Yarrowia lipolytica engineering bacterium for producing esterified astaxanthin as well as construction method and application thereof

Abstract

The invention discloses yarrowia lipolytica engineering bacteria for producing esterified astaxanthin as well as a construction method and application of the yarrowia lipolytica engineering bacteria. According to the invention, acyltransferase ScDGAT2-1 from fission vibrio is integrated in a genome of a yarrowia lipolytica engineering strain DN20, diacylglycerol acyltransferase DGA1 and phosphatidic acid phosphatase PAH1 are knocked out, and the corn oil lipoprotein Oleosin and the acyltransferase ScDGAT2-1 from fission vibrio are subjected to fusion expression, so that the yarrowia lipolytica engineering strain DN20 is obtained. According to the present invention, the Yarrowia lipolytica engineering bacterium capable of producing esterified astaxanthin at a high yield is constructed by using SBT06-005 as a raw material and integrating beta-carotene hydroxylase CrtZ, endogenous truncated 3-hydroxy3-methylglutaryl coenzyme A reductase tHMG1, phytoene synthase CarRP and beta-isopropyl malate dehydrogenase LEU2, and the SBT06-005 can produce 166.6 mg / L esterified astaxanthin and 1097.0 mg / L total astaxanthin in a 5L fermentation tank.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering bacteria technology, and relates to a Yersinia lipophila engineered strain that produces esterified astaxanthin, its construction method, and its application. Background Technology

[0002] Astaxanthin (3,3'-dihydroxy-4,4'-diketo-β,β'-carotene) is an orange-red carotenoid widely found in animals, plants, and microorganisms. Astaxanthin possesses strong antioxidant properties and is considered one of the strongest known antioxidants in nature, thus finding widespread use in skincare and health supplements.

[0003] Although astaxanthin has broad market prospects and application value, its instability due to photosensitivity and thermosensitivity, as well as its low solubility and bioavailability, increase the cost of its production and transportation, limiting its further promotion and application. Esterification modification of astaxanthin can effectively improve its photothermal stability, solubility, and bioavailability, making it more beneficial for the production and application of related products.

[0004] Esterified astaxanthin can be obtained through three methods: chemical synthesis, natural biosynthesis, and heterologous biosynthesis. Chemically synthesized esterified astaxanthin is inexpensive, but its potential for use in humans is limited by the presence of toxic intermediates in the reaction process. Natural biosynthesis primarily utilizes Haematococcus pluvialis, but its large-scale cultivation is mainly outdoors and dependent on stable light and temperature, making it subject to significant site limitations. Heterologous biosynthesis utilizes high-density fermentation by microorganisms; bioreactor technology is mature, not limited by plant site, and has lower costs, making it one of the ideal methods for producing esterified astaxanthin.

[0005] Yarrowia lipolytica is an oil-producing yeast with high acetyl-CoA production, providing a sufficient precursor for esterified astaxanthin. Furthermore, Yarrowia lipolytica is a recognized safe strain and is therefore widely used in carotenoid synthesis. However, while it has been demonstrated that Yarrowia lipolytica can synthesize esterified astaxanthin, there are no reports on metabolic engineering modifications or specific yields of esterified astaxanthin. Therefore, it is necessary to develop Yarrowia lipolytica strains specifically for the synthesis of esterified astaxanthin. Summary of the Invention

[0006] This invention provides an engineered strain of Yersinia lipophila that produces esterified astaxanthin, its construction method, and its application.

[0007] The *Yersinia lipolytica* engineered strain producing esterified astaxanthin described in this invention is an engineered *Yersinia lipolytica* strain obtained by any of the following genetic modification methods, starting from strain DN20:

[0008] (1) Integrate the acyltransferase ScDGAT2-1 from Schizochytrium sp. and knock out the diacylglycerol acyltransferase DGA1;

[0009] (2) Integrate the acyltransferase ScDGAT2-1 from Schizobacterium and knock out the diacylglycerol acyltransferase DGA1, and fuse and express the corn oil protein Oleosin and the acyltransferase ScDGAT2-1 from Schizobacterium.

[0010] (3) Integrate the acyltransferase ScDGAT2-1 from Schizobacterium and knock out the diacylglycerol acyltransferase DGA1 and phosphatidyl phosphatase PAH1, and fuse and express the corn oil protein Oleosin and the acyltransferase ScDGAT2-1 from Schizobacterium.

