cultivate Rhodaff yeast to produce astaxanthin. However, because the phenolic compounds in SSB inhibit Rhodaff yeast, Therefore, the inhibition of red yeast rice by SSB was alleviated by treating SSB with activated carbon and laccase, and the cell dry weight of the bacteria was finally increased from 15.6 g/L to 23.6 g/L, and the astaxanthin production was increased from 9.55 mg/L to 48.9 mg/L.

In synthetic biology, the most common means of increasing the content of target products is still through mutation and metabolic engineering. Gassel et al. [40] obtained a strain of red yeast with high astaxanthin content through random mutagenesis. On this basis, the flux of astaxanthin synthesis was further increased by expressing the lycopene cyclase gene crtYB and the astaxanthin synthesis gene ASY. Finally, through a fermentation tank amplification experiment, the maximum astaxanthin content reached 9.7 mg/g DCW. Another study has shown that the ergosterol synthesis pathway can feedback to inhibit the MVA pathway. Therefore, deleting genes involved in ergosterol synthesis is an effective strategy for improving terpene production. Yomamoto et al. [43] sequentially deleted the two CYP61 genes encoding C-22 sterol desaturase involved in ergosterol biosynthesis, and then verified by fermentation that the astaxanthin production of recombinant red yeast Rhodotorula glutinis was increased by 1.4 times.

Although red yeast is one of the chassis cells that naturally produce astaxanthin, the yield of wild-type red yeast is low and it is prone to degradation, so there are still some challenges to achieving large-scale industrial production. Therefore, the selection of astaxanthin-high-yielding strains has become the primary goal of current research. In the future, the yield of astaxanthin can be further increased by mutagenesis to select high-yielding strains, metabolic engineering, combined with optimization of the culture medium formula and fermentation conditions.

2.2.2 Saccharomyces cerevisiae

Saccharomyces cerevisiae is the first eukaryotic microorganism to undergo whole genome sequencing. It is easy to genetically manipulate, has a clear mechanism of gene expression regulation, and mature high-density fermentation technology. In particular, in recent years, the development of a series of tools suitable for the assembly of pathways in Saccharomyces cerevisiae has made it an ideal chassis cell for synthetic biology research [44]. However, like many engineered yeasts, the wild-type Saccharomyces cerevisiae cannot synthesize astaxanthin. It needs to be genetically engineered by introducing key genes for astaxanthin synthesis to achieve astaxanthin synthesis. And the astaxanthin synthesized is mostly in the (3S, 3'S) configuration. Table 2 shows the latest progress in astaxanthin production by Saccharomyces cerevisiae.

The crtZ and crtW of different species have a significant effect on the synthesis of astaxanthin by Saccharomyces cerevisiae, so the compatibility of exogenous astaxanthin synthesis genes with the chassis cells is particularly important. Wang Ruizhao et al. [45] expressed different species of crtZ and crtW in Saccharomyces cerevisiae in combination, and selected crtW from Brevundimonas vesicularis and crtZ from Alcaligenes from 30 combinations and crtZ from Alcaligenes, and the resulting engineered strain SyBE-Sc118076 produced up to 81.0 mg/L astaxanthin. Screening for mutants of key genes is also an effective way to increase astaxanthin production.

For example, the construction of a fusion enzyme can effectively reduce the accumulation of intermediate metabolites. Based on the fusion of crtW and crtZ using a linker, nine fusion mutants with increased astaxanthin production were obtained through directed evolution. Combining these dominant mutants resulted in the high-yield astaxanthin yeast strain L95S+1206L, which has 3.8 times the astaxanthin content of the control strain [49-50].

In order to improve the efficiency of β-carotene conversion to astaxanthin, Zhou Pingping et al. [46] obtained superior yeast mutants of crtZ and crtW through directed coevolution, and at the same time introduced a Gal4M9-based temperature-responsive regulatory system [51], decoupling astaxanthin synthesis from cell growth coupling, i.e., the temperature was kept at 30 °C in the first phase to allow rapid cell growth; when the cells entered the log phase, the culture temperature was changed to 24 °C to promote astaxanthin synthesis; finally, 235 mg/L astaxanthin was synthesized through two-phase high-density fermentation.

