A high astaxanthin-producing yarrowia lipolytica engineering strain and a construction method and application thereof

Abstract

The application provides a high astaxanthin yield Yarrowia lipolytica engineering strain and a construction method and application thereof. Through genetic engineering means, the Yarrowia lipolytica is subjected to multi-target metabolic engineering modification, four gene clusters of McCarRP-McCarB, PnCrtW-linker-HpCrtZ, SpHMGR-DGA1 and ZWF1-GND1 are overexpressed on the basis of a wild type Yarrowia lipolytica strain, and finally an engineering strain yyYLA004 is obtained. Overexpression of the coding genes of the proteins in the Yarrowia lipolytica can directly improve the astaxanthin synthesis capacity of the cells, and the application has large-scale production level and good industrial application prospect.

Description

Technical Field

[0001] This invention belongs to the field of microbial genetic engineering and metabolic engineering technology, specifically relating to a high-yield astaxanthin-producing Yersinia lipolyticis strain and its construction method and application. Background Technology

[0002] Astaxanthin is a carotenoid with extremely strong antioxidant activity, possessing various physiological functions such as scavenging free radicals, enhancing immunity, protecting the cardiovascular system, anti-inflammation, and anti-tumor activity. It is widely used in food, health products, cosmetics, and animal feed. Currently, the main methods for producing astaxanthin include chemical synthesis, animal and plant extraction, and microbial fermentation. Chemical synthesis suffers from problems such as simple structure, low biological activity, and potential for toxic byproducts; animal and plant extraction is limited by resources, resulting in low yields and high costs. Microbial fermentation, due to its advantages of short production cycles, relatively low costs, and the potential for large-scale industrial production, has become a research hotspot in astaxanthin production.

[0003] Microorganisms capable of synthesizing astaxanthin mainly include *Phaffia rhodozyma* and *Haematococcus pluvialis*. *Phaffia rhodozyma* has a rapid growth rate and short fermentation cycle, but its astaxanthin yield is relatively low; *Haematococcus pluvialis* has a high astaxanthin content, but its cultivation cycle is long, it is sensitive to environmental conditions, and its cultivation cost is high. Therefore, constructing high-yield astaxanthin microbial engineered strains is of significant practical importance.

[0004] *Yarrowia lipolytica* is an unconventional yeast with a strong ability to utilize hydrophobic carbon sources and accumulate lipids. Its endogenous synthetic pathway can synthesize substances such as farnesyl pyrophosphate, serving as precursors for astaxanthin synthesis. Wild-type *Yarrowia lipolytica* cannot synthesize astaxanthin itself, requiring metabolic engineering to introduce and optimize its astaxanthin synthesis pathway. In the astaxanthin biosynthesis pathway, the β-carotene hydroxylase encoded by the *crtZ* gene and the β-carotene ketolase encoded by the *crtW* gene are key rate-limiting enzymes. During the biosynthesis of β-carotene to astaxanthin, the sequence of ketation and hydroxylation reactions is diverse, resulting in the synthesis of various byproducts such as zeaxanthin, ursolic acid, and canthaxanthin, thus affecting the yield of the final product, astaxanthin. Furthermore, the heterologous astaxanthin synthesis pathway requires the participation of reduced coenzyme II NADPH, and the NAD(P)H / NAD(P) ratio in the late fermentation system is crucial. + The ratio has a significant impact on the catalytic activity of enzymes. When the ratio of cofactors is not within the appropriate range for the target enzyme, the catalytic efficiency will be reduced, thereby negatively affecting the synthesis efficiency of the target product. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a high-astaxanthin-producing *Yarrowia lipolytica* engineered strain, its construction method, and its applications. This invention utilizes genetic engineering techniques to modify *Yarrowia lipolytica* through multi-target metabolic engineering. By overexpressing four gene clusters—McCarRP-McCarB, PnCrtW-linker-HpCrtZ, SpHMGR-DGA1, and ZWF1-GND1—on a wild-type *Yarrowia lipolytica* strain, the engineered strain yyYLA004 was ultimately obtained. The high-astaxanthin-producing *Yarrowia lipolytica* strain obtained by this invention directly enhances the cell's ability to synthesize astaxanthin by overexpressing the encoding of multiple key enzymes in the astaxanthin synthesis pathway.

[0006] To address the metabolic engineering bottleneck in astaxanthin synthesis using *Yersinia lipolytica*, this invention first expresses the McCarB and McCarRP genes in *Yersinia lipolytica*, introducing a β-carotene synthesis pathway. A flexible linker sequence (amino acid sequence shown in SEQ ID NO. 89: GGGGSGGGGS) is introduced between the CrtW and CrtZ genes, and a tandem expression vector is constructed and successfully integrated into the *Yersinia lipolytica* genome. This flexible linker shortens the distance between the enzyme and its substrate, eliminating metabolic bottlenecks and reducing the synthesis of byproducts. Furthermore, expressing the 3-hydroxy-3-methylglutaryl-CoA reductase gene SpHMGR1 from *Silicibacter pomeroyi*, and the endogenous ZWF1 and GND1 genes from *Yersinia lipolytica*, can increase the cell's NADPH regeneration capacity, thereby improving astaxanthin production. Currently, no prior art has been disclosed that simultaneously addresses both of these bottlenecks by optimizing the metabolic engineering of *Yersinia lipolytica* strains that synthesize astaxanthin through metabolic engineering.

[0007] To achieve the above objectives, the present invention provides a high-yield astaxanthin-producing Yarrowia lipolytica engineered strain. The engineered strain is based on wild-type Yarrowia lipolytica and is genetically engineered to overexpress multiple key enzyme genes in the astaxanthin synthesis pathway.

[0008] Preferably, the high-astaxanthin-producing *Yersinia lipolytica* engineered strain is obtained by integrating key enzyme genes in the astaxanthin synthesis pathway into the starting strain. The key enzyme genes in the astaxanthin synthesis pathway include: McCarRP, a bifunctional enzyme derived from *Mucor* (a type of mold); McCarB, a phytoene dehydrogenase derived from *Mucor*; PnCrtW, a β-carotene ketolase derived from *Paracococcus*; HpCrtZ, a β-carotene hydroxylase derived from *Haematococcus pluvialis*; SpHMGR, a 3-hydroxy-3-methylglutaryl-CoA reductase derived from *Roseobacterium*; DGA1, a diacylglycerol acyltransferase derived from *Yersinia lipolytica*; ZWF1, a glucose-6-phosphate dehydrogenase derived from *Yersinia lipolytica*; and GND1, a 6-phosphate gluconate dehydrogenase derived from *Yersinia lipolytica*.