[0011] (4) Integrate the acyltransferase ScDGAT2-1 from Schizobacterium and knock out diacylglycerol acyltransferase DGA1 and phosphatidylphosphatase PAH1, fuse and express corn oil protein Oleosin and acyltransferase ScDGAT2-1 from Schizobacterium, and integrate β-carotene hydroxylase CrtZ, endogenous truncated 3-hydroxy-3-methylglutaryl-CoA reductase tHMG1, phytopenic oleoresin synthase CarRP and β-isopropylmalate dehydrogenase LEU2.

[0012] The nucleotide sequence of the acyltransferase ScDGAT2-1 derived from *Schizophyllum commune* described in this invention is shown in SEQ ID No. 4; the nucleotide sequence of the fusion gene of acyltransferase ScDGAT2-1 and corn oil protein Oleosin derived from *Schizophyllum commune* is shown in SEQ ID No. 22; the nucleotide sequence of the β-carotene hydroxylase CrtZ gene is shown in SEQ ID No. 38; the nucleotide sequence of the phytoene synthase CarRP gene is shown in SEQ ID No. 39; the nucleotide sequence of the endogenous truncated 3-hydroxy-3-methylglutaryl-CoA reductase tHMG1 gene is shown in SEQ ID No. 40; and the nucleotide sequence of the β-isopropylmalate dehydrogenase LEU2 gene is shown in SEQ ID No. 45.

[0013] The method for constructing the above-mentioned engineered *Yarrowia lipolytica* strain that produces esterified astaxanthin includes the following steps:

[0014] The coding gene for the acyltransferase ScDGAT2-1 from *Schizophyllum commune* was integrated into the genome of the starting strain *Yersinia lipophila* engineered strain DN20 to obtain *Yersinia lipophila* engineered strain SBT06-001. Based on *Yersinia lipophila* engineered strain SBT06-001, the coding gene for the diacylglycerol acyltransferase DGA1 was knocked out to obtain *Yersinia lipophila* engineered strain SBT06-002.

[0015] Alternatively, based on the engineered Yersinia lipophila SBT06-002, the fusion gene of acyltransferase ScDGAT2-1 from Schizobacterium and corn oil protein Oleosin was integrated into the genome of the engineered Yersinia lipophila SBT06-002 to obtain engineered Yersinia lipophila SBT06-003.

[0016] Alternatively, based on the engineered Yersinia lipolytica strain SBT06-003, the gene encoding phosphatidylcholine phosphatase PAH1 can be knocked out to obtain engineered Yersinia lipolytica strain SBT06-004.

[0017] Alternatively, based on the engineered *Yersinia lipolytica* strain SBT06-004, the coding genes for β-carotene hydroxylase CrtZ, endogenous truncated 3-hydroxy-3-methylglutaryl-CoA reductase tHMG1, phytoene synthase CarRP, and β-isopropylmalate dehydrogenase LEU2 were integrated into the genome of the engineered *Yersinia lipolytica* strain SBT06-004 to obtain engineered *Yersinia lipolytica* strain SBT06-005.

[0018] The application of the above-mentioned engineered Yersinia lipolytica strain that produces esterified astaxanthin in the production of esterified astaxanthin.

[0019] Furthermore, the specific method for the above application is as follows: the above-mentioned engineered Yersinia lipolyticis is inoculated into YPD medium for fermentation to produce esterified astaxanthin.

[0020] The YPD medium described in this invention is the YPD medium commonly used in the culture of Yersinia lipolytica, and the medium composition is: 2% glucose, 2% peptone and 2% yeast extract.

[0021] This invention integrates the acyltransferase ScDGAT2-1 from Vibrio lipase into the genome of the engineered Yersinia lipolytica strain DN20, which produces free astaxanthin, to obtain the engineered strain SBT06-001. Then, the diacylglycerol acyltransferase DGA1 and phosphatidylphosphatase PAH1 are knocked out in SBT06-001, and ScDGAT2-1 is localized into liposomes by fusion expression of corn oil protein Oleosin to obtain the engineered strain SBT06-004, which has the highest reported proportion of esterified astaxanthin in microbial systems to date. By integrating β-carotene hydroxylase CrtZ, endogenous truncated 3-hydroxy-3-methylglutaryl-CoA reductase tHMG1, phytopenic oleoresin synthase CarRP, and β-isopropylmalate dehydrogenase LEU2 into SBT06-004, the engineered strain SBT06-005 was obtained. This strain can produce 166.6 mg / L of esterified astaxanthin and 1097.0 mg / L of total astaxanthin in a 5L fermenter, and has broad application prospects in the large-scale bio-fermentation production of esterified astaxanthin. Attached Figure Description

[0022] Figure 1 The astaxanthin peaks were detected by HPLC in the fermentation extracts of engineered strains SBT06-000 and SBT06-001.

