Candida tropicalis engineering bacterium for producing astaxanthin

Abstract

The invention discloses a candida tropicalis engineering bacterium for producing astaxanthin, and belongs to the field of metabolic engineering. Candida tropicalis ANT-06 is used as a starting strain to integrally express a heterologous gene CrtZ and a gene CrtW, the heterologous gene CrtZ and the gene CrtW are subjected to fusion expression with a peroxisome signal peptide ePTS and a lipid droplet signal peptide HD2, and the yield of astaxanthin can be obviously increased through a combined strategy of knocking out a phosphatidic acid phosphatase gene PAH1 and a functional homolog gene FLD1 of human seipin. And after combined fermentation optimization, fermentation culture at 22 DEG C and addition of 10% of dodecane, 2.5% of DMSO and 2.5 mM of betaine, the shake flask fermentation yield of the astaxanthin is increased to 320.1 mg / L, and the amplification fermentation yield of a 5L fermentation tank is increased to 3096.2 mg / L. The preparation method has the characteristics of low cost, high efficiency, simplicity in operation, wide prospect and the like.

Description

Technical Field

[0001] This invention relates to an engineered strain of *Candida tropicalis* that produces astaxanthin, belonging to the field of metabolic engineering. Background Technology

[0002] Astaxanthin is a ketocarotene, deep red in color, naturally occurring in algae, shrimp, crabs, shellfish, and other organisms (Lim KC, Yusoff FM, Shariff M, et al. Astaxanthin as feed supplement in aquatic animals. Reviews in Aquaculture, 2018, 10: 738-73). Astaxanthin exists primarily in free and esterified forms. Free astaxanthin is extremely unstable and easily oxidized. Natural astaxanthin consists of four isoprene units (eight in total) linked together, forming rings at both ends, with a total of 11 conjugated double bonds. This is a typical carbon chain structure of carotenoids, characterized by a carbonyl group and a hydroxyl group on each of its unsaturated rings. This structure is the basis for astaxanthin's powerful antioxidant activity. The hydroxyl groups on the two six-membered rings form chiral centers, resulting in three different configurations: levorotatory (3S-3S'), dextrorotatory (3R-3R'), and meso (3S-3R'). Research on the synthesis of human health products using astaxanthin has been conducted abroad for some time. Its efficacy is positioned in strengthening system function, anti-cancer, protecting the retina from ultraviolet radiation, anti-inflammation, and preventing oxidative damage to low-density lipoprotein (LDL) cholesterol in the blood (Novoveská L, Ross ME, Stanley MS, et al. Microalgal carotenoids: A review of production, current markets, regulations, and future direction. Mar Drugs, 2019, 17(11): 640). Astaxanthin has a variety of biological activities, mainly manifested in coloring function, high antioxidant properties, immune enhancement, anti-inflammatory effects, and photoprotective effects.

[0003] Natural astaxanthin can be obtained from crustaceans, fish, birds, and poultry. However, the yield of this naturally sourced astaxanthin is extremely low, the process is complex and costly, and the extraction process is easily contaminated, making it economically unfeasible. Marine organisms often contain natural astaxanthin. The method of extracting astaxanthin from astaxanthin-rich processing byproducts such as crushed shrimp and crab, removing limestone, and using organic solvents is called natural extraction. This method can promote the development of aquaculture while reducing environmental pollution from waste aquatic products. However, the high ash and chitin content in the shells of discarded shrimp and crab, coupled with the low astaxanthin content, makes the extraction process complex and costly. Chemical synthesis has a long production cycle and is complex. The synthesized product is a mixture of multiple configurations and accumulates various byproducts. Furthermore, chemically synthesized astaxanthin is in a free state and is extremely unstable. Its absorption and utilization rate in the body is lower than that of naturally extracted astaxanthin, therefore it is not approved for human use.

[0004] In these contexts, metabolically engineered microorganisms demonstrate significant potential in terms of low cost and high purity, offering a broad production prospect and a huge market for the industrial production of astaxanthin through microbial fermentation. Research on astaxanthin metabolic engineering has made groundbreaking progress in recent years, evolving from traditional single-enzyme modification to a systematic and intelligent metabolic network reconstruction stage through multidisciplinary integration. In the fields of synthetic biology and metabolic engineering, researchers have successfully developed various innovative strategies to improve astaxanthin yield and production efficiency. Optimization of key enzyme fusion expression technologies has significantly improved catalytic efficiency, while the development of novel host systems provides more options for industrial production. Microbial hosts have achieved efficient heterologous synthesis through synthetic biology modification, while natural producers have seen substantial yield increases through mutagenesis and gene editing. The dynamic regulation of metabolic networks and the introduction of cofactor engineering have enabled a better balance between cell growth and product accumulation. Simultaneously, the development of low-cost culture strategies has laid an important foundation for industrial applications. Therefore, producing astaxanthin through microbial metabolic engineering technology is a novel approach and method. Microbial transformation methods offer advantages such as high conversion rates, mild reaction conditions, low carbon footprint, and sustainable development, thus possessing broad development prospects.

[0005] tropical candidiasis ( Candida tropicalis As a diploid, non-conventional yeast, it possesses characteristics such as rapid cell growth, large biomass, broad substrate range, and strong environmental tolerance, exhibiting immense industrial potential and commercial application prospects (Li Y, Zhang L, Yang H, et al. Development of a gRNA expression and processing platform for efficient CRISPR-Cas9-based geneediting and gene silencing in Candida tropicalis Microbiology Spectrum, 2022, 10(3): e00059-22). It has been reported that *Candida tropicalis* has achieved industrial and commercial production of long-chain dicarboxylic acids and xylitol. Furthermore, *Candida tropicalis* possesses a high capacity for lipid production and is a potential chassis cell for efficient terpene synthesis.

[0006] There are currently no reports on the synthesis of astaxanthin by Candida tropicalis. Therefore, providing engineered strains capable of efficiently synthesizing astaxanthin would be of great value for the further production and application of astaxanthin. Summary of the Invention

[0007] To address the shortcomings of the existing technologies, this invention systematically constructs engineered strains that enhance astaxanthin potency through genetic engineering, effectively increasing the accumulation of astaxanthin in Candida tropicalis and promoting its application in pharmaceutical and chemical fields. The aim is to solve the problem of the lack of existing technology for synthesizing astaxanthin using Candida tropicalis.

[0008] The first technical solution provided by this invention is a genetically engineered bacterium that produces astaxanthin. The genetically engineered bacterium uses Candida tropicalis as the starting strain. The starting strain is transformed with a gene expression cassette that synthesizes astaxanthin. The gene expression cassette includes one or more “promoter-target gene-transcription terminator” structural units from upstream to downstream. The target genes mentioned above include any one of the following groups: (1) Haematococcus pluvialis β-carotene hydroxylase gene CrtZ Haematococcus pluvialis β-carotene ketolase gene CrtW (2) The ePTS nucleotide sequences shown in SEQ ID NO. 1 are ligated to the 3' ends of each gene. CrtZ-ePTS Genes and CrtW-ePTS (3) Link the HD2 nucleotide sequence as shown in SEQ ID NO.2 to the 3' end of each gene. CrtZ-linker-HD2 Genes and CrtW-linker-HD2 .