[0009] Preferably, the key enzyme gene in the astaxanthin synthesis pathway is selected from at least one of the following astaxanthin synthesis pathway key enzyme gene expression cassettes (referred to as gene expression cassettes or gene clusters): SpHMGR-DGA1, McCarRP-McCarB, PnCrtW-linker-HpCrtZ, and ZWF1-GND1. These eight genes are derived from: McCarRP, a bifunctional enzyme of phytoene synthase / lycopene β-cyclase, from *Mucor lusitanicus*; McCarB, a phytoene dehydrogenase from *Mucor lusitanicus*; PnCrtW, a β-carotene ketolase from *Paracoccus sp.*; HpCrtZ, a β-carotene hydroxylase from *Haematococcus lacustris*; SpHMGR, a 3-hydroxy-3-methylglutaryl-CoA reductase from *Silicibacter pomeroyi*; and DGA1, ZWF1, and GND1, a diacylglycerol acyltransferase from *Yarrowia lipolytica*.

[0010] Preferably, the key enzyme gene in the astaxanthin synthesis pathway is driven by a strong constitutive promoter of *Yersinia lipolytica*, wherein the strong constitutive promoter of *Yersinia lipolytica* is at least one of pTEF1, pTEF1in, or pGPD1.

[0011] Preferably, the starting strain is wild-type Yersinia lipophila Po1f.

[0012] In a preferred embodiment of the present invention, a high-astaxanthin-producing engineered Yersinia lipolytica strain yyYLA004 is provided, which uses wild-type Yersinia lipolytica Po1f as the starting strain and overexpresses four gene clusters: McCarRP-McCarB, PnCrtW-linker-HpCrtZ, SpHMGR-DGA1, and ZWF1-GND1.

[0013] Preferably, the nucleotide sequence of McCarRP is shown in SEQ ID NO.1;

[0014] Preferably, the nucleotide sequence of McCarB is shown in SEQ ID NO.2;

[0015] Preferably, the nucleotide sequence of PnCrtW is as shown in SEQ ID NO.3;

[0016] Preferably, the nucleotide sequence of HpCrtZ is as shown in SEQ ID NO.4;

[0017] Preferably, the nucleotide sequence of spHMGR is as shown in SEQ ID NO.5;

[0018] Preferably, the nucleotide sequence of DGA1 is as shown in SEQ ID NO.6;

[0019] Preferably, the nucleotide sequence of ZWF1 is as shown in SEQ ID NO.7;

[0020] Preferably, the nucleotide sequence of GND1 is as shown in SEQ ID NO.8.

[0021] The present invention also provides a method for preparing the high-astaxanthin-producing *Yersinia lipophila* engineered strain as described in any of the above claims, comprising the following steps:

[0022] Step 1: Construct a recombinant plasmid EcYL001 containing expression cassettes of McCarRP and McCarB genes and an expression cassette of the URA3 selection marker gene. Transform it into wild-type Yersinia lipophila. After verification screening, the correct transformants are obtained. Then, the URA3 selection marker is removed to obtain the engineered strain yyYLA001.

[0023] Step 2: Construct recombinant plasmid EcYL002 containing expression cassettes of PnCrtW and HpCrtZ genes and expression cassette of URA3 selection marker gene, transform it into the engineered yeast strain yyYLA001, obtain the correct transformant through verification screening, and then remove the URA3 selection marker to obtain engineered strain yyYLA002.

[0024] Step 3: Construct recombinant plasmid EcYL003 containing expression cassettes of the SpHMGR gene, DGA1 gene, and URA3 selection marker gene, and transform it into the Yeast lipolyticis engineered strain yyYLA002. After verification screening, the correct transformants were obtained, and then the URA3 selection marker was removed to obtain engineered strain yyYLA003.

[0025] Step 4: Construct a recombinant plasmid EcYL004 containing the ZWF1 gene expression cassette, the GND1 gene expression cassette, and the URA3 selection marker gene. Transform this plasmid into the *Yersinia lipolytica* engineered strain yyYLA003. After verification and screening to obtain the correct transformants, the transformants were then introduced with the UP-LEU2-DN fragment for LEU2 nutrient marker replenishment, resulting in the engineered strain yyYLA004. This engineered strain yyYLA004 is the high-astaxanthin-producing *Yersinia lipolytica* engineered strain described in this invention.

[0026] Preferably, the recombinant plasmid EcYL002 contains homologous arms above and below the integration site; the recombinant plasmid EcYL003 contains homologous arms above and below the integration site; the recombinant plasmid EcYL004 contains homologous arms above and below the integration site; preferably, the URA3 selection marker gene has the NCBI accession number XM_066094358.2 and is an endogenous selection reporter gene of the strain.

[0027] Preferably, the UP-LEU2-DN fragment is obtained as follows: using the *Yersinia lipolytica* Po1f genome as a template, the LEU2-UP sequence is amplified using primers LEU2-UP-F and LEU2-UP-R; using the pYLXP'-URA3 plasmid as a template, the LEU2 gene sequence is amplified using primers LEU2-F and LEU2-R; using the *Yersinia lipolytica* Po1f genome as a template, the LEU2-DN sequence is amplified using primers LEU2-DN-F and LEU2-DN-R; and LEU2-UP, LEU2, and LEU2-DN are spliced ​​into the UP-LEU2-DN sequence by overlap extension PCR.

[0028] In a preferred embodiment of the present invention, a method for constructing a high-astaxanthin-producing engineered Yersinia lipolytica strain is provided, comprising the following steps:

[0029] 1. Using overlap extension PCR technology, the promoter, target gene, and terminator were spliced ​​into two gene expression cassettes, pTEF1-McCarRP-tXPR2 and pGPD1-McCarB-tLIP1. Then, the two gene expression cassettes were assembled with the homologous arms above and below the integration site, the URA3 expression cassette, and the pUC19 plasmid backbone through seamless cloning to construct the recombinant plasmid EcYL001. After linearization by PmeI restriction enzyme digestion, it was transformed into wild-type Yersinia lipolytica. The correct transformants were obtained through verification screening and then transformed into the plasmid pYL-Cre carrying Cre recombinase to remove the URA3 selection marker, thus obtaining the engineered strain yyYLA001.