Since astaxanthin is fat-soluble, this conflicts with the limited lipid storage capacity of model microorganisms such as Saccharomyces cerevisiae [52]. Therefore, the astaxanthin content can be increased by promoting lipid synthesis to expand lipid droplets and provide more storage space for astaxanthin synthesis. Therefore, Li Ming et al. used a three-functional CRISPR system to screen a library of genes related to lipid metabolism [47, 53], and moderately up-regulated the expression levels of opi3 and hrd1 to promote lipid synthesis. After balancing the expression of crtZ and crtW, the astaxanthin content was increased to 10.21 mg/g DCW. Finally, by combining spatial regulation of lipid synthesis and temporal regulation of temperature response, 446.4 mg/L astaxanthin was synthesized in the fed-batch fermentation.

2.2.3 Lipolytic yeast

Lipolytic yeast is one of the most widely studied and used unconventional yeasts. Astaxanthin produced by lipolytic yeast is mostly in the (3S, 3'S) configuration. Compared with conventional yeast Saccharomyces cerevisiae, lipolytic yeast has a variety of unique biochemical and metabolic characteristics. It is a typical type II yeast that is an aerobic bacterium that basically does not produce ethanol, which is toxic to cells [54]. It also has an efficient acetyl coenzyme A metabolic pathway and a high tricarboxylic acid cycle flux in its cells, and the accumulation of lipids can reach 77%. This makes it ideal for industrial production of organic acids, lipids and their derivatives [55-57]. In addition, Y. lipolytica can grow at lower pH and higher osmotic pressure, and can utilize a variety of carbon sources, proteins and hydrophobic substrates, such as sugars, hydrocarbons, alcohols, lipids, etc. Therefore, it is not strict in terms of growth environment and can grow under various environmental conditions, which has good industrial application prospects [58]. Table 3 shows the latest progress in astaxanthin production by Y. lipolytica.

The astaxanthin biosynthetic genes from different sources are still the key factors affecting the astaxanthin production in Y. lipolytica. Kildegaard et al. [59] expressed the crtW from Paracoccus sp. N81106 and the crtZ from P. ananatis in a high-yield β-carotene-producing Y. lipolytica strain, and optimized the copy number of the relevant genes copies were optimized, and 3.5 mg/g DCW (54.6 mg/L) astaxanthin was obtained. In another study, three pairs of crtW and crtZ from different sources were expressed, and it was pointed out that HpCrtW and HpCrtZ from Haematococcus pluvialis had the strongest activity in converting β-carotene to astaxanthin [65]. Through modular assembly of the relevant genes by the RIAD-RIDD short peptide [62], and at the same time increasing the copy number to 20, the astaxanthin production of recombinant lipase yeast reached 3.3 g/L under batch culture conditions with a supplement, which is the highest level of astaxanthin synthesis in a microbial chassis to date.

Most metabolic regulation methods for producing high value-added chemicals in microorganisms use the cytoplasm as a reaction vessel [66]. However, the isolation of enzymes and substrates and metabolic crosstalk often hinder the effective synthesis of target compounds in the cytoplasm. The regionalization of organelles in eukaryotic cells provides a solution to this bottleneck. For example, Ma Yongshuo et al. [60] not only accelerated the conversion of β-carotene to astaxanthin, but also significantly reduced the accumulation of carotenoid intermediates by localizing the astaxanthin synthesis pathway in the endoplasmic reticulum and peroxisomes, thereby increasing astaxanthin production 141 times. In fed-batch fermentation, 858 mg/L astaxanthin can be synthesized.

Astaxanthin is a fat-soluble terpene compound, and its strong hydrophobicity leads to low bioavailability. The addition of an external oil phase can promote the dissolution of astaxanthin and prevent the formation of crystals, thereby increasing the yield of astaxanthin. Yuzbasheva et al. [61] used a modular engineering approach and fusion technology, constructed a recombinant lipase-deficient Saccharomyces cerevisiae strain with astaxanthin production of 587.3 mg/L, and astaxanthin production could be increased to 973.4 mg/L with the addition of an oil overlay.