[0009] In one embodiment of the present invention, the gene expression cassette includes any one of the following groups: Expression Box CrtZ and CrtW 1: Using P ENO1 Weak promoters and T ENO1 Termination subexpression CrtZ Genes, using P GAP1 Strong promoters and T PGK1 Termination subexpression CrtW T gene ENO1 - CrtZ -P ENO1 -P GAP1 - CrtW -T PGK1 ; Expression Box CrtZ and CrtW 2: Using P ENO1 Weak promoters and T ENO1 Termination subexpression CrtZ Genes, using P FBA1 Medium-strength promoters and T PGK1 Termination subexpression CrtW T gene ENO1 - CrtZ -P ENO1 -P FBA1 - CrtW -T PGK1 ; Expression Box CrtZ and CrtW 3: Using P ENO1 Weak promoters and T ENO1 Termination subexpression CrtZ Genes, using P ENO1 Weak promoters and T PGK1 Termination subexpression CrtW P of the gene ENO1 - CrtZ -T ENO1 -P ENO1 - CrtW -T PGK1 ; Expression Box CrtZ and CrtW 4: Use P FBA1 Medium-strength promoters and T ENO1 Termination subexpression CrtZ Genes, using P GAP1 Strong promoters and T PGK1 Termination subexpression CrtW T gene ENO1 - CrtZ -P FBA1 -P GAP1 - CrtW -TPGK1 ; Expression Box CrtZ and CrtW 5: Use P FBA1 Medium-strength promoters and T ENO1 Termination subexpression CrtZ Genes, using P FBA1 Medium-strength promoters and T PGK1 Termination subexpression CrtW P of the gene FBA1 - CrtZ -T ENO1 -P FBA1 - CrtW -T PGK1 ; Expression Box CrtZ and CrtW 6: Use P FBA1 Medium-strength promoters and T ENO1 Termination subexpression CrtZ Genes, using P ENO1 Weak promoters and T PGK1 Termination subexpression CrtW T gene ENO1 - CrtZ -P FBA1 -P ENO1 - CrtW -T PGK1 ; Expression Box CrtZ and CrtW 7: Use P GAP1 Strong promoters and T ENO1 Termination subexpression CrtZ Genes, using P GAP1 Strong promoters and T PGK1 Termination subexpression CrtW P of the gene GAP1 - CrtZ -T ENO1 -P GAP1 - CrtW -T PGK1 ; Expression Box CrtZ and CrtW 8: Use P GAP1 Strong promoters and T ENO1 Termination subexpression CrtZ Genes, using P FBA1 Medium-strength promoters and T PGK1 Termination subexpression CrtW T gene ENO1 - CrtZ -P GAP1 -P FBA1 - CrtW -T PGK1 ; Expression Box CrtZ and CrtW 9: Use P GAP1 Strong promoters and T ENO1 Termination subexpression CrtZ Genes, using P ENO1 Weak promoters and T PGK1 Termination subexpression CrtW T gene ENO1 - CrtZ -P GAP1 -P ENO1- CrtW -T PGK1 ; Expression Box CrtZ-ePTS and CrtW-ePTS Connect ePTS to each CrtZ and CrtW After the 3' end of the gene, P was used ENO1 Weak promoters and T ENO1 Terminator overexpression CrtZ-ePTS Genes, using P GAP1 Strong promoters and T PGK1 Terminator overexpression CrtW-ePTS T gene ENO1 - CrtZ-ePTS -P ENO1 -P GAP1 - CrtW-ePTS -T PGK1 ; Expression Box CrtZ-linker-HD2 and CrtW-linker-HD2 The linker has the amino acid sequence GGGGS and the nucleotide sequence GGTGGCGGAGGTAGT (5'→3', as shown in SEQ ID NO.3). HD2 is linked to the linker via the linker. CrtZ and CrtW After the 3' end of the gene, P was used ENO1 Weak promoters and T ENO1 Termination subexpression CrtZ-linker-HD2 Genes, using P GAP1 Strong promoters and T PGK1 Termination subexpression CrtW-linker-HD2 T gene ENO1 - CrtZ-linker-HD2 -P ENO1 -P GAP1 - CrtW-linker-HD2 -T PGK1 .

[0010] In one embodiment of the present invention, the Haematococcus pluvialis β-carotene hydroxylase gene CrtZ The Genbank accession number is KP866868.1, and the nucleotide sequence is shown in SEQ ID NO.4; the Haematococcus pluvialis β-carotene ketolase gene. CrtW The Genbank accession number is D45881.1, and the nucleotide sequence is shown in SEQ ID NO.5.

[0011] In one embodiment of the present invention, the gene expression cassette is integrated and expressed in the starting strain. DPP1 site, DPP3 site, DLD35 Site and / or GPP1 Site.

[0012] In one embodiment of the present invention, the genetically engineered bacteria also overexpress a gene. INO2 .

[0013] Furthermore, expression box INO2 Using P GAP1 Strong promoters and T ENO1 Terminator expresses genes of key regulators of lipid metabolism and membrane homeostasis. INO2 P GAP1 - INO2 -T ENO1 .

[0014] In one implementation, the expression is P GAP1 Strong promoters and T ENO1 Termination subexpression INO2 The gene, the nucleotide sequence of which is shown in SEQ ID NO.6.

[0015] In one embodiment, the genetically engineered bacteria also overexpress a gene. DGA1 .

[0016] In one implementation, the expression box DGA1 Using P GAP1 Strong promoters and T ENO1 Terminator expresses diacylglycerol kinase gene DGA1 P GAP1 - DGA1 -T ENO1 .

[0017] In one implementation, the expression is P GAP1 Strong promoters and T ENO1 Termination subexpression DGA1 The gene, the nucleotide sequence of which is shown in SEQ ID NO.7.

[0018] In one embodiment of the present invention, the genetically engineered bacteria also have the phosphatidylphosphatase gene knocked out. PAH1 The nucleotide sequence is shown in SEQ ID NO.8; and / or the functional homolog gene of human seipin. FLD1 The nucleotide sequence is shown in SEQ ID NO.9.

[0019] In one embodiment of the present invention, genes PAH1 ,Gene FLD1 Two copies of each copy were knocked out.

[0020] In one embodiment of the present invention, the genetically engineered bacteria also knocked out transcription repressors. OPI1 The nucleotide sequence is shown in SEQ ID NO. 10.

[0021] In one embodiment of the present invention, the genetically engineered bacteria also knock out ubiquitin ligases in the endoplasmic reticulum-related degradation pathway. HRD1 The nucleotide sequence is shown in SEQ ID NO.11.

[0022] In one implementation, the expression is an expression box. CrtZ and CrtW 1 Integration into the genome DPP3 site, DPP1 Site.

[0023] In one implementation, the expression is an expression box. CrtZ and CrtW 2 Integration into the genome DPP3 Site.

[0024] In one implementation, the expression is an expression box. CrtZ and CrtW 3 Integration into the genome DPP3 Site.

[0025] In one implementation, the expression is an expression box. CrtZ and CrtW 4 Integration into the genome DPP3 Site.

[0026] In one implementation, the expression is an expression box. CrtZ and CrtW 5 Integration into the genome DPP3 Site.

[0027] In one implementation, the expression is an expression box. CrtZ and CrtW 6 Integration into the genome DPP3 Site.

[0028] In one implementation, the expression is an expression box. CrtZ and CrtW 7 Integration into the genome DPP3 Site.

[0029] In one implementation, the expression is an expression box. CrtZ and CrtW 8 Integration into the genome DPP3 Site.

[0030] In one implementation, the expression is an expression box. CrtZ and CrtW 9 Integration into the genome DPP3 Site.

[0031] In one implementation, the expression is an expression box. CrtZ-ePTS and CrtW-ePTS Integration into the genome DLD35 Site.

[0032] In one implementation, the expression is an expression box. CrtZ-linker-HD2 and CrtW-linker-HD2 Integration into the genome GPP1 Site.

[0033] In one implementation, the expression is an expression box. INO2 Integration into the genome DOS2 Site.

[0034] In one implementation, the expression is an expression box. DGA1 Integration into the genome DOS2 Site.

[0035] In one implementation, the reusable screening marker is URA3; the weak promoter is the endogenous promoter P of *Candida tropicalis*. ENO1 The terminator is endogenous T from Candida tropicalis. ENO1 The strong promoter is the endogenous promoter P of Candida tropicalis. GAPDH The medium-strong promoter is the endogenous promoter P of Candida tropicalis. FBA1 The terminator is endogenous T from Candida tropicalis. PGK1 The homologous arm originates from any endogenous gene in Candida tropicalis that does not affect yeast growth.

[0036] In one implementation, the filter marker URA3 The nucleotide sequence of the gene is shown in SEQ ID NO.12; the weak promoter P ENO1 The nucleotide sequence is shown in SEQ ID NO.13; the terminator T ENO1 The nucleotide sequence is shown in SEQ ID NO. 14; strong promoter P GAPDH The nucleotide sequence is shown in SEQ ID NO.15; the terminator T PGK1The nucleotide sequence is shown in SEQ ID NO.16; the medium-strong promoter P FBA1 The nucleotide sequence is shown in SEQ ID NO.17.

[0037] In one embodiment, the starting strain is Candida tropicalis strain ANT-06.