[0030] 2. Using overlap extension PCR technology, the promoter, target gene, and terminator were spliced ​​into the pTEF1in-PnCrtW-linker-HpCrtZ-tXPR2 gene expression cassette. Then, the gene expression cassette was assembled with the homologous arms above and below the integration site, the URA3 expression cassette, and the pUC19 plasmid backbone through seamless cloning to construct the recombinant plasmid EcYL002. After linearization by PmeI restriction enzyme digestion, it was transformed into the engineered Yersinia lipolytica strain yyYLA001. After verification and screening, the correct transformants were obtained and then transformed into the plasmid pYL-Cre carrying Cre recombinase to remove the URA3 selection marker, thus obtaining the engineered strain yyYLA002.

[0031] 3. Using overlap extension PCR technology, the promoter, target gene, and terminator were spliced ​​into two gene expression cassettes, pTEF1-SpHMGR-tXPR2 and pGPD1-DGA1-tLIP1. Then, the two gene expression cassettes were assembled with the homologous arms above and below the integration site, the URA3 expression cassette, and the pUC19 plasmid backbone using a seamless cloning method to construct the recombinant plasmid EcYL003. After linearization by PmeI restriction enzyme digestion, it was transformed into the engineered Yeast Rice strain yyYLA002. After verification and screening, the correct transformants were obtained and then transformed into the plasmid pYL-Cre carrying Cre recombinase to remove the URA3 selection marker, thus obtaining the engineered strain yyYLA003.

[0032] 4. Using overlap extension PCR technology, the promoter, target gene, and terminator were spliced ​​into two gene expression cassettes, pTEF1-ZWF1-tXPR2 and pTEF1-GND1-tXPR2. Then, the two gene expression cassettes were assembled with the homologous arms above and below the integration site, the URA3 expression cassette, and the pUC19 plasmid backbone using a seamless cloning method to construct the recombinant plasmid EcYL004. After linearization by PmeI restriction enzyme digestion, it was transformed into the engineered Yersinia lipolytica strain yyYLA003. After verification and screening, the correct transformants were obtained, and then transformed into the strain carrying the UP-LEU2-DN fragment for LEU2 nutrient marker replenishment to obtain the engineered strain yyYLA004.

[0033] The present invention also provides a method for producing astaxanthin from a high-yield astaxanthin-producing *Yersinia lipolyticis* engineered strain according to any one of the preceding claims, comprising the following steps:

[0034] Step a: Initial fermentation;

[0035] Step b: After the initial carbon source is consumed, start adding 700 g / L glucose solution at a rate of 5 mL / hour until fermentation is complete;

[0036] Step c: From the initial fermentation in step a until 120 hours of fermentation, ammonia water is used to adjust the pH of the fermentation system to maintain a pH of 6; at 120 hours, the ammonia water is replaced with 3 M sodium hydroxide, and then fermentation continues for another 40 hours to end the fermentation.

[0037] In step c, sodium hydroxide serves to control nitrogen uptake, slow down cell growth rate, and increase product accumulation rate.

[0038] The ammonia water is a conventional reagent in the prior art. The ammonia water used in the fermentation system adjustment of the present invention is not limited to its concentration. Ammonia water obtained through commercial channels, including but not limited to those in the prior art, is applicable to the present invention.

[0039] Preferably, after fermentation, astaxanthin is extracted from the fermentation broth and / or the astaxanthin content is detected.

[0040] Preferably, in step a, the initial fermentation temperature is 30°C.

[0041] In any of the above-mentioned preferred embodiments, in step a, the stirring speed for the initial fermentation is 400-1200 rpm. More preferably, it is 400, 500, 600, 700, 800, 900, 1000, 1100, 1200 rpm and the range thereof.

[0042] Preferably, in step a, the aeration rate for the initial fermentation is 2 L / min.

[0043] In any of the above, it is preferred that, in step a, the initial fermentation culture system has a pH of 6.

[0044] In any of the above-mentioned preferred embodiments, in step a, the dissolved oxygen content in the initial fermentation system is above 30%.

[0045] In any of the above preferred embodiments, in step a, the initial fermentation culture medium consists of: 30 g / L yeast extract, 20 g / L ammonium sulfate, 10 g / L potassium dihydrogen phosphate, and 10 g / L magnesium sulfate heptahydrate.

[0046] In any of the above preferred embodiments, in step b, the 700 g / L glucose solution contains 0.05 g / L ferrous sulfate heptahydrate, 0.005 g / L copper sulfate pentahydrate, 0.04 g / L calcium chloride dihydrate, 0.005 g / L zinc sulfate, and 0.005 g / L potassium iodide.

[0047] Of the above, the preferred fermentation system is 2L.

[0048] In a preferred embodiment of the present invention, a 2 L fermenter was used for fermentation. The initial culture medium consisted of 30 g / L yeast extract, 20 g / L ammonium sulfate, 10 g / L potassium dihydrogen phosphate, and 10 g / L magnesium sulfate heptahydrate. Fermentation was carried out at 30°C, with a stirring speed of 400-1200 rpm, an aeration rate of 2 L / min, and under the conditions of pH 6 and dissolved oxygen levels above 30%. After the initial carbon source was depleted, 700 g / L glucose (containing 0.05 g / L ferrous sulfate heptahydrate, 0.005 g / L copper sulfate pentahydrate, 0.04 g / L calcium chloride dihydrate, 0.005 g / L zinc sulfate, and 0.005 g / L potassium iodide) was added at a rate of 5 mL / hour. At 120 h, the ammonia used for pH adjustment was replaced with 3 M sodium hydroxide, and fermentation continued for another 40 h, for a total of 160 h, at which point fermentation was completed.

[0049] Preferably, the astaxanthin extraction steps are as follows: Take the fermentation broth, centrifuge to collect the bacterial cells, add an appropriate amount of quartz sand and extraction solvent, shake to extract, centrifuge to collect the supernatant organic phase, and repeat the extraction steps until the bacterial cells turn white. Collect all organic phases and filter using an organic filter membrane.

[0050] Preferably, the extractant contains 50% ethyl acetate and 50% methanol.

[0051] Astaxanthin content was extracted and detected from the fermentation broth. This invention employs a novel fermentation process, reducing the fermentation time to 160 hours and increasing the yield to 3749 mg / L.

[0052] The present invention also provides the application of the high-astaxanthin-producing Yersinia lipolyticis engineered strain described in any of the above claims in the preparation of astaxanthin or astaxanthin-containing foods, health products, cosmetics or feed additives.