[0023] Figure 2 Astaxanthin yield of engineered strains SBT06-000 and SBT06-001.

[0024] Figure 3 Astaxanthin yield of engineered strains SBT06-002 and SBT06-003.

[0025] Figure 4 Astaxanthin yield of engineered strains SBT06-004 and SBT06-005.

[0026] Figure 5 Astaxanthin yield of engineered strain SBT06-005 in a 5L fermenter. Detailed Implementation

[0027] The technical solution of the present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. It should be understood that these embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the following embodiments, they are performed in accordance with the techniques or conditions described in the literature in the field or in accordance with the product manual.

[0028] The starting strain, Yersinia lipophila strain DN20, described in this invention was constructed with reference to Chinese patent application CN202210058092.3.

[0029] The construction methods of plasmids pINA1312_ScDGAT2-1, pINA1312_DLO, and pINA1312_RtZL used in the following examples can be found in the literature [Nicaud, JM, Madzak, C., Broek, P., Gysler, C., Duboc, P., Niederberger, P., Gaillardin, C. 2002 Protein expression and secretion in the yeast Yarrowia lipolytica. FEMS yeast research 2:371-379.].

[0030] The construction methods of plasmids pCRISPRyl_DGA1, pHR_DGA1, pCRISPRyl_PAH1, and pHR_PAH1 used in the following examples can be found in the literature [Schwartz.C., Shabbir-Hussain.M., Frogue.K., et al. Standardized markerless gene integration for pathway engineering in Yarrowia lipolytica. 2017 ACS synthetic biology 6:402-409.].

[0031] Comparative Example 1: Constructing the control strain SBT06-000

[0032] Primer pairs P1 (SEQ ID No. 1) and P2 (SEQ ID No. 2) were designed to amplify the expression cassette (SEQ ID No. 3) encoding orotidine 5-phosphate decarboxylase URA3, and the expression was performed using the Frozen EZ Yeast Transformation II yeast transformation kit. TM (Zymo Research) integrated the URA3 expression cassette into the genome of the Yersinia lipolytica strain DN20, which produces free astaxanthin, to obtain the control strain SBT06-000.

[0033] Example 1: Construction of an engineered strain of Yersinia lipophila producing esterified astaxanthin, SBT06-001

[0034] 1. The acyltransferase ScDGAT2-1 (SEQ ID No. 4) derived from *Schizophyllum commune* was optimized and synthesized by Beijing Qingke Biotechnology Co., Ltd. Primer pairs P3 (SEQ ID No. 5) and P4 (SEQ ID No. 6) were designed to amplify the corresponding fragment, and then ligated and transformed with the linearized plasmid fragment pINA1312 digested with BamHI to obtain the recombinant plasmid pINA1312_ScDGAT2-1.

[0035] 2. The recombinant plasmid pINA1312_ScDGAT2-1 was linearized by the NruI restriction site and integrated into the genome of Yersinia lipolytica strain DN20 to obtain the engineered Yersinia lipolytica strain SBT06-001.

[0036] Example 2: Construction of the lipophilic Yersinia spp. engineered strain SBT06-002 containing esterified astaxanthin

[0037] 1. Three primer pairs, P5 / P6 (SEQ ID No. 7 and SEQ ID No. 8), P7 / P8 (SEQ ID No. 9 and SEQ ID No. 10) and P9 / P10 (SEQ ID No. 11 and SEQ ID No. 12), were designed to amplify the pHR plasmid fragment, the upstream 1000bp (SEQ ID No. 13) and downstream 1000bp homologous arm (SEQ ID No. 14) of the endogenous diacylglycerol transferase DGA1, respectively, and the CRISPR plasmid pHR_DGA1 was constructed by seamless cloning.

[0038] 2. Primer pairs P11 and P12 (SEQ ID No. 15 and SEQ ID No. 16) were designed to amplify the sgRNA (SEQ ID No. 17) that targets the DGA1 gene, and the CRISPR plasmid pCRISPR_DGA1 was constructed by seamless cloning.