[0038] The second technical solution provided by this invention is a method for increasing the yield of astaxanthin produced by fermentation of recombinant Candida tropicalis, wherein the method involves at least one of the following improvements to the Candida tropicalis ANT-06 strain: (1) Through the weak promoter P ENO1 Transcription of Haematococcus pluvialis β-carotene hydroxylase gene CrtZ strong starter P GAP1 Transcriptional expression of Haematococcus pluvialis β-carotene ketolase gene CrtW ; (2) Through the weak promoter P ENO1 Transcription of Haematococcus pluvialis β-carotene hydroxylase gene CrtZ strong promoter P FBA1 Transcriptional expression of Haematococcus pluvialis β-carotene ketolase gene CrtW ; (3) Through the weak promoter P ENO1 Transcription of Haematococcus pluvialis β-carotene hydroxylase gene CrtZ weak promoter P ENO1 Transcriptional expression of Haematococcus pluvialis β-carotene ketolase gene CrtW ; (4) Through the strong promoter P FBA1 Transcription of Haematococcus pluvialis β-carotene hydroxylase gene CrtZ strong starter P GAP1 Transcriptional expression of Haematococcus pluvialis β-carotene ketolase gene CrtW ; (5) Through the strong promoter P FBA1 Transcription of Haematococcus pluvialis β-carotene hydroxylase gene CrtZ strong promoter P FBA1 Transcriptional expression of Haematococcus pluvialis β-carotene ketolase gene CrtW ; (6) Through the strong promoter P FBA1 Transcription of Haematococcus pluvialis β-carotene hydroxylase gene CrtZ weak promoter P ENO1 Transcriptional expression of Haematococcus pluvialis β-carotene ketolase gene CrtW ; (7) Through the strong promoter P GAP1Transcription of Haematococcus pluvialis β-carotene hydroxylase gene CrtZ strong starter P GAP1 Transcriptional expression of Haematococcus pluvialis β-carotene ketolase gene CrtW ; (8) Through the strong promoter P GAP1 Transcription of Haematococcus pluvialis β-carotene hydroxylase gene CrtZ strong promoter P FBA1 Transcriptional expression of Haematococcus pluvialis β-carotene ketolase gene CrtW ; (9) Through the strong promoter P GAP1 Transcription of Haematococcus pluvialis β-carotene hydroxylase gene CrtZ weak promoter P ENO1 Transcriptional expression of Haematococcus pluvialis β-carotene ketolase gene CrtW ; (10) Through the weak promoter P ENO1 Transcription of Haematococcus pluvialis β-carotene hydroxylase gene CrtZ strong starter P GAP1 Transcriptional expression of Haematococcus pluvialis β-carotene ketolase gene CrtW ; (11) Connect the ePTS to the following connections respectively CrtZ and CrtW After the 3' end of the gene, via the weak promoter P ENO1 Transcription of Haematococcus pluvialis β-carotene hydroxylase gene CrtZ-ePTS strong starter P GAP1 Transcriptional expression of Haematococcus pluvialis β-carotene ketolase gene CrtW-ePTS ; (12) The amino acid sequence of the linker is GGGGS, and the nucleotide sequence is GGTGGCGGAGGTAGT (5'→3', as shown in SEQ ID NO.3). HD2 is linked to the linker through the linker. CrtZ and CrtW After the 3' end of the gene, via the weak promoter P ENO1 Transcription of Haematococcus pluvialis β-carotene hydroxylase gene CrtZ-linker-HD2 strong starter P GAP1 Transcriptional expression of Haematococcus pluvialis β-carotene ketolase gene CrtW-linker-HD2 The two genes were located in the lipid droplets of the cell organelle; (13) Through the strong promoter P GAP1 Transcriptional expression of key regulatory genes of lipid metabolism and membrane homeostasis INO2 ; (14) Through the strong promoter P GAP1 Transcription of diacylglycerol kinase gene DGA1 ; (15) The phosphatidylphosphatase gene was knocked out. PAH1 Functional homolog of human seipin gene FLD1 Two copies of each; transcription repressor was also knocked out. OPI1 ubiquitin ligases in endoplasmic reticulum-related degradation pathways HRD1 .

[0039] The third technical solution provided by the present invention is a method for producing astaxanthin by fermentation, wherein the method utilizes the genetically engineered bacteria described in the first technical solution to prepare astaxanthin by fermentation.

[0040] In one embodiment, the seed culture of the genetically engineered bacteria is prepared at 1% (OD200). 600 The inoculum was transferred to 2×YPD fermentation medium and cultured at 30℃ and 200 rpm for 72 h.

[0041] In one embodiment, the fermentation is a shake-flask fermentation, in which the genetically engineered bacteria are activated and cultured at 30°C and 200 rpm until OD (dose-free survival). 600 Seed solution was obtained at 12-15°C, and then diluted with 1% (OD200) solution. 600 The seed culture was transferred to 2×YPD fermentation medium and fermented at 30℃ and 200 rpm for 72 h.

[0042] In one embodiment, the method involves inoculating the genetically engineered bacteria into a fermentation system containing 2 mM betaine, 10% dodecane, and 2.5% DMSO for fermentation.

[0043] The fourth technical solution provided by the present invention is the application of the genetically engineered bacteria described in the first technical solution, or the method described in the second technical solution, or the method described in the third technical solution in the preparation of astaxanthin or products containing astaxanthin.

[0044] This invention also provides applications of the above-mentioned recombinant Candida tropicalis in agriculture, food industry, medicine and health fields, or cosmetic fields.

[0045] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) This invention utilizes three types of strong, medium, and weak promoters to express the target gene, resulting in a total of 9 combinations. Engineered bacteria AST-01 (i.e., DPEG, hereinafter referred to as AST-01) express in the cytoplasm CrtZ and CrtW Astaxanthin production was 126.7 mg / L; the engineered strain DPEF expressed in the cytoplasm... CrtZ and CrtW Astaxanthin production was 90.4 mg / L; the engineered strain DPEE expressed in the cytoplasm. CrtZ and CrtW Astaxanthin production was 32.3 mg / L; the engineered DPFG strain expressed in the cytoplasm... CrtZ and CrtW Astaxanthin production was 32.1 mg / L; the engineered strain DPFF expressed in the cytoplasm. CrtZ and CrtW Astaxanthin production was 5.9 mg / L; the engineered strain DPFE expressed in the cytoplasm. CrtZ and CrtW Astaxanthin production was 9.8 mg / L; the engineered bacteria DPGG expressed in the cytoplasm CrtZ and CrtW Astaxanthin production was 9.3 mg / L; DPGF expression in the cytoplasm of the engineered bacteria was observed. CrtZ and CrtW Astaxanthin production was 32.7 mg / L; the engineered bacteria expressed DPGE in the cytoplasm. CrtZ and CrtW The astaxanthin yield was 10.2 mg / L; (2) This invention employs a subcellular organelle compartmentalized approach to coordinate the expression of target genes, expressing them separately in the cytoplasm, peroxisomes, and lipids, followed by combined expression: Engineered bacteria AST-02 in peroxidase expression CrtZ and CrtW The astaxanthin yield was 147.9 mg / L; Engineered bacteria AST-03 expressed in lipid droplets CrtZ and CrtW The astaxanthin yield was 47.0 mg / L; Engineered strain AST-04 expresses cytoplasmic and peroxidase expression CrtZ and CrtW The astaxanthin yield was 153.3 mg / L; Engineered bacteria AST-05 expressed in cytoplasm CrtZ and CrtW Two copies, in peroxidase expression CrtZ and CrtW Four copies were used, and the astaxanthin yield was 132.8 mg / L. Engineered bacteria AST-06 expressed in cytoplasm CrtZ and CrtW Four copies in peroxidase expression CrtZ and CrtW Two copies were obtained, and the astaxanthin yield was 153.8 mg / L; Engineered bacteria AST-07 expressed in cytoplasm CrtZ and CrtWFour copies in peroxidase expression CrtZ and CrtW Two copies, in lipid droplet expression CrtZ and CrtW Two copies were obtained, and the astaxanthin yield was 192.0 mg / L; (3) This invention employs an endoplasmic reticulum expansion strategy to increase astaxanthin production. Engineered strain AST-08 expresses cytoplasmic, peroxidase, and lipid content. CrtZ and CrtW Knock out two copies of the phosphatidylphosphatase gene PAH1 The astaxanthin yield was 229.0 mg / L; Engineered strain AST-09 expresses cytoplasmic, peroxidase, and lipid content. CrtZ and CrtW Knock out two copies of the transcriptional repressor OPI1 The astaxanthin yield was 190.7 mg / L; Engineered strain AST-10 expresses cytoplasmic, peroxidase, and lipid content. CrtZ and CrtW The cytoplasm expresses the key regulatory factor gene INO2, which is related to lipid metabolism and membrane homeostasis. The copy number is 2, and the astaxanthin production is 203.2 mg / L. Engineered strain AST-11 expresses cytoplasmic, peroxidase, and lipid content. CrtZ and CrtW Knock out two copies of the phosphatidylphosphatase gene PAH1 and two copies of transcription repressor OPI1 The astaxanthin yield was 201.4 mg / L; Engineered strain AST-12 expresses cytoplasmic, peroxidase, and lipid content. CrtZ and CrtW Knock out two copies of the phosphatidylphosphatase gene PAH1 The cytoplasm expresses the key regulatory factor gene INO2, which is related to lipid metabolism and membrane homeostasis, with a copy number of 2 and an astaxanthin production of 202.1 mg / L. (4) This invention employs an enhanced lipid synthesis strategy to improve astaxanthin synthesis. Engineered strain AST-13 expresses cytoplasmic, peroxidase, and lipid content. CrtZ and CrtW Knock out two copies of the phosphatidylphosphatase gene PAH1 Expressing diacylglycerol kinase gene DGA1 The astaxanthin yield was 203.3 mg / L; Engineered strain AST-14 expresses cytoplasmic, peroxidase, and lipid content. CrtZ and CrtW Knock out two copies of the phosphatidylphosphatase genePAH1 Knock out ubiquitin ligases in endoplasmic reticulum-related degradation pathways HRD1 The astaxanthin yield was 195.1 mg / L; Engineered strain AST-15 expresses cytoplasmic, peroxidase, and lipid content. CrtZ and CrtW The phosphatidylphosphatase gene has a copy number of 8, and two copies of the gene have been knocked out. PAH1 Knockout of functional homolog genes of human seipin FLD1 The astaxanthin yield was 249.8 mg / L; (5) This invention optimizes the fermentation conditions (dodecane addition amount, betaine addition amount, DMSO addition amount, and temperature). When the temperature is 22℃, with the addition of 2.5mM betaine, 10% dodecane, and 2.5% DMSO, the astaxanthin yield after fermentation with engineered bacteria AST-15 is 320.1mg / L. Attached Figure Description

[0046] Figure 1 A schematic diagram illustrating the metabolic modification pathway for astaxanthin synthesis in Candida tropicalis. Figure 2 Engineered expression of Haematococcus pluvialis β-carotene hydroxylase gene to promote its growth. CrtZ Haematococcus pluvialis β-carotene ketolase gene CrtW The yield of astaxanthin produced by the engineered bacteria is shown in the figure. Figure 3 The figure shows the yield of astaxanthin synthesized by the subcellular organelle compartmentalization. Figure 4 Figure showing the yield of astaxanthin produced by engineered strains of Candida tropicalis modified with endoplasmic reticulum expansion strategy. Figure 5 The yield of astaxanthin produced by engineered strains of Candida tropicalis to enhance lipid synthesis is shown in the figure. Figure 6 The yield results of astaxanthin production after optimizing fermentation conditions; Figure 7 The graph shows the yield results of astaxanthin production using the fed-batch fermentation method. Detailed Implementation

[0047] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0048] 1. Culture medium: YPD medium (g / L): glucose 20, yeast extract 10, peptone 20.