[0053] The beneficial effects of this invention are as follows:

[0054] This invention, through genetic engineering of *Yersinia lipolytica*, overexpresses four gene clusters—McCarRP-McCarB, PnCrtW-linker-HpCrtZ, SpHMGR-DGA1, and ZWF1-GND1—into the *Yersinia lipolytica* genome via homologous recombination, thereby enhancing the astaxanthin production capacity of the engineered strain. By integrating the McCarRP and McCarB genes into the *Yersinia lipolytica* genome, a heterologous β-carotene synthesis pathway is introduced. Under the catalysis of phytoene synthase (CarRP), geraniol-geraniol pyrophosphate (GGPP) is converted to phytoene, which is then catalyzed by phytoene dehydrogenase (CarB) through four dehydrogenation processes to generate lycopene. Lycopene then undergoes cyclization to generate β-carotene. By integrating the PnCrtW-linker-HpCrtZ gene into the *Yersinia lipolytica* genome, a heterologous astaxanthin synthesis pathway is introduced. β-Carotene hydroxylase (CrtZ) hydroxylates β-carotene at the C-3 and C-3' positions to generate zeaxanthin, while β-carotene ketolase (CrtW) introduces ketone groups at the C-4 and C-4' positions of zeaxanthin, ultimately synthesizing astaxanthin. Furthermore, a flexible linker (amino acid sequence shown in SEQ ID NO. 89: GGGGSGGGGS) was constructed between CrtW and CrtZ enzymes. The two proteins were expressed as a translational fusion, separated by a linker peptide spacer, maintaining close proximity between the two enzymes and further enhancing the engineered strain's ability to synthesize astaxanthin. By integrating the 3-hydroxy-3-methylglutaryl-CoA reductase gene SpHMGR1 into the genome of *Yarrowia lipolytica*, the key rate-limiting step of the mevalonate pathway was enhanced, effectively increasing the synthesis efficiency and intracellular supply level of upstream isoprene precursors, providing a sufficient substrate basis for downstream carotenoid synthesis, while alleviating specific dependence on NADPH as a reducing cofactor. Since carotenoids are hydrophobic compounds and are mainly stored in cell membranes and lipid organelles, integrating and expressing the DGA1 gene into the *Yarrowia lipolytica* genome can regulate intracellular lipid levels, promoting carotenoid accumulation and further increasing the yield of the substrate β-carotene and the product astaxanthin. Integrating and expressing the ZWF1 and GND1 genes into the *Yarrowia lipolytica* genome can enhance the flux of the pentose phosphate pathway, increasing NADPH regeneration capacity and thus improving astaxanthin production.

[0055] The high-yield astaxanthin engineered yeast strain provided by this invention can achieve a fermentation tank yield of approximately 3749.02 mg / L within 160 hours, which is fully capable of commercial production and has good prospects for industrial application. Attached Figure Description

[0056] Figure 1 This is a schematic diagram of the structure of the recombinant plasmid EcYL001-pUC19-D1-URA3-McCarRP-McCarB in the preferred embodiment of the present invention.

[0057] Figure 2 This is a schematic diagram of the structure of the recombinant plasmid EcYL002-pUC19-E14-URA3-PnCrtW-HpCrtZ provided in preferred embodiment 2 of the present invention.

[0058] Figure 3 This is a schematic diagram of the structure of the recombinant plasmid EcYL003-pUC19-A2-URA3-SpHMGR-DGA1 provided in the preferred embodiment 3 of the present invention.

[0059] Figure 4 This is a schematic diagram of the structure of the recombinant plasmid EcYL004-pUC19-C2-URA3-ZWF1-GND1 provided in the preferred embodiment 4 of the present invention.

[0060] Figure 5 The fermentation results of the engineered strain yyYLA004 in a 2L fermenter system are shown in the preferred embodiment of the present invention, 6. Detailed Implementation

[0061] The technical solution of the present invention will be clearly and completely described below with reference to the accompanying drawings and specific embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0062] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the instruments, reagents, and materials involved in the following embodiments are all conventional instruments and reagents already available in the prior art and can be obtained through legitimate commercial channels. The gene synthesis, primer synthesis, and sequencing described in this invention were all performed by Suzhou Genewiz Biotechnology Co., Ltd.

[0063] Tables 1 to 6 below show the primers used in the preferred embodiments of the present invention.

[0064] Table 1 Primer List

[0065] Table 2 Primer List (continued)

[0066] Table 3 Primer List (continued)

[0067] Table 4 Primer List (continued)

[0068] Table 5 Primer List (continued)

[0069] Table 6 Primer List (continued)

[0070] Table 7 below shows the source, NCBI accession number, and sequence number of each gene used in the preferred embodiments of the present invention. The D1 site is selected from the DPP1 gene; the E14, A2, and C2 sites are selected from blank sequences that do not encode specific genes and are marked as intergenetic sequences in Table 7. The nucleotide sequence shown in SEQ ID NO. 90 is a knockout sequence from the D1 site sequence shown in SEQ ID NO. 9, and the flanking sequences of the nucleotide sequence shown in SEQ ID NO. 90 are homologous arms. The nucleotide sequence shown in SEQ ID NO. 91 is a knockout sequence from the E14 gene sequence shown in SEQ ID NO. 10, and the flanking sequences of the nucleotide sequence shown in SEQ ID NO. 91 are homologous arms. The nucleotide sequence shown in SEQ ID NO. 92 is a knockout sequence from the A2 gene sequence shown in SEQ ID NO. 11, and the flanking sequences of the nucleotide sequence shown in SEQ ID NO. 92 are homologous arms. The nucleotide sequence shown in SEQ ID NO.93 is a knockout sequence from the C2 gene sequence shown in SEQ ID NO.12, and the flanking sequences of the nucleotide sequence shown in SEQ ID NO.93 are homologous arms.

[0071] Table 7. Gene Origin and Description

[0072]

[0073] In Table 7, the five heterologous gene sequences McCarRP, McCarB, PnCrtW, HpCrtZ, and SpHMGR were codon-optimized using *Yersinia lipolytica* as the host and synthesized by Suzhou Genewiz Biotechnology Co., Ltd., yielding plasmids pUC57-SpHMGR, pUC57-McCarRP, pUC57-McCarB, pUC57-PnCrtW, pUC57-HpCrtZ, and pUC57-GND1. Since the endogenous gene GND1 contains introns, this invention designed an intron-free GND1 sequence, which was synthesized by Suzhou Genewiz Biotechnology Co., Ltd., yielding the pUC57-GND1 plasmid. The DGA1 and ZWF1 genes were obtained by PCR amplification using the *Yersinia lipolytica* Po1f genome as a template.