Glycosylation can also significantly increase the water solubility of astaxanthin, thereby improving its bioavailability, light stability and biological activity [67-68]. Chen Jing et al. [63] constructed a plant-derived astaxanthin-producing strain by expressing the carotenoid 4-hydroxy-β-cyclodehydro-genase (HBFD) and carotenoid-β-cyclodehydro-genase (CBFD) genes from the summer marigold in Pichia pastoris, and the astaxanthin yield reached 3.46 mg/L. On this basis, by expressing the zeaxanthin glycosyltransferase (CrtX) gene from Pantoea ananatis (P. ananatis ATCC 19321), a glycosylated astaxanthin synthesis pathway was successfully constructed, and the yield reached 1.47 mg/L, which is the highest yield of glycosylated astaxanthin produced by microorganisms reported so far.

2.2.4 Kluyveromyces marxianus

In recent years, Kluyveromyces marxianus has been widely used in the synthesis of natural products. Compared with other traditional yeasts, it has the following advantages: Maxicruve yeast can increase cell production by providing an excess of carbon sources [69]; some Maxicruve yeasts are resistant to high temperatures and can ferment at temperatures between 25 and 52 °C [70]; it has appropriate glycosylation and strong signal peptides, and has a higher secretion capacity than Saccharomyces cerevisiae [71].

At present, based on metabolic engineering methods, Maxicruve yeast has been used as a chassis cell to synthesize astaxanthin, and the astaxanthin synthesized is mostly in the (3S, 3'S) configuration. For example, Lin Yuju et al. [72] constructed an astaxanthin heterologous synthesis pathway in Maxicruve yeast to achieve astaxanthin synthesis using glucose. They integrated the Hpchyb and bkt genes from Haematococcus pluvialis into the genome of the Saccharomyces cerevisiae and increased their copy number to obtain four modified strains. To further improve the yield, site-directed mutations were introduced into the Hpchyb gene to improve enzyme efficiency and prevent ubiquitination-induced degradation of heterologous proteins.

This ultimately resulted in the synthesis of 9972 μg/g DCW astaxanthin in a 5 L fermenter. In another study, the use of a xylose-inducible promoter and a temperature regulation system allowed for the decoupling of cell growth and product synthesis. Further optimization of the metabolic pathway and fermentation conditions resulted in astaxanthin production of 56.8 mg/L [73]. Although there are few reports on the use of Maxicruvin yeast as a chassis cell for the production of astaxanthin, its unique advantages provide a new technical option for the microbial fermentation and synthesis of astaxanthin.

2.2.5 Other yeasts

In addition to the typical engineered yeasts mentioned above, there are also relatively few reports on the production of astaxanthin by yeasts such as Rhodotorula mucilaginosa and marine red yeast. Rhodotorula mucilaginosa is a red yeast containing oil and is mainly used to produce β-carotene. A team obtained a strain of astaxanthin-producing Rhodotorula mucilaginosa RG-31 through mutagenesis screening, and finally optimized the fermentation conditions to achieve an astaxanthin content of 7.41 μg/mL. Marine red yeast is a type of unicellular yeast that naturally exists in the ocean. It has good salt tolerance and produces carotenoids, mainly astaxanthin. A strain of marine deep red yeast S8 with high carotenoid production was obtained by ultraviolet mutagenesis to obtain strain ST5, and by optimizing the fermentation conditions, the astaxanthin content can reach 520 μg/g.

2.3 Bacteria

Due to the low production of astaxanthin by bacteria, research on astaxanthin has mainly focused on algae and fungi at home and abroad, and relatively little research has been done on astaxanthin synthesis by bacteria. Although the astaxanthin content of most bacteria is much lower than that of some algae and fungi, the introduction of genes related to astaxanthin synthesis into bacteria [20] can greatly increase astaxanthin production. At the same time, compared with fungi and algae, the use of bacterial fermentation makes it easier to extract astaxanthin, which can greatly simplify the subsequent extraction process. In particular, Gram-negative bacteria have thin and easily broken cell walls, which facilitates the extraction of astaxanthin from the cells. Therefore, the production of astaxanthin by bacterial fermentation can greatly reduce the production cost of astaxanthin and is of great significance for future industrial production.