[0049] 2×YPD medium (g / L): glucose 40, yeast extract 20, peptone 40.

[0050] MM medium (g / L): glucose 20, yeast nitrogen base (YNB) 6.7, ammonium sulfate 10.

[0051] LB medium (g / L): peptone 10, yeast extract 5, sodium chloride 10.

[0052] 2. Fermentation method: 2.1 Shake-flask fermentation: The recombinant strain was streaked onto YPD plates, and single colonies were picked from the solid plates and transferred to 20 mL (50 mL Erlenmeyer flask) of YPD liquid medium. The culture was incubated at 200 r / min and 30℃ until OD500 was reached. 600 The seed culture was prepared with an initial OD value of 8-15, and then transferred to 15 mL (250 mL) of 2×YPD medium to allow the initial OD value to rise. 600 Astaxanthin was produced by culturing at 0.1°C, 30°C, and 200 rpm for 72 hours. Three biological replicates were set up for each single colony.

[0053] Biomass changes during fermentation were measured using a UV-Vis spectrophotometer at 600 nm light absorption intensity (OD). 600 To characterize the cells, the measurement range was kept between 0.2 and 0.8. If the bacterial concentration was too high, the cells were diluted and measured again for calculation.

[0054] 2.2, 5-L fermenter: Single colonies of engineered *Candida tropicalis* were obtained by streaking YPD solid medium in a 30°C incubator for 48 h. Each single colony was inoculated into a 100 mL flask containing 20 mL of YPD liquid medium and incubated at 30°C and 200 rpm for 24 h. The pre-culture was then inoculated into 50 mL of YPD medium (1% inoculation volume) and incubated at 30°C and 200 rpm for 14 h until OD (dose retardation) was achieved. 600 The culture temperature was increased to 12-15, and then seed culture was obtained. The seed culture was inoculated into a 5 L bioreactor (Shanghai Bailun Biotechnology Co., Ltd., China) containing 2 L of YPD60 medium (0.5 g / L MgSO4). The temperature was maintained at 22 °C, the pH at 5.5, the agitator speed at 400-800 rpm, and the aeration rate at 2-4 L / min. -1 Dissolved oxygen was maintained above 30%. When the glucose concentration dropped to <5 g / L, the fed-batch concentration was 800 g·L⁻¹. -1Glucose solution, control the sugar concentration of the fermentation broth at 1 g·L⁻¹ -1 After approximately 96 hours, the sugar concentration was controlled at 2-5 g·L. -1 Approximately. When the glucose in the culture medium is depleted (i.e., dissolved oxygen rebound): add 10% dodecane and 2mM betaine. After 108 hours of incubation, add 2.5% DMSO. During this process, adjust the pH with ammonia.

[0055] 3. Detection of astaxanthin content: 1. Extraction Transfer 1 mL of fermentation broth to a 1.5 mL centrifuge tube and centrifuge to collect cells. Wash the cells three times with sterile water. Then resuspend them in 1 mL of 3 mol / L HCl, heat at 99°C for 3 minutes, and then remove the HCl. Wash the cells again with sterile water. Resuspend the cells in 1 mL of acetone and place in a shaker for 10 minutes. After centrifuging at 5000×g for 5 minutes, collect the acetone. Repeat this process until the cells are colorless. Mix the collected acetone and determine the astaxanthin yield.

[0056] 2. Testing 500 μL of acetone extract was filtered through a 0.22 μm organic filter membrane and analyzed using Shimadzu liquid HPLC. The detection conditions were: XDB-C18 column (5 μm, 44.6 × 250 mm), detection wavelength 450 nm, mobile phase composition of 35% acetonitrile, 35% methanol, and 30% isopropanol, flow rate 1 mL / min, column temperature 25℃, and sample detection time 30 min. Unless otherwise specified, the experimental methods described in the following examples can be performed in accordance with conventional methods or the instructions of the manufacturer of the product used. Unless otherwise specified, the materials and reagents used can be obtained commercially.

[0057] 4. Plasmids plasmid Ts-TPGK1- carB -PFBA1-PGAPDH- carRP -TENO1 was published in the literature "Systematic metabolic engineering of terpenoid natural products produced by Candida tropicalis" (Zhang Lihua. Systematic metabolic engineering of terpenoid natural products produced by Candida tropicalis [D]. Jiangnan University, 2022.).

[0058] plasmid Ts- gda324-URA3 The findings are published in the literature “Establishment of the genetic operating system of Candida tropicalis and its application in the synthesis of dicarboxylic acids” (Zhang Lihua. Establishment of the genetic operating system of Candida tropicalis and its application in the synthesis of dicarboxylic acids [D]. Jiangnan University, 2017.).

[0059] Example 1: Construction of Nine Expression Boxes (1) Construction of expression box Expression Box CrtZ and CrtW 4: β-Carotene Ketoylase Gene from Haematococcus pluvialis CrtW The nucleotide sequence of the synthesized material was optimized by Tianlin Biotechnology Co., Ltd. based on the codon preference of Candida tropicalis (as shown in SEQ ID NO. 5), and then ligated into plasmid pUC57. The synthesized plasmid was named pUC57- CrtW Primers CrtW-F / R were designed, and PCR amplification was performed using this plasmid as a template to obtain the linear fragment "- CrtW The fragment, marked with a "-", contains Spe I and Xho I restriction sites at both ends. The linear fragment was purified using a gel extraction and purification kit for later use. The plasmid Ts-TPGK1- was previously constructed in our laboratory. carB -PFBA1-PGAPDH- carRP -TENO1 was used as a template (see the literature "Systematic Metabolic Engineering of Terpenoid Natural Product Production by Candida Tropicalis" for details), and the linear fragment "-TENO1-TPGK1-Ts-TENO1-" was obtained by digestion with Spe I and Xho I enzymes. carRP -PGAPDH-PFBA1-". The fragment was ligated to "-" using a one-step ligation kit (Takara). CrtW After ligation of the linear fragment, it was introduced into *E. coli* JM109 via chemical transformation. After culturing ampicillin-resistant LB agar plates for 10-12 hours, positive transformants were screened by colony PCR and inoculated into ampicillin-resistant LB liquid medium at 37°C and 200 rpm for another 10-12 hours. Finally, the plasmid was extracted and verified by enzyme digestion, yielding the correct plasmid Ts-TPGK1-. carB -PFBA1-PGAPDH- CrtW -TENO1. Next, the gene encoding β-carotene hydroxylase from Haematococcus pluvialis was analyzed. CrtZ (NCBI Accession Number: KP866868.1) The plasmid pUC57-CrtZ was optimized based on the codon preference of *Candida tropicalis* (nucleotide sequence shown in SEQ ID NO.4) and synthesized by Tianlin Biotechnology Co., Ltd. Primers CrtZ-F / R were designed, and PCR amplification was performed using this plasmid as a template to obtain the linear fragment "- CrtZ The fragment, marked with a "-", contains Sal I and Nhe I restriction sites at both ends. After purification using a gel extraction and purification kit, the linear fragment was used to construct the plasmid Ts-TPGK1-. carB -PFBA1-PGAPDH- CrtWUsing TENO1 as a template, the fragment "-TENO1-Ts-TPGK1-" was obtained by digestion with Sal I and Nhe I enzymes. CrtW -PGAPDH-PFBA1-". The fragment was ligated to "-" using a one-step ligation kit (Takara). CrtZ After ligation of the linear fragment, it was introduced into *E. coli* JM109 via chemical transformation. After culturing on ampicillin-resistant LB agar plates for 10-12 hours, positive transformants were screened by colony PCR and inoculated into ampicillin-resistant LB liquid medium at 37°C and 200 rpm for another 10-12 hours. Finally, the plasmid was extracted and verified by enzyme digestion to obtain the correct plasmid "Ts-TENO1-". CrtZ -PFBA1-PGAPDH- CrtW -TPGK1".