[0074] The CarRP gene encodes a bifunctional enzyme, phytopenic lycopene synthase / lycopene β-cyclase, while the CarB gene encodes phytopenic lycopene dehydrogenase. Integrating the CarRP and CarB genes into the Yersinia lipolytica genome can enhance the ability of engineered strains to synthesize β-carotene.

[0075] The CrtW gene encodes β-carotene ketolase, and the CrtZ gene encodes β-carotene hydroxylase. Integrating the CrtW and CrtZ genes into the *Yarrowia lipolytica* genome enhances the engineered strain's ability to synthesize astaxanthin. A flexible linker (GGGGSGGGGS) was constructed between the CrtW and CrtZ enzymes, and the two proteins are expressed as a translational fusion, separated by a linker peptide spacer. This maintains close proximity between the two enzymes while allowing for their interaction, further improving the engineered strain's ability to synthesize astaxanthin.

[0076] The SpHMGR gene encodes 3-hydroxy-3-methylglutaryl-CoA reductase, and the DGA1 gene encodes diacylglycerol acyltransferase. Integrating the NADH-dependent HMGR (SpHMGR) derived from *Silicibacter pomeroyi* into the *Yarrowia lipolytica* genome directs metabolic flux to the mevalonate pathway while alleviating specific dependence on NADPH as a reducing cofactor. Overexpression of the diacylglycerol acyltransferase (DGA1) in the liposynthesis pathway further increases β-carotene production.

[0077] The ZWF1 gene encodes glucose-6-phosphate dehydrogenase, and the GND1 gene encodes 6-phosphate gluconate dehydrogenase. Integration of the ZWF1 and GND1 genes into the *Yarrowia lipolytica* genome can enhance the flux of the pentose phosphate pathway, thereby increasing NADPH regeneration capacity and ultimately improving astaxanthin production.

[0078] Example 1

[0079] In this embodiment, wild-type Yersinia lipolyticis Po1f was used as the starting strain to construct the engineered strain yyYLA001. The construction method is as follows:

[0080] 1. Using pUC19 plasmid as a template, the pUC19 backbone sequence was amplified using primers PUC19-F and PUC19-R; using the *Yersinia lipolytica* Po1f genome as a template, the upper and lower homologous arm sequences of D1-UP and D1-DN were amplified using primers D1-UP-F and D1-UP-R, and D1-DN-F and D1-DN-R; using pYLXP'-URA3 synthetic plasmid as a template, the URA-D1 sequence was amplified using primers URA-D1-F and URA-D1-R; using the *Yersinia lipolytica* Po1f genome as a template, the pTEF1 sequence was amplified using primers TEF1-McCarRP-F and TEF1-McCarRP-R; using pUC57-McCarRP plasmid as a template, the *McCarRP* sequence was amplified using primers McCar... The McCarRP gene sequence was obtained by amplification using RP-F and McCarRP-R primers; the tXPR2 sequence was obtained by amplification using the *Yersinia lipolytica* Po1f genome as a template with primers XPR2-McCarRP-F and XPR2-McCarRP-R primers; the pGPD1 sequence was obtained by amplification using the *Yersinia lipolytica* Po1f genome as a template with primers GPD1-McCarB-F and GPD1-McCarB-R primers; the McCarB sequence was obtained by amplification using the pUC57-McCarB template with primers McCarB-F and McCarB-R primers; and the tLIP1 sequence was obtained by amplification using the *Yersinia lipolytica* Po1f genome as a template with primers LIP1-McCarB-F and LIP1-McCarB-R primers.

[0081] 2. The promoter, target gene, and terminator were spliced ​​into two gene expression cassettes, pTEF1-McCarRP-tXPR2 and pGPD1-McCarB-tLIP1, by overlap extension PCR. Using a seamless ligation kit (Novizan ClonExpress UltraOne Step Cloning Kit V3), the pTEF1-McCarRP-tXPR2 and pGPD1-McCarB-tLIP1 gene expression cassettes, along with the upper and lower homologous arms of the integration site D1 (i.e., D1-UP and D1-DN homologous arms), the URA3 expression cassette (i.e., URA-D1), and the pUC19 plasmid backbone, were assembled according to the manufacturer's instructions at 50℃ for 30 min. The assembled products were transformed into *E. coli* NEB-10β. PCR verification and sequencing confirmed correctness, yielding the recombinant plasmid EcYL001 (i.e., pC1-D1). Figure 1The recombinant plasmid shown is EcYL001-pUC19-D1-URA3-McCarRP-McCarB (hereinafter referred to as EcYL001 in this invention).

[0082] 3. Next, the PmeI-digested and linearized EcYL001 was transformed into wild-type Yersinia lipolytica Po1f. The transformation procedure was performed according to the Frozen EZ Yeast Transformation II kit. TM (Purchased from Zymo Research) The product was plated on Sc-URA auxotrophic plates and incubated at 30°C. The correct transformants were obtained by PCR screening. The transformants were then transformed into plasmid pYL-Cre carrying the Cre recombinase expression cassette. The URA3 selection marker was successfully recovered by PCR and plate printing to obtain the engineered strain yyYLA001.

[0083] The pYLXP'-URA3 synthetic plasmid uses pYLXP' as its backbone, inserts the selection marker URA3 between pTEF1in and tXPR2, and adds a pair of unidirectional loxP sites upstream of pTEF1in and downstream of tXPR2. The modified loxP-pTEF1in-URA3-tXPR2-loxP expression cassette is located between the AvrII and NheI restriction sites on the pYLXP' backbone and is used for screening positive single clones.

[0084] The pYLXP'-URA3 synthetic plasmid was designed and synthesized by Suzhou Genewiz Biotechnology Co., Ltd. The nucleotide sequence of the loxP-pTEF1in-URA3-tXPR2-loxP expression cassette is shown in SEQ ID NO.94, and the fragment shown in SEQ ID NO.94 contains partial sequences of the backbone plasmid pYLXP' on both sides.

