Recombinant yarrowia lipolytica strain with high astaxanthin production and construction method and application thereof

Abstract

The present application relates to a recombinant Yarrowia lipolytica strain with high astaxanthin yield and its construction method and application, which is obtained by expressing the expression cassette of geranylgeranyl diphosphate synthase CrtE, phytoene synthase / phytoene cyclase CrtYB, phytoene desaturase CrtI, 3-hydroxy-3-methylglutaryl CoA reductase tHMGR, beta-carotene ketolase CrtW, beta-carotene hydroxylase CrtZ, ATP citrate lyase ACL, acetyl-CoA synthase ACS, choline kinase CK, inositol polyphosphate kinase IPK, farnesyl diphosphate synthase ERG20 and isopentenyl pyrophosphate isomerase ID in the host strain. At the same time, the photocatalytic material O-g-C3N4 / PEI is added to accept visible light excitation and produce reducing electrons, promote the synthesis of intracellular NADPH, and further promote the accumulation of astaxanthin. Continuous feeding fermentation is carried out in a 5 L fermenter, which greatly improves the production performance of Yarrowia lipolytica, and lays a foundation for the industrialized production of high-value terpenoids.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to recombinant lipophilic yeast with high astaxanthin production, its construction method, and its application. Background Technology

[0002] The demand for natural products in food, pharmaceuticals, and chemicals continues to rise, but traditional preparation methods have long been limited by technological bottlenecks. Plant extraction methods rely on specific geographical and climatic conditions, making it difficult to meet large-scale supply needs; chemical synthesis methods face problems such as cumbersome steps, mixed isomers, and catalyst contamination, and the synthesis yield of most complex natural products is extremely low.

[0003] Microbial fermentation is considered an alternative, but traditional host strains have significant drawbacks: *Aspergillus niger* and *Penicillium* lack food-grade safety certifications for their compounds; *Saccharomyces cerevisiae* and *Escherichia coli* suffer from insufficient acetyl-CoA supply, weak intracellular storage capacity, and poor tolerance to hydrophobic products in the synthesis of lipid-soluble products. Against this backdrop, the unique advantages of *Yersinia lipolytica* are gradually emerging. *Yersinia lipolytica* is a non-traditional yeast strain listed as GRAS by the US FDA. It exhibits tolerance to high salt and acidic environments, high acetyl-CoA metabolic flux, and is naturally adapted to the synthetic pathways of lipids and terpenes. Furthermore, a mature molecular manipulation technology platform provides strong support for its further development and application, giving it broad prospects in the field of biotechnology.

[0004] Astaxanthin is a natural carotenoid with the chemical structure 3,3'-dihydroxy-β,β'-carotene-4,4'-diketone. Its molecule contains 11 conjugated double bonds, 2 hydroxyl groups, and 2 ketone functional groups. The conjugated double bonds endow it with strong antioxidant capabilities; its free radical scavenging ability is 550 times that of vitamin E and 10 times that of β-carotene. It can efficiently scavenge reactive oxygen species (ROS) such as singlet oxygen and hydroxyl radicals, reducing lipid peroxidation damage to cell membranes and other physiological effects. Therefore, it has broad development prospects in health products, pharmaceuticals, cosmetics, food additives, and aquaculture. In recent years, with the continuous development of synthetic biology, the use of metabolic engineering and genetic engineering techniques to modify *Yersinia lipolytica* yeast to synthesize astaxanthin has become a new research hotspot. However, insufficient precursor supply and the imbalance of cofactors during synthesis are key factors hindering the improvement of astaxanthin yield. Summary of the Invention

[0005] The purpose of this invention is to construct a complete astaxanthin synthesis pathway in Yersinia lipophila, enabling it to synthesize astaxanthin de novo, and to further improve the ability of recombinant bacteria to synthesize astaxanthin by enhancing the supply of precursor acetyl-CoA, reconstructing the IUP pathway, and simultaneously enhancing the precursor FPP. The present invention also provides the application of the above-mentioned recombinant Yersinia lipolyticis strain in the synthesis of the natural product astaxanthin.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: A recombinant Yersinia lipolytica strain producing astaxanthin was obtained by expressing, in a host strain, geranyl-geranyl diphosphate synthase CrtE, phytoene synthase / lycopene cyclase CrtYB, and phytoene desaturase CrtI from Pharbitis rubescens, 3-hydroxy-3-methylglutaryl-CoA reductase tHMGR from Saccharomyces cerevisiae, and β-carotene ketolase CrtW and β-carotene hydroxylase CrtZ from Haematococcus pluvialis. The host bacteria is *Yarrowia lipophila*. po1f .