[0039] 3. The CRISPR plasmids pHR_DGA1 and pCRISPR_DGA1 were simultaneously transformed into the engineered strain SBT06-001 of Yersinia lipolytica to obtain the engineered strain SBT06-002 of Yersinia lipolytica.

[0040] Example 3: Construction of the lipophilic Yersinia spp. engineered strain SBT06-003 containing esterified astaxanthin

[0041] 1. Two primer pairs, P13 / P14 (SEQ ID No. 18 and SEQ ID No. 19) and P15 / P16 (SEQ ID No. 20 and SEQ ID No. 21), were designed. The fusion gene fragment of ScDGAT2-1 and Oleosin (SEQ ID No. 22) was obtained by overlap PCR and ligated with the linearized plasmid fragment pINA1312 digested with BamHI for transformation to obtain the recombinant plasmid pINA1312_DLO.

[0042] 2. The recombinant plasmid pINA1312_DLO was linearized by the NruI restriction site and transformed into the engineered strain SBT06-002 of Yersinia lipolytica to obtain the engineered strain SBT06-003 of Yersinia lipolytica.

[0043] Example 4: Construction of the lipophilic Yersinia spp. engineered strain SBT06-004 containing esterified astaxanthin

[0044] 1. Three primer pairs, P5 / P6 (SEQ ID No. 7 and SEQ ID No. 8), P17 / P18 (SEQ ID No. 23 and SEQ ID No. 24) and P19 / P20 (SEQ ID No. 25 and SEQ ID No. 26), were designed to amplify the pHR vector fragment, the upstream 1000bp (SEQ ID No. 27) and downstream 1000bp homologous arm (SEQ ID No. 28) of the endogenous phosphatidylphosphatase PAH1, respectively, and the CRISPR plasmid pHR_PAH1 was constructed by seamless cloning.

[0045] 2. Primer pairs P21 and P22 (SEQ ID No. 29 and SEQ ID No. 30) were designed to amplify the sgRNA (SEQ ID No. 31) that targets the PAH1 gene, and the CRISPR plasmid pCRISPR_PAH1 was constructed by seamless cloning.

[0046] 3. The CRISPR plasmids pHR_PAH1 and pCRISPR_PAH1 were simultaneously transformed into the engineered strain SBT06-003 of Yersinia lipolytica to obtain the engineered strain SBT06-004 of Yersinia lipolytica.

[0047] Example 5: Construction of the lipophilic Yersinia spp. engineered strain SBT06-005 containing esterified astaxanthin

[0048] 1. Using the genome of the engineered Yersinia lipolytica strain DN20 as a template, three primer pairs, P23 / P24 (SEQ ID No. 32 and SEQ ID No. 33), P25 / P26 (SEQ ID No. 34 and SEQ ID No. 35) and P27 / P28 (SEQ ID No. 36 and SEQ ID No. 37), were designed to amplify expression cassettes of β-carotene hydroxylase CrtZ (SEQ ID No. 38), phytopenic oleoresin synthase CarRP (SEQ ID No. 39), and endogenous truncated 3-hydroxy-3-methylglutaryl-CoA reductase tHMG1 (SEQ ID No. 40) by overlap PCR.

[0049] 2. Design two primer pairs, P29 / P30 (SEQ ID No. 41 and SEQ ID No. 42) and P31 / P32 (SEQ ID No. 43 and SEQ ID No. 44), to amplify the expression cassette encoding β-isopropylmalate dehydrogenase LEU2 (SEQ ID No. 45) and the pINA1312 plasmid fragment.

[0050] 3. The recombinant plasmid pINA1312_RtZL was constructed by seamless cloning of the CrtZ expression cassette, CarRP expression cassette, tHMG1 expression cassette, leu2 expression cassette and plasmid fragment.

[0051] 4. The recombinant plasmid pINA1312_RtZL was linearized into the engineered strain SBT06-004 of Yersinia lipolytica through the restriction enzyme site NruI, to obtain the engineered strain SBT06-005 of Yersinia lipolytica.

[0052] Example 6: Esterified Astaxanthin Yield from Engineered Strains of Yersinia lipophila

[0053] The engineered strains of *Yarrowia lipolyticis* from Example 1, SBT06-001, SBT06-002, SBT06-003, SBT06-004, and SBT06-005, were inoculated into test tubes containing 1 mL of YPD medium and cultured for 1 day. 10 μL of the bacterial culture was then transferred to deep-well plates and cultured for 4 days. The YPD medium consisted of 2% glucose, 2% peptone, and 2% yeast extract. After fermentation, the bacterial culture was collected and astaxanthin was extracted using DMSO and acetone. The extract was enzymatically hydrolyzed with esterase and then extracted with ethyl acetate. The enzymatically hydrolyzed and unhydrolyzed astaxanthin extracts were filtered and analyzed by HPLC using a YMC C30 carotenoid column.