[0085] Example 2

[0086] In this embodiment, the engineered strain yyYLA001 of *Yarrowia lipolyticis* was used as the starting strain to construct the engineered strain yyYLA002. The construction method is as follows:

[0087] 1. Using pUC19 plasmid as a template, the pUC19 backbone sequence was amplified using primers PUC19-F and PUC19-R; using the *Yersinia lipolytica* Po1f genome as a template, the E14-UP and E14-DN homologous arm sequences were amplified using primers E14-UP-F and E14-UP-R, E14-DN-F and E14-DN-R; using pYLXP'-URA3 synthetic plasmid as a template, the URA-E14 sequence was amplified using primers URA-E14-F and URA-E14-R; using the *Yersinia lipolytica* Po1f genome as a template, the *Yersinia lipolytica* Po1f genome was amplified using primer TEF1i. The pTEF1in sequence was obtained by amplification using n-PnCrtW-F and TEF1in-PnCrtW-R; the PnCrtW gene sequence was obtained by amplification using pUC57-PncrtW plasmid as template and primers PncrtW-F and PncrtW-R; the HpCrtZ gene sequence was obtained by amplification using pUC57-HpCrtZ plasmid as template and primers HpCrtZ-F and HpCrtZ-R; and the tXPR2 sequence was obtained by amplification using Yersinia lipolytica Po1f genome as template and primers XPR2-HpCrtZ-F and XPR2-HpCrtZ-R.

[0088] 2. The promoter, target gene, and terminator were spliced ​​into the pTEF1in-PnCrtW-HpCrtZ-tXPR2 gene expression cassette using overlap extension PCR. Using a seamless ligation kit (Novizan ClonExpress Ultra One Step Cloning Kit V3), the pTEF1in-PnCrtW-HpCrtZ-tXPR2 gene expression cassette, along with the homologous arms of the integration site E14 (E14-UP and E14-DN), the URA3 expression cassette (URA-E14), and the pUC19 plasmid backbone, were assembled according to the manufacturer's instructions at 50℃ for 30 min. The assembled product was transformed into *E. coli* NEB-10β. PCR verification and sequencing confirmed correctness, yielding the recombinant plasmid EcYL002 (i.e.,...). Figure 2 The recombinant plasmid shown is EcYL002-pUC19-E14-URA3-PnCrtW-HpCrtZ (hereinafter referred to as EcYL002 in this invention).

[0089] 3. Next, the PmeI-digested and linearized EcYL002 strain was transformed into the engineered bacterium yyYLA001. The transformation procedure was performed according to the Frozen EZ Yeast Transformation II kit. TM(Purchased from Zymo Research) The product was plated on Sc-URA auxotrophic plates and incubated at 30°C. The correct transformants were obtained by PCR screening. The transformants were then transformed into plasmid pYL-Cre carrying the Cre recombinase expression cassette. The URA3 selection marker was successfully recovered by PCR and plate printing to obtain the engineered strain yyYLA002.

[0090] Example 3

[0091] In this embodiment, the engineered strain yyYLA002 of *Yarrowia lipolyticis* was used as the starting strain to construct the engineered strain yyYLA003. The construction method is as follows:

[0092] 1. Using pUC19 plasmid as a template, the pUC19 backbone sequence was amplified using primers PUC19-F and PUC19-R; using the *Yersinia lipolytica* Po1f genome as a template, the A2-UP and A2-DN homologous arm sequences were amplified using primers A2-UP-F and A2-UP-R, A2-DN-F and A2-DN-R; using pYLXP'-URA3 synthetic plasmid as a template, the URA-A2 sequence was amplified using primers URA-A2-F and URA-A2-R; using the *Yersinia lipolytica* Po1f genome as a template, the pTEF1 sequence was amplified using primers TEF1-SpHMGR-F and TEF1-SpHMGR-R; using pUC57-SpHMGR plasmid as a template, the sequence was amplified using primers TEF1-SpHMGR-F and TEF1-SpHMGR-R. The SpHMGR gene sequence was obtained by amplification using primers SpHMGR-F and SpHMGR-R; the tXPR2 sequence was obtained by amplification using primers XPR2-SpHMGR-F and XPR2-SpHMGR-R using the Yersinia lipolytica Po1f genome as a template; the pGPD1 sequence was obtained by amplification using primers GPD1-DGA1-F and GPD1-DGA1-R using the Yersinia lipolytica Po1f genome as a template; the DGA1 gene sequence was obtained by amplification using primers DGA1-F and DGA1-R using the Yersinia lipolytica Po1f genome as a template; and the tLIP1 sequence was obtained by amplification using primers LIP1-DGA1-F and LIP1-DGA1-R using the Yersinia lipolytica Po1f genome as a template.

[0093] 2. The promoter, target gene, and terminator were spliced ​​into two gene expression cassettes, pTEF1-SpHMGR-tXPR2 and pGPD1-DGA1-tLIP1, by overlap extension PCR. Using a seamless ligation kit (Novizan ClonExpress Ultra OneStep Cloning Kit V3), the pTEF1-SpHMGR-tXPR2 and pGPD1-DGA1-tLIP1 gene expression cassettes, along with the upper and lower homologous arms of the integration site A2 (i.e., A2-UP and A2-DN homologous arms), the URA3 expression cassette (i.e., URA-A2), and the pUC19 plasmid backbone, were assembled according to the manufacturer's instructions at 50℃ for 30 min. The assembled products were transformed into *E. coli* NEB-10β. PCR verification and sequencing confirmed correctness, yielding the recombinant plasmid EcYL003 (i.e., pTEF1-SpHMGR-tXPR2 and pGPD1-DGA1-tLIP1). Figure 3 The recombinant plasmid shown is EcYL003-pUC19-A2-URA3-SpHMGR-DGA1, which is referred to as EcYL003 in this invention.

[0094] 3. Next, the PmeI-digested and linearized EcYL003 strain was transformed into the engineered bacterium yyYLA002. The transformation procedure was performed according to the Frozen EZ Yeast Transformation II kit. TM (Purchased from Zymo Research) The product was plated on Sc-URA auxotrophic plates and incubated at 30°C. The correct transformants were obtained by PCR screening. The transformants were then transformed into plasmid pYL-Cre carrying the Cre recombinase expression cassette. The URA3 selection marker was successfully recovered by PCR and plate printing to obtain the engineered strain yyYLA003.