2.3.1 Escherichia coli

Escherichia coli is a Gram-negative, facultatively anaerobic bacterium. It is easy to cultivate, simple to operate, inexpensive, and has mature molecular genetic modification tools. It has become one of the best hosts for metabolic engineering and synthetic biology. As a non-carotenoid-producing strain, it can synthesize the terpene compound precursors IPP and DMAPP through the MEP pathway. In wild-type E. coli, the FPP synthase can be produced in vivo, which can condense IPP and DMAPP to generate GPP and FPP, but lacks the enzymes that convert FPP to the final astaxanthin. Therefore, by introducing an exogenous astaxanthin synthesis module into E. coli, it is relatively easy to synthesize astaxanthin in E. coli, and the astaxanthin synthesized is mostly in the levorotatory (3S, 3'S) configuration. Table 4 shows the latest progress in E. coli astaxanthin production.

The identification of key genes in the metabolic synthesis pathway of astaxanthin has made it possible to construct engineered bacteria with high astaxanthin production. Jeong et al. [75] used the MEP pathway from Kocuria gwangallensis to co-express dxs, ispC, ispD , ispE , ispF , ispG , ispH and idi and the genes involved in the conversion of astaxanthin synthesis (crtI, crtE, crtYB, crtW, crtZ) were co-expressed in E. coli to increase astaxanthin production. This engineered E. coli containing the genes of the non-mevalonate pathway was able to synthesize 1100 μg/g DCW of astaxanthin. The lytB, ispA and idi genes in the isoprenoid pathway are essential for the synthesis of IPP, DMAPP and FPP. However, since E. coli itself can only synthesize these precursors to meet its own growth needs and cannot divert them to the astaxanthin synthesis pathway, the astaxanthin yield is too low. Therefore, Lee et al. [76] overexpressed lytB, ispA and idi in E. coli carrying the astaxanthin synthesis genes (crtI, crtE, crtYB, crtW, crtZ), and ultimately engineered E. coli to synthesize 1200 μg/g DCW astaxanthin. Screening for crtW and crtZ from different sources is still one of the routine methods for improving astaxanthin production.

For example, Lu et al. [78] compared crtW and crtZ from different sources and concluded that crtW from Brevundimonas sp. SD212 and crtZ from Pantoea agglomerans were the best combination for astaxanthin production. By balancing the expression activity of crtW and crtZ, a strain of E. coli ASTA-1 was constructed that carried neither a plasmid nor an antibiotic marker. Without the addition of an inducer, the recombinant strain synthesized 96.6% of the carotenoids as astaxanthin, reaching a content of 7.4 mg/g DCW. Wu Yuanqing et al. [79] screened 9 crtZ and crtW from different sources and introduced them into engineered E. coli with high β-carotene production, and the astaxanthin content reached 0.49–8.07 mg/L. Subsequently, the crtZ and crtW were fused using an optimized peptide linker, which further increased the astaxanthin production by 127.6%.

Li Shun et al. [80] expressed the HpCHY gene from Haematococcus pluvialis and the CrBKT gene from Chlamydomonas reinhardtii in Escherichia coli by selecting the GadE promoter. The final engineered strain was able to synthesize 24.16 mg/L astaxanthin, which is 40 times higher than the original strain. This shows that selecting the appropriate gene elements for astaxanthin synthesis can significantly affect the expression level of astaxanthin.

Increasing the copy number of key genes is a simple, direct and effective way to increase astaxanthin production. For example, in E. coli, the bottleneck of insufficient astaxanthin accumulation was eliminated by increasing the copy number of crtYB, and by regulating the expression of the promoter, the final astaxanthin production reached 1.18 g/L under fed-batch fermentation conditions [20].