[0060] Expression cassettes CrtZ and CrtW1: Using the genome of *Candida tropicalis* ATCC20336 as a template, primers PENO1-F1 / R1 were designed for PCR amplification to obtain the linear fragment "-Not I-PENO1- Sal I-", which contains Spe I and Not I restriction sites at both ends. After digestion with Sal I and Not I, the fragment "-PENO1-" was obtained. The expression cassettes were then used to express the expression fragment "-PENO1-". CrtZ -PFBA1-PGAPDH- CrtW After double digestion with Sal I and Not I, the "-TENO1" fragment was ligated to the linear fragment "-PENO1-" using a one-step ligation kit (Takara), and then introduced into *E. coli* JM109 via chemical transformation. After culturing on ampicillin-resistant LB agar plates for 10-12 hours, positive transformants were screened by colony PCR and inoculated into ampicillin-resistant LB liquid medium at 37°C and 200 rpm for another 10-12 hours. Finally, the plasmid "Ts-TPGK1-" was extracted and verified by enzyme digestion to obtain the correct plasmid. CrtZ -PENO1-PGAPDH- CrtW -TENO1". Primers DPP3-PEG-F / R were designed to obtain the fragment "-TENO1-". CrtZ -PENO1-PGAPDH- CrtW "-TPGK1-" was purified using a gel recovery and purification kit and then prepared for use.

[0061] Expression cassettes CrtZ and CrtW1: Primers PGF-F / R were designed to amplify the expression cassettes CrtZ and CrtW1 using them as templates, yielding the linearized fragment "-PGAPDH-PFBA1-". The expression cassettes CrtZ and CrtW1 were then digested with SalI and SpeI enzymes to obtain the linearized fragment "". -CrtW- TENO1-TPGK1 -CrtZ- The two fragments were ligated in one step and introduced into *E. coli* JM109 via chemical transformation. After culturing on ampicillin-resistant LB agar plates for 10-12 hours, positive transformants were screened by colony PCR and inoculated into ampicillin-resistant LB liquid medium at 37°C and 200 rpm for another 10-12 hours. Finally, the plasmid was extracted and verified by enzyme digestion to obtain the correct plasmid "Ts-TENO1-". CrtZ -PGAPDH-PFBA1- CrtW -TPGK1".

[0062] Expression cassettes CrtZ and CrtW9: Primers PENO1-F2 / R2 were designed to amplify CrtZ and CrtW9 using them as templates via PCR, yielding the linearized fragment "-PENO1-". CrtZ and CrtW9 were then digested with Xho I and Spe I enzymes to obtain the linearized fragment "-". CrtW- TPGK1-TENO1 -CrtZ- The two fragments, "PGAPDH-", were ligated in one step and introduced into *E. coli* JM109 via chemical transformation. After culturing on ampicillin-resistant LB agar plates for 10-12 hours, positive transformants were screened by colony PCR and inoculated into ampicillin-resistant LB liquid medium at 37°C and 200 rpm for another 10-12 hours. Finally, the plasmid "Ts-TENO1-" was extracted and verified by enzyme digestion to obtain the correct plasmid. CrtZ -PGAPDH-PENO1- CrtW -TENO1".

[0063] Expression cassettes CrtZ and CrtW6: Primers PENO1-F2 / R2 were designed to perform PCR amplification using expression cassettes CrtZ and CrtW1 as templates, obtaining the linearized fragment "-PENO1-". Expression cassettes CrtZ and CrtW6 were then used... Xho I and Spe I. Enzyme digestion to obtain linearized fragment "- CrtW -TPGK1-TENO1- CrtZThe two fragments, "-PFBA1", were ligated in one step and introduced into *E. coli* JM109 via chemical transformation. After culturing on ampicillin-resistant LB agar plates for 10-12 hours, positive transformants were screened by colony PCR and inoculated into ampicillin-resistant LB liquid medium at 37°C and 200 rpm for another 10-12 hours. Finally, the plasmid "Ts-TENO1-" was extracted and verified by enzyme digestion to obtain the correct plasmid. CrtZ -PFBA1-PENO1- CrtW -TENO1".

[0064] Expression cassettes CrtZ and CrtW2: Primers PENO1-F1 / R1 were designed to perform PCR amplification using expression cassettes CrtZ and CrtW1 as templates to obtain the linear fragment "-Not I". - PENO1-Sal Ⅰ-”, the fragment contains at both ends Not I and Sal Ⅰ Enzyme cleavage site, via Not I and Sal I. Enzyme digestion yielded the fragment "-PENO1-", which was then used to express the cassettes CrtZ and CrtW1. Not I and Sal After double enzyme digestion, the fragment was ligated to the linear fragment "-PENO1-" using a one-step ligation kit (Takara), and then introduced into *E. coli* JM109 via chemical transformation. After culturing on ampicillin-resistant LB agar plates for 10-12 hours, positive transformants were screened by colony PCR and inoculated into ampicillin-resistant LB liquid medium at 37°C and 200 rpm for another 10-12 hours. Finally, the plasmid "Ts-TENO1-" was extracted and verified by enzyme digestion to obtain the correct plasmid. CrtZ -PENO1-PFBA1- CrtW -TPGK1".

[0065] Expression cassettes CrtZ and CrtW 7: Primers PGG-F / R were designed to perform PCR amplification using expression cassettes CrtZ and CrtW 1 as templates to obtain the linear fragment "-PGAPDH-". CrtW After double digestion of the expression cassettes CrtZ and CrtW8 with Not I and Xho I, the fragment was ligated with "-TPGK1-" using a one-step ligation kit (Takara). CrtWAfter ligation of the linear fragment "-TPGK1-", it was introduced into *E. coli* JM109 via chemical transformation. After culturing ampicillin-resistant LB agar plates for 10-12 hours, positive transformants were screened by colony PCR and inoculated into ampicillin-resistant LB liquid medium at 37°C and 200 rpm for another 10-12 hours. Finally, the plasmid "Ts-PGAPDH-" was extracted and verified by enzyme digestion to obtain the correct plasmid. ​ -TENO1-PGAPDH- ​ -TPGK1".

[0066] Expression cassettes CrtZ and CrtW 5: Primers PFF-F / R were designed to perform PCR amplification using expression cassettes CrtZ and CrtW 8 as templates to obtain the linear fragment "-PFBA1-". ​ After double digestion of the expression cassettes CrtZ and CrtW4 with Not I and Xho I, the fragment was ligated with "-TPGK1-" using a one-step ligation kit (Takara). ​ After ligation of the linear fragment "-TPGK1-", it was introduced into *E. coli* JM109 via chemical transformation. After culturing ampicillin-resistant LB agar plates for 10-12 hours, positive transformants were screened by colony PCR and inoculated into ampicillin-resistant LB liquid medium at 37°C and 200 rpm for another 10-12 hours. Finally, the plasmid "Ts-PFBA1-" was extracted and verified by enzyme digestion to obtain the correct plasmid. ​ -TENO1-PFBA1- ​ -TPGK1".

[0067] Expression cassettes CrtZ and CrtW 3: Primers PEE-F / R were designed to perform PCR amplification using expression cassettes CrtZ and CrtW 6 as templates to obtain the linear fragment "-PENO1-CrtW-TPGK1-". After double digestion of expression cassettes CrtZ and CrtW 1 with Not I and Xho I, the fragment was ligated to "-PENO1-" using a one-step ligation kit (Takara). ​ After ligation of the linear fragment "-TPGK1-", it was introduced into *E. coli* JM109 via chemical transformation. After culturing ampicillin-resistant LB agar plates for 10-12 hours, positive transformants were screened by colony PCR and inoculated into ampicillin-resistant LB liquid medium at 37°C and 200 rpm for another 10-12 hours. Finally, the plasmid "Ts-PENO1-" was extracted and verified by enzyme digestion to obtain the correct plasmid. ​ -TENO1-PENO1- ​ -TPGK1".

[0068] (2) Knockout box Ts- ​Build -gda324-URA3 Using the genome of Candida tropicalis ATCC20336 as a template, primers DPP3-F / R were designed for PCR amplification to obtain... ​ Fragment. Cloned via TA. ​ The fragment was inserted into the commercial vector T-vector9 (Simple) (the nucleotide sequence of the DPP3 fragment is shown in SEQ ID NO.18), and after chemical transformation into *E. coli* JM109, it was plated on ampicillin-resistant LB agar plates for screening of correct transformants. After culturing in LB liquid medium, plasmids were extracted to obtain Ts- ​ Plasmid. Design RDPP3-F / R primers to use Ts- ​ Using the plasmid as a template, a linear fragment "-DDPP3-Ts-UDPP3-" was obtained by inverse PCR amplification. This fragment has ligands at both ends. ​ I and ​ I. Restriction sites, prepared by restriction enzyme digestion and purification. Using a plasmid (Ts-) previously constructed in the laboratory. ​ () as a template, through ​ I and ​ Ⅰ. Enzyme digestion and gel recovery to obtain "- ​ - ​ - "fragment. The fragment "-DDPP3-Ts-UDPP3-" will be combined with "- ​ - ​ - The fragment was ligated with Solution I and transformed into E. coli JM109. Correct transformants were screened by colony PCR on ampicillin-resistant LB agar plates. After culturing in liquid medium (37℃, 200 rpm), Ts- was extracted using a plasmid extraction kit. ​ - ​ - ​ The plasmid was verified to be the correct product after enzyme digestion.