[0095] Example 4

[0096] In this embodiment, the engineered strain yyYLA003 of *Yarrowia lipolyticis* was used as the starting strain to construct the engineered strain yyYLA004. The construction method is as follows:

[0097] Using pUC19 plasmid as a template, the pUC19 backbone sequence was amplified using primers PUC19-F and PUC19-R; using the *Yersinia lipolytica* Po1f genome as a template, the upper and lower homologous arm sequences of C2-UP and C2-DN were amplified using primers C2-UP-F and C2-UP-R, and C2-DN-F and C2-DN-R; using the pYLXP'-URA3 synthetic plasmid as a template, the URA-C2 sequence was amplified using primers URA-C2-F and URA-C2-R; using the *Yersinia lipolytica* Po1f genome as a template, the pTEF1 sequence was amplified using primers TEF1-ZWF1-F and TEF1-ZWF1-R; and using pUC57-ZWF1 plasmid as a template... The ZWF1 gene sequence was obtained by amplification using primers ZWF1-F and ZWF1-R; the tXPR2 sequence was obtained by amplification using primers XPR2-ZWF1-F and XPR2-ZWF1-R using the Yersinia lipolytica Po1f genome as a template; the pTEF1 sequence was obtained by amplification using primers TEF1-GND1-F and TEF1-GND1-R using the Yersinia lipolytica Po1f genome as a template; the GND1 gene sequence was obtained by amplification using primers GND1-F and GND1-R using the pUC57-GND1 plasmid as a template; and the tXPR2 sequence was obtained by amplification using primers XPR2-GND1-F and XPR2-GND1-R using the Yersinia lipolytica Po1f genome as a template.

[0098] 2. The promoter, target gene, and terminator were spliced ​​into two gene expression cassettes, TEF1-ZWF1-tXPR2 and TEF1-GND1-tXPR2, by overlap extension PCR. Using a seamless ligation kit (Novizan ClonExpress Ultra One Step Cloning Kit V3), the two gene expression cassettes pTEF1-ZWF1-tXPR2 and pTEF1-GND1-tXPR2, along with the homologous arms of the integration site C2 (i.e., C2-UP and C2-DN homologous arms), the URA3 expression cassette (i.e., URA-C2), and the pUC19 plasmid backbone, were assembled according to the manufacturer's instructions at 50℃ for 30 min. The assembled products were transformed into *E. coli* NEB-10β. PCR verification and sequencing confirmed correctness, yielding the recombinant plasmid EcYL004 (i.e.,...). Figure 4 The recombinant plasmid shown is EcYL004-pUC19-C2-URA3-ZWF1-GND1, which is referred to as EcYL004 in this invention.

[0099] 3. Next, the PmeI-digested and linearized EcYL004 was transformed into the engineered bacterium yyYLA003. The transformation procedure was performed according to the Frozen EZ Yeast Transformation II kit. TMFollowing the instructions (purchased from Zymo Research), the transformants were plated on Sc-URA auxotrophic plates and incubated at 30°C. Correct transformants were obtained through PCR verification and screening. Subsequently, the UP-LEU2-DN fragment was introduced for LEU2 nutrient labeling recovery. Successfully transformed strains were screened using Sc-LEU medium, and the correctness of the strain was verified by PCR, yielding the engineered strain yyYLA004.

[0100] The UP-LEU2-DN fragment was obtained as follows: using the *Yersinia lipolytica* Po1f genome as a template, the LEU2-UP sequence was amplified using primers LEU2-UP-F and LEU2-UP-R; using the pYLXP'-URA3 plasmid as a template, the LEU2 gene sequence was amplified using primers LEU2-F and LEU2-R; using the *Yersinia lipolytica* Po1f genome as a template, the LEU2-DN sequence was amplified using primers LEU2-DN-F and LEU2-DN-R; and LEU2-UP, LEU2, and LEU2-DN were spliced ​​into the UP-LEU2-DN sequence by overlap extension PCR.

[0101] Example 5

[0102] This embodiment verifies the astaxanthin production of the engineered bacterium yyYLA004, which can enhance astaxanthin secretion, through fermentation tank cultivation. The cultivation method is as follows:

[0103] 1) Cultivation of primary seed culture: Take strain yyYL004 preserved in a glycerol tube at -80℃, streak it on a solid YPD plate, and incubate it at 30℃ for 48 hours. Inoculate a single colony of strain yyYL004 on the solid YPD plate into a test tube containing 5 mL of YPD liquid medium, and incubate at 30℃ and 200 rpm for 24 hours.

[0104] 2) Cultivation of secondary seed culture: Transfer the primary seed culture at a 5% inoculum to a 500 mL Erlenmeyer flask containing 100 mL of YPD liquid medium, and incubate at 30°C and 200 rpm for 8 hours. 600 Achieving a score of 10 yields a secondary seed solution;

[0105] The YPD plates and YPD liquid culture medium were prepared according to conventional methods of existing technology or purchased through commercial channels.

[0106] 3) Transfer the secondary seed culture at an inoculation rate of 10% to a 2 L fermenter containing 1 L of fermentation medium for fermentation.

[0107] More preferably, the detailed process of step 3) is as follows: the secondary seed culture is transferred to a 2 L fermenter containing 1 L of fermentation medium at an inoculation rate of 10% for fermentation. The initial medium composition is 30 g / L yeast extract, 20 g / L ammonium sulfate, 10 g / L potassium dihydrogen phosphate, and 10 g / L magnesium sulfate heptahydrate. The fermentation is carried out at a temperature of 30℃, a stirring speed of 400-1200 rpm, an aeration rate of 2 L / min, a pH of 6, and dissolved oxygen of 30% or higher. After the initial carbon source is exhausted, 700 g / L glucose is added at a rate of 5 mL / hour until fermentation ends after 200 h.

[0108] Example 6

[0109] This embodiment provides a method for the extraction and determination of astaxanthin after fermentation, as detailed below:

[0110] Astaxanthin extraction: Take 200 μL of fermentation broth, centrifuge to collect the bacterial cells, add appropriate amount of quartz sand and extraction solvent (containing 50% ethyl acetate and 50% methanol), shake to extract, centrifuge to collect the supernatant organic phase, repeat the extraction step until the bacterial cells turn white. Collect all organic phases, filter with an organic filter membrane, and determine the astaxanthin yield by high performance liquid chromatography.

[0111] Astaxanthin determination: The measurement parameters are set, with the wavelength set to 475 nm (astaxanthin).

[0112] The chromatographic column was a C18 column (Φ4.6x150 mm, 5 μm). Mobile phase A consisted of acetonitrile and water in a ratio of 9:1; mobile phase B consisted of methanol and isopropanol in a ratio of 3:2. The column temperature was 25℃. Chromatographic conditions were as follows: initial conditions: 0% B phase; 0-15 min; 0-90% B phase; 15-30 min: 90% B phase; 30-35 min: 90-0% B phase; 35-40 min: 0% B phase.