Li Di et al. [82] first performed random mutations on crtW to increase its activity in converting β-carotene to astaxanthin, and then increased the copy number of crtW and crtZ by the Cre-loxP method to construct an E. coli strain with high astaxanthin production. Finally, by fed-batch fermentation, the astaxanthin production reached 0.88 g/L [84]. Zhang Meng et al. [83] found that co-expression of crtZ from Paracoccus sp. PC1 and crtZ from Pantoea agglomerans can increase the accumulation of astaxanthin and intermediates. Finally, an engineered E. coli strain an engineered E. coli strain with two copies of PAcrtZ and one copy of PCcrtZ. After 70 h of fed-batch fermentation, astaxanthin production reached 1.82 g/L.

In addition, strategies for enhancing the astaxanthin metabolic pathway through unconventional technical means can also be used to achieve the goal of increasing astaxanthin production. For example, by co-expressing the partner genes ApcpnA and ApcpnB in E. coli expressing astaxanthin synthesis genes (crtI, crtE, crtYB, crtW, crtZ), the final engineered bacteria can produce 890 μg/g DCW of astaxanthin [74]. Lemuth et al. [77] constructed the first plasmid-free E. coli, and stably integrated the genes of the astaxanthin biosynthesis pathway into the E. coli chromosome through the γ-Red recombination technology, so that the pathway only pointed to astaxanthin synthesis. The final astaxanthin content reached 1.4 mg/g DCW [85]. Morphology-based and oxidative stress engineering are also effective strategies for improving astaxanthin synthesis in E. coli. For example, deleting genes related to morphology and membranes can result in larger, longer cells, which in turn increase astaxanthin accumulation.

Oxidative stress refers to an imbalance between the production of reactive oxygen species and antioxidants in cells, which leads to cell damage. Therefore, deleting genes related to oxidative stress can increase the level of reactive oxygen species in cells to protect the cells [81]. At the same time, cell morphology will still change after the temperature rises, and the level of reactive oxygen species will also be higher. Therefore, by establishing a complementary expression system of temperature-sensitive plasmids, the astaxanthin content of this strain of E. coli can ultimately be increased to 11.92 mg/g DCW. In addition, enzyme localization strategies can also be used to increase the production of astaxanthin in engineered E. coli. For example, Ye Lijun et al. [86-88] used an E. coli localization tag to localize the β-carotene cleavage dioxygenase PhCCD1 to different cell compartments and found that its catalytic efficiency was optimal at the membrane. Finally, after fusing CrtW and CrtZ to the E. coli cell membrane through the GlpF protein, astaxanthin production increased by 215.4%.

2.3.2 Paracoccus

Paracoccus (Paracoccus sp.) is an aerobic, Gram-negative bacterium that contains astaxanthin and other rare carotenoids in its cells. However, there are few research papers on astaxanthin synthesis in Paracoccus [89-90]. Natural carotenoids usually exist in the E configuration, which is less bioavailable to humans and animals. “Z-isomerization” is an effective means of increasing their bioavailability [91-93]. For example, Honda et al. [94] used the coccolithophore as the chassis cell to isomerize carotenoids such as astaxanthin under subcritical water conditions (heating water above the boiling point, below the critical point, and controlling the system pressure to keep the water in a liquid state). It was found that adding ethanol under heating and pressurizing conditions significantly improved the efficiency of “Z-isomerization”. and pressurized conditions, the efficiency of “Z-isomerization” was significantly improved. Finally, after 30 minutes of subcritical water treatment, while inhibiting carotenoid degradation, astaxanthin with a “Z-isomerization” ratio of more than 50% was obtained.

Appropriate regulation of the composition of the culture medium and the parameters during fermentation is an effective strategy for increasing astaxanthin production. Adding tricarboxylic acid intermediates to the culture medium can enhance the supply of precursors. The activity of key enzymes can also be enhanced by adding cofactors for key enzymes in astaxanthin synthesis (ferrous sulfate, ascorbic acid, NADPH, ATP and 2-oxoglutarate), thereby increasing astaxanthin accumulation. For example, when producing astaxanthin in a Bacillus subtilis chassis, the activity of the Crt enzyme can be increased by adding 5 mmol/L malate and 1 mmol/L ferrous sulfate, increasing astaxanthin production from 177 μg/L to 3750 μg/L [95]. Although the production of astaxanthin by bacteria themselves is lower than that by algae and yeast, it provides alternative gene elements for the subsequent construction and modification of engineered strains.