[0007] As a preferred embodiment, the nucleotide sequences of the gerany-gerany diphosphate synthase CrtE, phytoene synthase / lycopene cyclase CrtYB, phytoene desaturase CrtI, 3-hydroxy-3-methylglutaryl-CoA reductase tHMGR, β-carotene ketolase CrtW, and β-carotene hydroxylase CrtZ are shown in SEQ ID NO: 1-6, respectively; the genes for CrtE, CrtYB, and CrtI are derived from *Phaefflera heliotropium* (Red Pharf yeast). Xanthophyllomyces denrorhous The tHMGR gene is derived from Saccharomyces cerevisiae (Saccharomyces cerevisiae). Saccharomyces cerevisiae The genes for CrtW and CrtZ are derived from Haematococcus pluvialis (…). Haematococcus pluvialis ).

[0008] A recombinant lipophilic yeast strain that enhances the supply of precursor acetyl-CoA and produces astaxanthin.

[0009] The astaxanthin-producing recombinant Yersinia lipolytica, which enhances the supply of precursor acetyl-CoA, was obtained by further expressing the ATP citrate lyase ACL and acetyl-CoA synthase ACS genes in the above-mentioned recombinant Yersinia lipolytica strain that produces astaxanthin.

[0010] The ATP citrate lyase ACL and acetyl-CoA synthase ACS were derived from Yersinia lipolytica. Yarrowia lipolytica The nucleotide sequences are shown in SEQ ID NO: 7-8.

[0011] A recombinant Pichia pastoris strain that produces astaxanthin via the isopentenol utilization pathway (IUP).

[0012] The astaxanthin-producing recombinant Pichia pastoris utilizing the isopentenol pathway was obtained by expressing choline kinase CK and inositol polyphosphokinase IPK in the aforementioned astaxanthin-producing recombinant Pichia pastoris host strain supplied with the enhanced precursor acetyl-CoA.

[0013] Through the action of two enzymes, CK and IPK, isopentenol can be converted into IPP in two steps, increasing the content of IPP, the precursor of astaxanthin synthesis.

[0014] As an optimized implementation, the choline kinase CK is derived from Saccharomyces cerevisiae, and the inositol polyphosphokinase IPK is derived from Arabidopsis thaliana. The nucleotide sequences of the choline kinase CK and inositol polyphosphokinase IPK are shown in SEQ ID NO: 9-10, respectively.

[0015] A recombinant lipophilic yeast strain that enhances the precursor FPP and produces astaxanthin.

[0016] The astaxanthin-producing recombinant Yersinia lipolytica, which enhances the precursor FPP, was obtained by expressing farnesyl diphosphate synthase ERG20 and isopentenyl pyrophosphate isomerase IDI in a host bacterium that enhances the supply of precursor acetyl-CoA and reconstructs the IUP pathway.

[0017] The nucleotide sequences of farnesyl diphosphate synthase ERG20 and isopentenyl pyrophosphate isomerase IDI are shown in SEQ ID NO: 11-12, respectively, and are derived from Yersinia lipolytica.

[0018] The present invention also provides a method for constructing the above-mentioned recombinant Yersinia lipophila strain.

[0019] Using BB3aK-AG as the vector plasmid, a co-expression recombinant plasmid of CrtI, CrtE, CrtYB, tHMGR, CrtW, and CrtZ was constructed. The recombinant plasmid was integrated into the intE site of the po1f genome to obtain the astaxanthin-producing strain PA. Using BB3rN-AC as the vector plasmid, a recombinant plasmid for co-expression of ACL and ACS was constructed. The recombinant plasmid was integrated into the intB site of the PA genome to obtain the recombinant Yersinia lipolytica strain P-AC with enhanced precursor acetyl-CoA. Using BB3rN-AE as the vector plasmid, a co-expression recombinant plasmid of ACL, ACS, IPK, and CK was constructed. The recombinant plasmid was integrated into the intB site of the PA genome to obtain the recombinant Yersinia lipophila strain P-ACI of the isopentenol utilization pathway.

[0020] Using BB3rN-AH as the vector plasmid, a co-expression recombinant plasmid of ACL, ACS, IPK, CK, ERG20, and IDI was constructed. The recombinant plasmid was integrated into the intB site of the PA genome to obtain the recombinant Yersinia lipophila strain P-ACIE of the isopentenol utilization pathway.

[0021] In a preferred embodiment, the fermentation culture uses glucose as the carbon source.

[0022] Application of the above-mentioned recombinant Yersinia lipophila strain in astaxanthin production.