[0069] (3) Construction of gene-editing recombinant plasmids Firstly, through ​ R-I enzyme digestion and purification of plasmid Ts- ​ -gda324-URA3, yielding a linear fragment "-DDPP3-Ts-UDPP3-gda324-URA3-" containing 20bp specific sequences at each end. This linearized fragment was then coupled with the expression cassettes CrtZ and CrtW1 "-TPGK1- ​ -PENO1-PGAPDH- ​ The "-TENO1-" linear fragment was ligated using a one-step ligation kit and transformed into *E. coli* JM109 plasmids coated with ampicillin-resistant LB agar plates. Positive transformants were screened by colony PCR. After culturing in liquid medium, Ts- was obtained using a plasmid extraction kit.​ - ​ - ​ -TPGK1- ​ -PENO1-PGAPDH- ​ The TENO1 plasmid was analyzed and verified by restriction endonuclease digestion. Finally, sequencing confirmed the correctness of the target gene.

[0070] Using a similar strategy, primers DPP3-PEG-F / R were designed to obtain fragments using expression cassettes CrtZ and CrtW 2, 4, 6, 8, and 9 as templates. Primer DPP3-PGG-F / R was used to obtain fragments using expression cassettes CrtZ and CrtW 3, 5, and 7 as templates. These fragments were purified using a gel extraction and purification kit for use. ​ R-I enzyme digestion and purification of plasmid Ts- ​ -gda324-URA3 was used to obtain a linear fragment "-DDPP3-Ts-UDPP3-gda324-URA3-" containing 20bp specific sequences at both ends. This fragment was ligated using a one-step ligation kit and transformed into *E. coli* JM109 cultured on ampicillin-resistant LB agar plates. Positive transformants were screened by colony PCR. After culturing in liquid medium, Ts- was obtained using a plasmid extraction kit. ​ - ​ - ​ -TENO1- ​ -PENO1-PFBA1- ​ -TPGK1 plasmid, Ts- ​ - ​ - ​ -TENO1- ​ -PFBA1-PGAPDH- ​ -TPGK1 plasmid, Ts- ​ - ​ - ​ -TENO1- ​ -PFBA1-PENO1- ​ -TPGK1 plasmid, Ts- ​ - ​ - ​ -TENO1- ​ -PGAPDH-PFBA1- ​ -TPGK1 plasmid, Ts- ​ - ​ - ​ -TENO1- ​ -PGAPDH-PENO1 ​ -TPGK1 plasmid, Ts- ​ - ​ - ​ -PENO1-​ -TENO1-PENO1- ​ -TPGK1 plasmid, Ts- ​ - ​ - ​ -PFBA1- ​ -TENO1-PFBA1- ​ -TPGK1 plasmid, Ts- ​ - ​ - ​ -PGAPDH- ​ -TENO1-PGAPDH- ​ The TPGK1 plasmid was validated by restriction endonuclease digestion. Finally, sequencing confirmed the correctness of the target gene.

[0071] (4) Strains ​ Construction of AST-01 Using the β-carotene-producing engineered strain ANT-06, previously constructed in our laboratory, as the starting strain (specific methods can be found in the literature "Systematic Metabolic Engineering of Terpenoid Natural Product Production by Candida tropicalis"), the marker gene URA3 was screened using the LiCl transformation method to obtain the uracil-deficient strain ANT-07. The plasmid Ts- obtained in Example 1 for Candida tropicalis transformation was then used. ​ - ​ - ​ -TPGK1- ​ -PENO1-PGAPDH- ​ -TENO1 via restriction endonuclease ​ I. After linearization, purification was performed using the LiCl transformation method with uracil-deficient strains. ​ ANT-07 was the starting strain, with ​ Genes are used as selection markers, ​ - ​ - ​ -TPGK1- ​ -PENO1- PGAPDH- ​ The linearized TENO1 fragment was transferred into the host cell, and the transformants were plated on MM solid medium and incubated at 30°C for 3 days. Transformants from the MM solid medium were picked, their genomes were extracted, and PCR verification and sequencing were performed to identify the correct recombinant transformants, which were then named... C. ​ AST-01.

[0072] strain ​The construction methods for DPEF (expression boxes CrtZ and CrtW 2), DPEE (expression boxes CrtZ and CrtW 3), DPFG (expression boxes CrtZ and CrtW 4), DPFF (expression boxes CrtZ and CrtW 5), DPFE (expression boxes CrtZ and CrtW 6), DPGG (expression boxes CrtZ and CrtW 7), DPGF (expression boxes CrtZ and CrtW 8), and DPGE (expression boxes CrtZ and CrtW 9) are the same as above.

[0073] Table 1 Primer Sequences

[0074] (5) Results of shake-flask fermentation of recombinant bacteria Engineered bacteria AST-01 expressed in cytoplasm ​ and ​ Astaxanthin production was 126.7 mg / L; the engineered strain DPEF expressed in the cytoplasm... ​ and ​ Astaxanthin production was 90.4 mg / L; the engineered strain DPEE expressed in the cytoplasm. ​ and ​ The astaxanthin yield was 32.7 mg / L; the engineered DPFG strain expressed in the cytoplasm... ​ and ​ Astaxanthin production was 32.1 mg / L; the engineered strain DPFF expressed in the cytoplasm. ​ and ​ Astaxanthin production was 5.9 mg / L; the engineered strain DPFE expressed in the cytoplasm. ​ and ​ Astaxanthin production was 35 mg / L; the engineered strain DPGG expressed in the cytoplasm. ​ and ​ Astaxanthin production was 9.3 mg / L; DPGF expression in the cytoplasm of the engineered bacteria was observed. ​ and ​ Astaxanthin production was 32.3 mg / L; the engineered bacteria expressed DPGE in the cytoplasm. ​ and ​ The astaxanthin yield was 10.2 mg / L ( ​ ).

[0075] Example 2: Subcellular organelle compartmentalization Following the knockout frame construction method described above, the recombinant plasmid pMD19T- was obtained via DPP1-F / DPP1-R and RDPP1-F / RDPP1-R, DLD35-F / DLD35-R and RDLD35-F / RDLD35-R, GPP1-F / GPP1-R and RGPP1-F / RGPP1-R, and primers, respectively. ​-gda324- ​ pMD19T- ​ -gda324- ​ pMD19T- ​ -gda324- ​ After linearization, the result is a deletion box, which is used for subsequent gene knockout or integration.

[0076] For expression box ​ and ​ Expression: The linear fragment was amplified by PCR using CrtZ-F and CrtZ-ePTS-R primers. ​ (The two ends of this segment contain respectively) ​ and Nhe Ⅰ (Enzyme restriction sites), after digestion with Sal I and Nhe I, and the plasmid "Ts-TPGK1-" after digestion with Sal I and Nhe I. CrtZ -PENO1-PGAPDH- CrtW -TENO1" fragment "-PENO1-PGAPDH-" CrtW The recombinant plasmid "TENO1-TPGK1-" was obtained by ligation using a one-step ligation kit (Takara). CrtZ - ePTS -PENO1-PGAPDH- CrtW -TENO1". Next, primers CrtW-ePTS-F and CrtW-ePTS-R were designed to amplify the linear fragment by PCR. CrtW-ePTS Primers WF and WR were used to amplify the linear fragment "-TENO1-TPGK1-" by PCR. CrtZ - ePTS "-PENO1-PGAPDH-" was purified using a gel extraction and purification kit, then ligated using a one-step ligation kit and transformed into *E. coli* JM109 plated on ampicillin-resistant LB agar plates. Positive transformants were screened by colony PCR. After culturing in liquid medium, "Ts-TPGK1-" was obtained using a plasmid extraction kit. CrtZ - ePTS -PENO1-PGAPDH- CrtW - ePTS The plasmid "-TENO1" was verified by restriction endonuclease digestion. Sequencing confirmed its correctness. Primers DPP3-PEG-F / DLD35-PEG-R were designed to amplify the linear fragment "-TPGK1-" by PCR. CrtZ - ePTS -PENO1-PGAPDH- CrtW -e PTS-TENO1- (20bp specific sequences at both ends) was purified using a gel extraction and purification kit, and compared with pMD19T- after digestion with EcoRI enzyme. DLD35-gda - URA3 Vector fragment ligation yielded recombinant plasmid pMD19T- DLD35-gda - URA3 -TPGK1- CrtZ - ePTS -PENO1-PGAPDH- CrtW - ePTS -TENO1.