[0054] like Figure 1As shown, the free astaxanthin content of the control strain SBT06-000 decreased slightly after enzymatic hydrolysis, indicating that the enzymatic hydrolysis process depletes astaxanthin. Conversely, the free astaxanthin content of the engineered strain SBT06-001 increased significantly after enzymatic hydrolysis, indicating that some astaxanthin in the engineered strain exists in esterified form and is converted into free astaxanthin after the ester bond breaks. The yield of esterified astaxanthin was calculated using the following formula:

[0055] C 酯化虾青素 =C 酶解后虾青素 -C 酶解前虾青素 ·A, where C 酯化虾青素 Represents the content of esterified astaxanthin, C 酶解后虾青素 To determine the yield of free astaxanthin in the enzymatically hydrolyzed astaxanthin extract, C 酶解前虾青素 The yield of free astaxanthin in the un-enzymatically hydrolyzed astaxanthin extract solution is given by A, where A is the recovery ratio of free astaxanthin before and after enzymatic hydrolysis of the fermentation extract.

[0056] The results are as follows Figure 2 As shown, the esterified astaxanthin yield of engineered strain SBT06-001 was 33.7 mg / L, with a yield of 1.9 mg / g DCW, accounting for 38.4% of the total astaxanthin. This indicates that integrating the acyltransferase ScDGAT2-1 derived from Schizobacterium into the genome of the engineered Yersinia lipolytica strain DN20 can increase the proportion of esterified astaxanthin in the total astaxanthin, thereby increasing the yield of esterified astaxanthin.

[0057] like Figure 3 As shown, the esterified astaxanthin yield of engineered strain SBT06-002 was 34.4 mg / L, with a yield of 2.6 mg / g DCW, accounting for 37.4% of the total astaxanthin. The yield of free astaxanthin was 57.5 mg / L, indicating that knocking out the diacylglycerol acyltransferase DGA1 significantly improved the yield of esterified astaxanthin. The esterified astaxanthin yield of engineered strain SBT06-003 was 37.2 mg / L, with a yield of 2.5 mg / g DCW, accounting for 38.9% of the total astaxanthin. The yield of free astaxanthin was 58.5 mg / L. The advantage of strain 003 over strain 002 lies in the higher proportion of total astaxanthin to carotenoids, i.e., lower byproducts.

[0058] like Figure 4 As shown, the engineered strain SBT06-004 achieved a higher esterified astaxanthin content of 39.6%, with an esterified astaxanthin yield of 40.5 mg / L and a yield of 2.9 mg / g DCW; while the engineered strain SBT06-005 achieved a higher total astaxanthin content, with a total astaxanthin yield of 92.9 mg / L.

[0059] Example 7: Esterified Astaxanthin Yield of Engineered Strains SBT06-005 in a 5L Fermenter

[0060] The engineered strain SBT06-005 was inoculated into a test tube containing 1 mL of YPD medium and cultured for 1 day. 10 μL of the bacterial culture was then transferred to a 250 mL shake flask containing 50 mL of YPD medium and cultured for 1 day. The YPD medium consisted of 2% glucose, 2% peptone, and 2% yeast extract. The seed culture was inoculated into a fermenter to achieve an initial bacterial concentration of OD0.05. 600 =0.8~1, fermentation began, and samples were taken for testing every 12 hours. The fermentation tank volume was 2L, the fermentation medium was 5 times the concentration of YPD, the pH was controlled at 6.8, the agitator speed was 800rpm, the aeration rate was 2vvm, and glucose was continuously supplemented to maintain dissolved oxygen at 20±5%. The esterified astaxanthin yield was extracted from the samples using the method described in Example 6, and the results are as follows: Figure 5 As shown, the highest yield of esterified astaxanthin (166.6 mg / L) was obtained after 84 hours of fermentation, and the highest yield of total astaxanthin (1097.0 mg / L) was obtained after 144 hours of fermentation.