[0113] like Figure 5 The image shows the fermentation results of the engineered strain yyYLA004 in a 2L fermenter system.

[0114] The final astaxanthin yield at the fermentation endpoint was measured to be 3749.02 mg / L.

[0115] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

[0116] The above embodiments are merely preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A high-astaxanthin-producing engineered strain of *Yersinia lipolytica*, using wild-type *Yersinia lipolytica* as the starting strain, characterized in that... The starting strain integrates key enzyme genes in the astaxanthin synthesis pathway, which include: McCarRP, a bifunctional enzyme of phytoene synthase / lycopene β-cyclase from *Mucor*; McCarB, a phytoene dehydrogenase from *Mucor*; PnCrtW, a β-carotene ketolase from *Paracococcus*; HpCrtZ, a β-carotene hydroxylase from *Haematococcus pluvialis*; SpHMGR, a 3-hydroxy-3-methylglutaryl-CoA reductase from *Roseobacterium*; DGA1, a diacylglycerol acyltransferase from *Yersinia lipolytica*; ZWF1, a glucose-6-phosphate dehydrogenase from *Yersinia lipolytica*; and GND1, a 6-phosphate gluconate dehydrogenase from *Yersinia lipolytica*.

2. The engineered strain of *Yersinia lipophila* as described in claim 1, characterized in that, The key enzyme genes in the astaxanthin synthesis pathway are constructed as gene expression cassettes, which include at least one of the following: SpHMGR-DGA1 gene expression cassette, McCarRP-McCarB gene expression cassette, PnCrtW-linker-HpCrtZ gene expression cassette, or ZWF1-GND1 gene expression cassette.

3. The engineered strain of *Yersinia lipophila* as described in claim 1, characterized in that, The key enzyme gene in the astaxanthin synthesis pathway is driven by a strong constitutive promoter of Yersinia lipolytica, wherein the strong constitutive promoter of Yersinia lipolytica is at least one of pTEF1, pTEF1in, or pGPD1.

4. The engineered strain of *Yersinia lipophila* as described in claim 1, characterized in that, The starting strain was wild-type Yersinia lipophila Po1f.

5. The engineered strain of *Yersinia lipophila* as described in claim 1, characterized in that, The starting strain integrates one or more of the following nucleic acid sequences a to h: a. The nucleic acid sequence of the McCarRP gene is shown in SEQ ID NO.1; b. The nucleic acid sequence of the McCarB gene is shown in SEQ ID NO.2; c. The nucleic acid sequence of the PnCrtW gene is shown in SEQ ID NO.3; d. The nucleic acid sequence of the HpCrtZ gene is shown in SEQ ID NO.4; e. The nucleic acid sequence of the spHMGR gene is shown in SEQ ID NO.5; f. The nucleic acid sequence of the DGA1 gene is shown in SEQ ID NO.6; g. The nucleic acid sequence of the ZWF1 gene is shown in SEQ ID NO.7; h. The nucleic acid sequence of the GND1 gene is shown in SEQ ID NO.

8.

6. The method for preparing a high-astaxanthin-producing *Yersinia lipophila* engineered strain according to any one of claims 1 to 5, characterized in that, Includes the following steps: Step 1: Construct a recombinant plasmid EcYL001 containing expression cassettes of McCarRP and McCarB genes and an expression cassette of the URA3 selection marker gene. Transform it into wild-type Yersinia lipophila. After verification screening, the correct transformants are obtained. Then, the URA3 selection marker is removed to obtain the engineered strain yyYLA001. Step 2: Construct recombinant plasmid EcYL002 containing expression cassettes of PnCrtW and HpCrtZ genes and expression cassette of URA3 selection marker gene, transform it into the engineered yeast strain yyYLA001, obtain the correct transformant through verification screening, and then remove the URA3 selection marker to obtain engineered strain yyYLA002. Step 3: Construct recombinant plasmid EcYL003 containing expression cassettes of the SpHMGR gene, DGA1 gene, and URA3 selection marker gene, and transform it into the Yeast lipolyticis engineered strain yyYLA002. After verification screening, the correct transformants were obtained, and then the URA3 selection marker was removed to obtain engineered strain yyYLA003. Step 4: Construct a recombinant plasmid EcYL004 containing the ZWF1 gene expression cassette, the GND1 gene expression cassette, and the URA3 selection marker gene. Transform it into the Yersinia lipolytica engineered strain yyYLA003. After verification and screening, the correct transformant is obtained. Then, the transformant is transformed with the UP-LEU2-DN fragment to perform LEU2 nutrient marker replenishment, and the engineered strain yyYLA004 is obtained. The engineered strain yyYLA004 is the Yersinia lipolytica engineered strain that produces high astaxanthin.

7. The method for preparing astaxanthin from the high-yield astaxanthin-producing *Yersinia lipolyticis* engineered strain according to any one of claims 1 to 5, characterized in that, Includes the following steps: Step a: Initial fermentation; Step b: After the initial carbon source is consumed, start adding 700 g / L glucose solution at a rate of 5 mL / hour until fermentation is complete; Step c: From the initial fermentation in step a to 120 hours of fermentation, ammonia water was used to adjust the pH of the fermentation system to maintain pH=6; at 120 hours of fermentation, the ammonia water was replaced with 3 M sodium hydroxide, and then fermentation continued for another 40 hours. After fermentation, astaxanthin was extracted from the fermentation broth.

8. The method for preparing astaxanthin according to claim 7, characterized in that, In step a, the initial fermentation temperature is 30℃; the initial fermentation stirring speed is 400-1200 rpm; the initial fermentation aeration rate is 2 L / min; the initial fermentation culture system pH is 6; and the dissolved oxygen in the initial fermentation system is above 30%.

9. The method for preparing astaxanthin according to claim 7, characterized in that, In step a, the initial fermentation medium contains 30 g / L yeast extract, 20 g / L ammonium sulfate, 10 g / L potassium dihydrogen phosphate, and 10 g / L magnesium sulfate heptahydrate.

10. The use of the high-astaxanthin-producing Yersinia lipolyticis engineered strain according to any one of claims 1 to 5 in the preparation of astaxanthin or astaxanthin-containing foods, health products, cosmetics or feed additives.

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