[0077] For expression box CrtZ-linker-HD2 and CrtW-linker-HD2 Expression: The linear fragment was amplified by PCR using CrtZ-F and CrtZ-linker-HD2-R as primers. [[ID=Z=27]]CrtZ- The linker-HD2 fragment (containing Sal I and Nhe I restriction sites at both ends) was digested with Sal I and Nhe I, and then combined with the Sal I and Nhe I-digested plasmid "Ts-TPGK1- CrtZ -PENO1-PGAPDH- CrtW -TENO1" fragment "-PENO1-PGAPDH-" CrtW The recombinant plasmid “Ts-TPGK1-” was obtained by ligation using a one-step ligation kit (Takara). CrtZ - linker-HD2 -PENO1-PGAPDH- CrtW -TENO1". Next, primers CrtW-ePTS-F and CrtW-linker-HD2-R were designed to amplify the linear fragment by PCR. CrtW- linker- HD2 Primers WF and WR were used to amplify the linear fragment "-TENO1-TPGK1-" by PCR. CrtZ - linker - HD2 "-PENO1-PGAPDH-" was purified using a gel extraction and purification kit, then ligated using a one-step ligation kit and transformed into *E. coli* JM109 plated on ampicillin-resistant LB agar plates. Positive transformants were screened by colony PCR. After culturing in liquid medium, Ts-TPGK1- was obtained using a plasmid extraction kit. CrtZ - linker-HD2 -PENO1-PGAPDH- CrtW- linker-HD2The plasmid "-TENO1" was verified by restriction endonuclease digestion. Sequencing confirmed its correctness. Primers DPP3-PEG-F / GPP1-PEG-R were designed to amplify the linear fragment "-TPGK1-Crt" by PCR. Z - linker-HD2 -PENO1-PGAPDH- CrtW - linker -HD2-TENO1- (20bp specific sequences at each end) was purified using a gel extraction and purification kit, and compared with pMD19T- after digestion with EcoRI enzyme. GPP1-gda - URA3 Vector fragment ligation yielded recombinant plasmid pMD19T- GPP1-gda - URA3 -TPGK1- CrtZ - linker - HD2 -PENO1-PGAPDH- CrtW - linker - HD2 -TENO1.

[0078] On genes CrtZ and CrtW Overexpression was performed: primers DPP1-PEG-F / R were designed, and the linear fragment "-TPGK1-" was amplified by PCR. CrtZ -PENO1-PGAPDH- CrtW -TENO1- (20bp specific sequences at each end), purified using a gel extraction and purification kit, and compared with pMD19T- after digestion with Xba I enzyme. DPP1-gda - URA3 Vector fragment ligation yielded recombinant plasmid pMD19T- DPP1 - gda - URA3 -TPGK1- CrtZ -PENO1-PGAPDH- CrtW -TENO1.

[0079] C. tropicalis AST-02: Following the steps above, the fragment obtained by linearizing BglⅠ is... DLD35-gda - URA3 -TPGK1- CrtZ - ePTS -PENO1-PGAPDH- CrtW - ePTS -TENO1 transformed into the strain constructed above C. tropicalis Two copies of the strain were obtained from AST-01. C. tropicalis AST-02.

[0080] C. tropicalis AST-03: Following the steps above, the fragment obtained by linearizing BglⅠ is... GPP1-gda - URA3 -TPGK1- CrtZ - linker - HD2 -PENO1-PGAPDH- CrtW - linker - HD2 -TENO1 transformed into the strain constructed above C. tropicalis Two-copy strains were obtained from ANT-07. C. tropicalis AST-03.

[0081] C. tropicalis AST-04: Following the steps above, the fragment obtained by linearizing BglⅠ is... DPP1-gda - URA3 -TPGK1- CrtZ -PENO1-PGAPDH- CrtW -TENO1 transformed into the strain constructed above C. tropicalis Two copies of the strain were obtained from AST-01. C. tropicalis AST-04.

[0082] C. tropicalis AST-05: Following the steps above, the fragment obtained by linearizing BglⅠ is... DLD24-gda - URA3 -TPGK1- CrtZ -PENO1-PGAPDH- CrtW -TENO1 transformed into the strain constructed above C. tropicalis Two copies of the strain were obtained from AST-04. C. tropicalis AST-05.

[0083] C. tropicalis AST-06: Following the steps above, the fragment obtained by linearizing BglⅠ is... DLD35-gda - URA3 -TPGK1- CrtZ - ePTS -PENO1-PGAPDH- CrtW - ePTS -TENO1 transformed into the strain constructed above C. tropicalis Two copies of the strain were obtained from AST-04. C. tropicalis AST-06.

[0084] C. tropicalis AST-07: Following the steps above, linearize BglⅠ to obtain the fragment. GPP1-gda - URA3-TPGK1- CrtZ - linker - HD2 -PENO1-PGAPDH- CrtW - linker - HD2 -TENO1 transformed into the strain constructed above C. tropicalis Two-copy strains were obtained from AST-06. C. tropicalis AST-07.

[0085] Table 2 Primer Sequences

[0086] Shake-flask fermentation results showed that the engineered strain AST-02 expressed cytoplasmic peroxidase... CrtZ and CrtW Astaxanthin production was 158.0 mg / L; cytoplasmic expression of peroxidase in engineered strain AST-03 was [data missing]. CrtZ and CrtW The astaxanthin yield was 46.4 mg / L; the engineered strain AST-04 expressed [data missing] in the cytoplasm and peroxidase. CrtZ and CrtW The astaxanthin yield was 152.0 mg / L; the engineered strain AST-05 expressed [data missing] in the cytoplasm and peroxidase. CrtZ and CrtW The astaxanthin yield was 131.4 mg / L; the engineered strain AST-06 expressed [data missing] in the cytoplasm and peroxidase. CrtZ and CrtW The astaxanthin yield was 156.3 mg / L; the engineered strain AST-07 showed improved expression in the cytoplasm, peroxidase, and lipids. CrtZ and CrtW The astaxanthin yield was 191.6 mg / L. Figure 3 ).

[0087] Example 3: Endoplasmic Reticulum Expansion Strategy for Astaxanthin Production Following the knockout box construction method described above, the recombinant plasmid pMD19T- was obtained using primers DOS2-F / DOS2-R and RDOS2-F / RDOS2-R, PAH1-F / PAH1-R and RPAH1-F / RPAH1-R, and OPI1-F / OPI1-R and ROPI1-F / ROPI1-R, respectively. DOS2 -gda324- URA3, pMD19T- PAH1 -gda324- URA3 pMD19T- OPI1 -gda324- URA3 After linearization, the result is a deletion box, which is used for subsequent gene knockout or integration.

[0088] Expression Box INO2 Using primers PGAPDH-F / R and expression cassettes CrtZ and CrtW8 as templates, PCR amplification was performed to obtain the linearized fragment "PGAPDH-". CrtZ -TENO1 was ligated into plasmid pUC57, and the synthesized plasmid was named pUC57-PGAPDH. Using the genome of *Candida tropicalis* ATCC20336 as a template, primers INO2-F / R were designed for PCR amplification to obtain... INO2 The fragment was cloned into plasmid pUC57-PGAPDH in one step. Sal I and Nhe Site I, plasmid Ts-PGAPDH- was obtained. INO2 -TENO1, use Mlu Ⅰ It was digested with enzymes and compared with... Eco pMD19T- after RⅠ enzyme digestion DOS2-gda - URA3 Vector fragment ligation yielded recombinant plasmid pMD19T- DOS2-gda - URA3 -PGAPDH- INO2 -TENO1.

[0089] C. tropicalis AST-08: Following the steps above, construct a knockout phosphatase gene with two copies removed. PAH1 The linearized fragment pMD19T- PAH1 - gda - URA3 Transformation into the strains constructed above C. tropicalis Recombinant strains were obtained from AST-07. C. tropicalis AST-08.

[0090] C. tropicalis AST-09: Following the steps above, a transcriptional repressor with two copies knocked out was constructed. OPI1 The linearized fragment pMD19T- OPI1 - gda - URA3 Transformation into the strains constructed above C. tropicalis Recombinant strains were obtained from AST-07. C. tropicalis AST-09.

[0091] C. tropicalis AST-10: Refer to the steps above, The The fragment obtained by linearizing BglⅠ DOS2-gda - URA3 -PGAPDH- INO2 -TENO1 transformed into the strain constructed aboveC. tropicalis Recombinant strains were obtained from AST-07. C. tropicalis AST-10.

[0092] C. tropicalis AST-11: Following the steps above, a transcriptional repressor with two copies knocked out was constructed. OPI1 The linearized fragment pMD19T- OPI1 - gda - URA3 Transformation into the strains constructed above C. tropicalis Recombinant strains were obtained from AST-08. C. tropicalis AST-11.

[0093] C. tropicalis AST-12: Referring to the steps above, Bgl I. Fragments obtained through linearization DOS2-gda - URA3 -PGAPDH- INO2 -TENO1 transformed into the strain constructed above C. tropicalis Recombinant strains were obtained from AST-08. C. tropicalis AST-12.