2.3.3 Other bacteria

There are relatively few bacteria that can produce astaxanthin, and the yields are relatively low. The most studied is Escherichia coli as a chassis cell. In addition, Mycobacterium lacticola and Bevibacterium can also produce astaxanthin. However, Mycobacterium lacticola only produces astaxanthin on hydrocarbon media and does not produce astaxanthin on nutrient media. Bevibacterium grows in oil, and at the end of the fermentation, the biomass is only 3 g/L, and the astaxanthin content is even lower.

In summary, E. coli is currently the ideal chassis cell for astaxanthin production among bacteria.

3 Extraction process of astaxanthin

Astaxanthin is an intracellular product, so the extraction of astaxanthin from microorganisms is divided into two steps: cell disruption and astaxanthin collection. Compared with bacteria, the cell walls of algae and yeast are tough and thick, and are not easily broken, which makes the extraction of the product very difficult. Therefore, the focus of astaxanthin extraction is on cell wall disruption [96].

Traditional methods of cell wall disruption mainly include physical, chemical and enzymatic methods [97]. Physical methods include mechanical crushing, ultrasonic crushing and supercritical fluid extraction. Mechanical crushing is simple to operate, and the cell walls are torn by mechanical agitation, thereby releasing the astaxanthin inside the cells. However, mechanical crushing may cause high temperatures in some places, which may damage the astaxanthin to a certain extent. Although ultrasonic crushing can effectively break the cell walls in the solute, as the intensity and duration of the ultrasonic action increase, the production of highly oxidizing free radicals increases, which leads to a decrease in the extraction rate of astaxanthin. Supercritical fluid extraction is the most effective extraction method for extracting various types of algae products in recent years. Compared with traditional liquid solvents, it has some unique physical and chemical properties, such as high diffusivity, high compressibility, low surface tension, low viscosity, and easy penetration of cell walls, which improves the extraction efficiency of the product.

Chemical methods mainly include organic solvent extraction, acid-base treatment, and dimethyl sulfoxide (DMSO) methods. Astaxanthin is a fat-soluble natural product, so the choice of organic solvent must take into account whether astaxanthin is soluble and the polarity of the solvent. For example, Xing Tao et al. [99] used ethyl acetate as an organic solvent to extract astaxanthin from Haematococcus pluvialis, and the final yield was 98.51%. Although the use of organic solvents to extract astaxanthin has a high yield, the downside is that many solvents are toxic and may have a damaging effect on astaxanthin. Acid-base treatment involves the use of large quantities of acid and alkali reagents, which may not only damage astaxanthin but also cause corresponding environmental pollution. Dimethyl sulfoxide is a polar solvent that is soluble in both water and organic solvents. It is a commonly used solvent for breaking cell walls in the laboratory, as it can quickly and efficiently break the cell walls of bacteria without significantly affecting astaxanthin [100]. When mixed with acetone in the right proportions, it can also extract astaxanthin relatively completely [101].

Enzymatic extraction of astaxanthin has the advantages of mild conditions, low energy consumption and short processing time. It can not only quickly and efficiently break down cell walls and release astaxanthin from cells, but also inhibit cell activity to prevent denaturation of intracellular substances [102]. Therefore, astaxanthin extracted by enzymatic method is more stable than that obtained by other methods. For example, β-glucanase can hydrolyze β-glucan in the cell wall, which can prevent astaxanthin from spilling out of the bacteria and reduce losses [100]. However, the enzymatic method requires a large amount of enzyme, which undoubtedly increases production costs. At the same time, because enzymes are proteins by nature, they are prone to denaturation and therefore are not suitable for large-scale astaxanthin extraction.

Since astaxanthin is a strong antioxidant, if it is exposed to air for a long time, the oxygen in the air will react with astaxanthin and cause it to lose its antioxidant capacity. Therefore, an oxygen-free (nitrogen-filled) extraction process is also one of the steps that is currently unavailable in industrial production. This method involves filling with an inert gas or nitrogen to provide an oxygen-free environment for organic solvent extraction, which protects its activity and significantly improves the antioxidant activity of astaxanthin.