Claims

1. An engineered strain of *Yersinia lipophila* that produces esterified astaxanthin, characterized in that, To use Yersinia lipophila ( Yarrowia lipolytica The engineered strain DN20 is an engineered *Yarrowia lipolytica* strain obtained by any of the following genetic modification methods: (1) Integrated fissile vibrio ( Schizochytrium sp. Acyltransferases from ) Sc DGAT2-1 and knockout of diacylglycerol acyltransferase DGA1; (2) Integration of acyltransferases from Schizobacterium Sc DGAT2-1 with the diacylglycerol acyltransferase DGA1 knocked out and fused with expression of corn oil protein Oleosin and an acyltransferase derived from Schizobacterium. Sc DGAT2-1; (3) Integration of acyltransferases from Schizobacterium Sc DGAT2-1 was developed by knocking out diacylglycerol acyltransferase DGA1 and phosphatidylphosphatase PAH1, and fused with expression of maize oil protein Oleosin and acyltransferases derived from Schizobacterium. Sc DGAT2-1; (4) Integration of acyltransferases from Schizobacterium tumefaciens Sc DGAT2-1 with the diacylglycerol acyltransferase DGA1 and phosphatidylphosphatase PAH1 knocked out, fused to express maize oil protein Oleosin and acyltransferases derived from Schizobacterium. Sc DGAT2-1 integrates β-carotene hydroxylase CrtZ, endogenous truncated 3-hydroxy-3-methylglutaryl-CoA reductase tHMG1, phytoene synthase CarRP, and β-isopropylmalate dehydrogenase LEU2.

2. The engineered *Yarrowia lipophila* strain according to claim 1, characterized in that, Acyltransferases derived from Schizovibrio Sc The amino acid sequence of DGAT2-1 is shown in SEQ ID No. 4, an acyltransferase derived from *Schizophyllum commune*. Sc The nucleotide sequence of the DGAT2-1 and corn oil protein Oleosin fusion gene is shown in SEQ ID No. 22, β-carotene hydroxylase. CrtZ The nucleotide sequence of the gene is shown in SEQ ID No. 38, phytopene synthase. CarRP The nucleotide sequence of the gene is shown in SEQ ID No. 39, endogenous truncated 3-hydroxy-3-methylglutaryl-CoA reductase. tHMG1 The nucleotide sequence of the gene is shown in SEQ ID No. 40, β-isopropylmalate dehydrogenase. LEU2 The nucleotide sequence of the gene is shown in SEQ ID No.

45.

3. The method for constructing the engineered *Yersinia lipophila* strain producing esterified astaxanthin according to claim 1 or 2, characterized in that, Includes the following steps: Acyltransferases derived from Schizobacterium Sc The coding gene of DGAT2-1 was integrated into the genome of the starting strain, the engineered strain of Yersinia lipolytica, DN20, to obtain the engineered strain of Yersinia lipolytica SBT06-001. Based on the engineered strain of Yersinia lipolytica SBT06-001, the coding gene of diacylglycerol acyltransferase DGA1 was knocked out to obtain the engineered strain of Yersinia lipolytica SBT06-002. Alternatively, based on the engineered Yersinia lipophila strain SBT06-002, acyltransferases derived from Schizobacterium could be added. Sc The fusion gene of DGAT2-1 and corn oil protein Oleosin was integrated into the genome of the engineered yeast SBT06-002 to obtain engineered yeast SBT06-003. Alternatively, based on the engineered Yersinia lipolytica strain SBT06-003, the gene encoding phosphatidylcholine phosphatase PAH1 can be knocked out to obtain engineered Yersinia lipolytica strain SBT06-004. Alternatively, based on the engineered *Yersinia lipolytica* strain SBT06-004, the coding genes for β-carotene hydroxylase CrtZ, endogenous truncated 3-hydroxy-3-methylglutaryl-CoA reductase tHMG1, and phytoene synthase CarRP can be integrated into the genome of the engineered *Yersinia lipolytica* strain SBT06-004 to obtain engineered *Yersinia lipolytica* strain SBT06-005.

4. The application of the engineered Yersinia lipolytica strain for producing esterified astaxanthin as described in claim 1 or 2 in the production of esterified astaxanthin.

5. The application according to claim 4, characterized in that, The specific method is as follows: the engineered strain of Yersinia lipolytica that produces esterified astaxanthin is inoculated into YPD medium for fermentation to produce esterified astaxanthin.

6. The application according to claim 5, characterized in that, YPD medium consists of 2% glucose, 2% peptone and 2% yeast extract.

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