[0094] Table 3 Primer Sequences

[0095] Shake-flask fermentation results showed that the engineered strain AST-08 exhibited abnormalities in cytoplasmic expression, peroxidase activity, and lipid expression. CrtZ and CrtW Knock out two copies of the phosphatidylphosphatase gene PAH1 The astaxanthin yield was 229.0 mg / L; Engineered strain AST-09 expresses cytoplasmic, peroxidase, and lipid content. CrtZ and CrtW Knock out two copies of the transcriptional repressor OPI1 The astaxanthin yield was 190.7 mg / L; Engineered strain AST-010 expresses cytoplasmic, peroxidase, and lipid content. CrtZ and CrtW The cytoplasm expresses the key regulatory factor gene INO2, which is related to lipid metabolism and membrane homeostasis. The copy number is 2, and the astaxanthin production is 203.2 mg / L. Engineered strain AST-11 expresses cytoplasmic, peroxidase, and lipid content. CrtZ and CrtW Knock out two copies of the phosphatidylphosphatase gene PAH1 and two copies of transcription repressor OPI1 The astaxanthin yield was 201.4 mg / L; Engineered strain AST-12 expresses cytoplasmic, peroxidase, and lipid content. CrtZ and ​ Knock out two copies of the phosphatidylphosphatase gene ​ The cytoplasm expresses INO2, a key regulatory factor for lipid metabolism and membrane homeostasis, with a copy number of 2, and astaxanthin production of 202.1 mg / L. ​ ).

[0096] Example 4: Enhanced lipid synthesis strategy for astaxanthin production Following the knockout box construction method described above, the recombinant plasmid pMD19T- was obtained using primers HRD1-F / HRD1-R and RHRD1-F / RHRD1-R, FLD1-F / FLD1-R and RFLD1-F / RFLD1-R, respectively. ​ -gda324- ​ pMD19T- ​ -gda324- ​ After linearization, the result is a deletion box, which is used for subsequent gene knockout or integration.

[0097] Expression Box ​ Using the genome of Candida tropicalis ATCC20336 as a template, primers DGA1-F / R were designed for PCR amplification to obtain... ​ The fragment was cloned into plasmid pUC57-PGAPDH in one step. ​ I and ​ Site I, plasmid Ts-PGAPDH- was obtained. ​ -TENO1, use ​ I. It was digested with enzymes, and then compared with pMD19T- after digestion with EcoRI enzyme. ​ - ​ Vector fragment ligation yielded recombinant plasmid pMD19T- ​ - ​ -PGAPDH- ​ -TENO1.

[0098] ​ AST-13: Referring to the steps above, ​ I. Fragments obtained by linearization ​ - ​ -PGAPDH- ​ -TENO1 transformed into the strain constructed above ​ AST-12, to obtain recombinant strain C. ​ AST-13.

[0099] ​AST-14: Following the steps above, construct a ubiquitin ligase in the endoplasmic reticulum-related degradation pathway with two copies knocked out. ​ The linearized fragment pMD19T- ​ - ​ - ​ Transformation into the strains constructed above ​ Recombinant strains were obtained from AST-12. ​ AST-14.

[0100] ​ AST-15: Following the steps above, construct a functional homolog of human seipin with two copies knocked out. ​ The linearized fragment pMD19T- ​ - ​ - ​ Transformation into the strains constructed above ​ Recombinant strains were obtained from AST-12. ​ AST-15.

[0101] Table 4 Primer Sequences

[0102] Shake-flask fermentation results showed that the engineered strain AST-13 expressed [symptoms] in the cytoplasm, peroxisomes, and lipids. ​ and ​ Knock out two copies of the phosphatidylphosphatase gene ​ Expressing diacylglycerol kinase gene ​ The astaxanthin yield was 203.3 mg / L; the engineered strain AST-14 showed improved expression in the cytoplasm, peroxidase, and lipids. ​ and ​ Knock out two copies of the phosphatidylphosphatase gene ​ Knock out ubiquitin ligases in endoplasmic reticulum-related degradation pathways ​ The astaxanthin yield was 195.1 mg / L; the engineered strain AST-15 showed improved expression in the cytoplasm, peroxidase, and lipids. ​ and ​ The phosphatidylphosphatase gene has a copy number of 8, and two copies of the gene have been knocked out. ​ Knockout of functional homolog genes of human seipin ​ The astaxanthin yield was 249.8 mg / L ( ​ ).

[0103] Example 5: Optimization of Fermentation Conditions The next step is to explore the fermentation method of fed-batch culture in a 5-L fermenter, optimize the fermentation conditions of the engineered strain AST-15, and optimize the dodecane addition, betaine addition, DMSO, and temperature control. ① Adding different volumes (0, 2.5%, 5%, 10%, 15%) of dodecane: Fermentation results showed that when 10% dodecane was added, the accumulation of astaxanthin reached 319.5 mg / L; ② Adding different concentration gradients (0, 1, 1.5, 2, 2.5, 3, 3.5, 5, 10, 20 mM) of betaine: Fermentation results showed that when the betaine concentration was 2 mM, the accumulation of astaxanthin reached 268.3 mg / L; ③ Adding different volumes (0, 2.5%, 5%, 10%) of DMSO: Fermentation results showed that when 2.5% DMSO was added, the accumulation of astaxanthin reached 268.2 mg / L; ④ Setting different fermentation temperatures (15, 20, 22, 25, 28, 30, 35℃): Fermentation results showed that when the temperature was 22℃, the accumulation of astaxanthin reached 303.1 mg / L.

[0104] Next, different fermentation conditions were combined. When 10% dodecane, 2mM betaine, and 2.5% DMSO were added, and fermentation was carried out at 22℃, the astaxanthin yield of engineered strain AST-15 was 320.1 mg / L. ​ ) Example 6: Scale-up synthesis of astaxanthin in a 5-L fermenter The engineered strain AST-15 was cultured in a fed-batch fermenter at 22°C with 2 mM betaine, 10% dodecane, and 2.5% DMSO added. After 288 h of fermentation, the intracellular astaxanthin yield reached 3096.2 mg / L. ​ ) Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A genetically engineered bacterium that produces astaxanthin, wherein the genetically engineered bacterium is based on Candida tropicalis as the starting strain, and the starting strain is transformed with a gene expression cassette for synthesizing astaxanthin, wherein the gene expression cassette comprises one or more "promoter-target gene-transcription terminator" structural units from upstream to downstream; wherein the target gene includes any one of the following groups: (1) Haematococcus pluvialis β-carotene hydroxylase gene CrtZ Haematococcus pluvialis β-carotene ketolase gene CrtW (2) The ePTS nucleotide sequences shown in SEQ ID NO. 1 are ligated to the 3' ends of each gene. CrtZ-ePTS Genes and CrtW- ePTS (3) Link the HD2 nucleotide sequence as shown in SEQ ID NO.2 to the 3' end of each gene. CrtZ-linker- HD2 Genes and CrtW-linker-HD2.

2. The genetically engineered bacterium according to claim 1, characterized in that, The Haematococcus pluvialis β-carotene hydroxylase gene CrtZ The nucleotide sequence is shown in SEQ ID NO. 4; the Haematococcus pluvialis β-carotene ketolase gene. CrtW The nucleotide sequence is shown in SEQ ID NO.

5.

3. The genetically engineered bacterium according to claim 1, characterized in that, The gene expression cassette is integrated and expressed in the starting strain. DPP1 site, DPP3 site, DLD35 Site and / or GPP1 Site.

4. The genetically engineered bacterium according to claim 1, characterized in that, The genetically engineered bacteria also overexpress genes. INO2 The gene INO2 The nucleotide sequence is shown in SEQ ID NO.

6.

5. The genetically engineered bacterium according to claim 1, characterized in that, The genetically engineered bacteria also overexpress genes. DGA1 The gene DGA1 The nucleotide sequence is shown in SEQ ID NO.

7.

6. The genetically engineered bacterium according to claim 1, characterized in that, The genetically engineered bacteria also had at least one of the following genes knocked out: (1) Phosphatidic acid phosphatase gene PAH1 The nucleotide sequence is shown in SEQ ID NO.8; (2) Functional homolog genes of human seipin FLD1 The nucleotide sequence is shown in SEQ ID NO.9; (3) Transcription repressors OPI1 The nucleotide sequence is shown in SEQ ID NO. 10; (4) Ubiquitin ligases in endoplasmic reticulum-related degradation pathways HRD1 The nucleotide sequence is shown in SEQ ID NO.

11.

7. The genetically engineered bacterium according to claim 1, characterized in that, The starting strain was Candida tropicalis strain ANT-06.

8. A method for producing astaxanthin through fermentation, characterized in that, The method is to prepare astaxanthin by fermentation using the genetically engineered bacteria described in any one of claims 1 to 7.

9. The method according to claim 8, characterized in that, The method involves inoculating the genetically engineered bacteria into a fermentation system containing 2 mM betaine, 10% dodecane, and 2.5% DMSO for fed-batch fermentation.

10. The use of the genetically engineered bacteria according to any one of claims 1 to 7, or the method according to any one of claims 8 to 9, in the preparation of astaxanthin or products containing astaxanthin.

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