4 Summary and outlook

Due to the wide application of astaxanthin in the fields of food, medicine and cosmetics, the market demand for astaxanthin is expected to increase. At present, chemical synthesis is still the main method of astaxanthin production, but due to the limitations of chemically synthesized astaxanthin, countries around the world are becoming increasingly strict in their management of chemical synthesis of astaxanthin. However, astaxanthin extracted directly from natural resources can hardly meet consumer demand.

Therefore, the industrial production of astaxanthin through microbial fermentation provides a promising option. Yeast is the most promising chassis cell due to its advantages such as a broad substrate spectrum, easy growth and culture, short fermentation cycle, and relatively mature genetic modification tools. Among them, Rhodopseudomonas palustris is capable of naturally producing astaxanthin, grows fast, and can utilize a variety of carbon sources; Yarrowia lipolytica has high acetyl coenzyme A flux and tricarboxylic acid cycle metabolic pathway, and can grow at lower pH and high osmotic pressure; Saccharomyces cerevisiae is easy to genetically manipulate, has a clear mechanism for regulating gene expression, and mature high-density fermentation technology; and Pichia pastoris can withstand high temperatures, has appropriate glycosylation, and strong signal peptides.

With the rapid development of synthetic biology, protein engineering, metabolic engineering and fermentation engineering, many microorganisms have been used as chassis cells to produce astaxanthin. However, despite the significant breakthroughs in astaxanthin biosynthesis, challenges remain:

First, microorganisms that can naturally produce astaxanthin, such as Rhodotorula glutinis and Haematococcus pluvialis, still have problems with low yields, strict culture conditions and high costs. To solve this problem, future research should focus on the cultivation and screening of high-yielding strains and the optimization of culture conditions to increase yields and reduce costs.

Second, due to the complex anabolic pathway of astaxanthin synthesis, there are still problems such as low yields in metabolic engineering of model organisms such as Escherichia coli, Lipomyces starkeyi, Saccharomyces cerevisiae, etc. In this regard, the following strategies can be used to improve the efficiency of astaxanthin synthesis in chassis cells (Figure 4): (1) Optimize and modify the MVA metabolic pathway. For example, truncated tHMG (truncated by 500 amino acids at the N-terminus) has been shown to be a key gene in the MVA pathway. It can effectively prevent its own degradation, thereby increasing the production of carotenoids.

(2) Replacing promoters of different strengths improves the catalytic matching in metabolic pathways. For example, in the gene for β-carotene biosynthesis, a strong promoter PTEF replaced other weak promoters, significantly increasing the β-carotene production in Yarrowia lipolytica [103].

③ Increasing the copy number of key genes to improve the metabolic flux of rate-limiting steps. For example, when the copy number of crtYB was increased from 1 to 4, β-carotene production increased by 76% [104].

④ Modular assembly of key genes using different linkers or short peptides to improve carbon metabolic flux, thereby increasing the potency of astaxanthin.

⑤Locate in different subcellular organelles to increase the storage space for astaxanthin. Carotenoids are generally stored in the cell membrane, which can reduce the fluidity of the cell membrane and cause cell death. Localizing the metabolic pathway of carotenoids in subcellular organelles can reduce metabolic disorders metabolism disorder. At the same time, it can also expand the storage space of carotenoids in the cell, reducing its toxicity to the cell. For example, the endoplasmic reticulum, mitochondria, and peroxisomes in eukaryotic cells have unique physical environments that provide favorable conditions for the synthesis of carotenoids.

⑥ Fermentation condition optimization is the most common and most effective way to increase astaxanthin production. For example, optimizing the medium with different carbon and nitrogen sources, different carbon and nitrogen ratios, pH values, and temperature control.

It is also worth noting that even though astaxanthin produced through biosynthesis has far greater application value than that produced through chemical synthesis, its application is still restricted by the laws and regulations of many countries. For example, the US FDA prohibits the use of astaxanthin as a food additive because prolonged heating of astaxanthin can produce carcinogens. France clearly stipulates that astaxanthin can only be used in specific health products and cosmetics. Therefore, there is still a long way to go before astaxanthin can be produced through microbial fermentation.

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