[0023] The applications specifically include: The recombinant strain was taken out from the cryopreservation tube and inoculated into YPD test tubes at an inoculum of 1-10%, and cultured at 30 ℃ for 24 h to obtain seed culture; The seed culture was inoculated into the fermentation medium at an inoculation rate of 1-10%, and cultured at 30 °C with shaking for 7 days; the carbon source of the fermentation medium was glucose.

[0024] Fermentation medium formula: 10 g / L yeast extract, 20 g / L peptone, 20~60 g / L glucose.

[0025] Adding the photocatalyst Og-C3N4 / PEI (polyethyleneimine) to the fermentation medium can promote the synthesis of astaxanthin by Yersinia lipolyticis.

[0026] The photocatalyst Og-C3N4 / PEI (polyethyleneimine) was added at 24 h of fermentation, at a concentration of 10–80 mg / L; preferably 20–60 mg / L. LED light strips were used with a light intensity of 1500–2000 Lux, starting from 24 h of fermentation and continuing for 4 days.

[0027] After the addition of photocatalytic materials, the expression level of tHMGR, the rate-limiting enzyme in the astaxanthin synthesis pathway of recombinant Yersinia lipolytica, was significantly upregulated, and the transcription level of tHMGR was increased. At the same time, the level of intracellular NADPH was increased, which alleviated the NADH / NADPH imbalance caused by astaxanthin synthesis.

[0028] Beneficial effects:

[0029] The recombinant *Yersinia lipolytica* strain of this invention enables de novo synthesis of astaxanthin from glucose, achieving highly efficient synthesis of the natural product astaxanthin in *Yersinia lipolytica*. Furthermore, this invention enhances the precursor supply pathway and improves the astaxanthin production capacity of *Yersinia lipolytica* by constructing precursor acetyl-CoA synthesis pathways, IUP pathways, and RRG20 and IDI. Simultaneously, the photocatalyst Og-C3N4 / PEI is added to increase intracellular NADPH levels, thereby regulating intracellular cofactor balance. In addition, qRT-PCR verification was performed on the recombinant *Yersinia lipolytica* strain with added photocatalyst Og-C3N4 / PEI and the engineered strain without added photocatalyst. The addition of the photocatalyst significantly upregulated the expression level of the rate-limiting enzyme tHMGR in the astaxanthin synthesis pathway of the recombinant *Yersinia lipolytica*. Finally, the P-ACIE strain achieved an astaxanthin yield of 3.78 g / L and a content of 22.26 mg / g in a 5 L fermenter using glucose as the sole carbon source during continuous fed-batch fermentation, laying the foundation for further industrial-scale production. Attached Figure Description

[0030] Figure 1 The diagram shows the structure of plasmid BB3-A6-intE-IEYBtWZ, which carries the genimycin gene and is used as a selection marker for Yersinia lipophila.

[0031] Figure 2 The diagram shows the structure of plasmid BB3rN-AC-ACL-ACS, which carries the norsinoxin gene and is used as a selection marker for Yersinia lipophila.

[0032] Figure 3 The plasmid BB3rN-AE-ACL-ACS-IPK-CK is constructed, and the norsinoxin gene carried by this plasmid is used as a selection marker for Yersinia lipophila.

[0033] Figure 4 The plasmid BB3rN-AH-ACL-ACS-IPK-CK-ERG20-IDI is constructed, and the norsinoxin gene carried by this plasmid is used as a selection marker for Yersinia lipophila.

[0034] Figure 5 This is a graph showing the yield of astaxanthin synthesized by the engineered strain.

[0035] Figure 6 The effect of different concentrations of Og-C3NA / PEI on the production of astaxanthin by engineered bacteria.

[0036] Figure 7 Validation analysis of key genes in the metabolic pathway of recombinant Yersinia lipolytica with or without the addition of photocatalytic material Og-C3N4 / PEI.

[0037] Figure 8The intracellular NADPH content of recombinant Yersinia lipolytica was determined with or without the addition of the photocatalytic material Og-C3N4 / PEI.

[0038] Figure 9 This is a diagram showing the fermentation of engineered strain P-ACI in a 5 L fermenter using glucose as the sole carbon source. Detailed Implementation

[0039] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0040] The original strain used in the examples was *Yarrowia lipolytica* po1f. *Yarrowia lipolytica* po1f is commercially available.

[0041] Preparation method of photocatalytic material Og-C3N4 / PEI: Preparation of Og-C3N4: A mixture of melamine (7.2 g), H2O2 (5 mL), and deionized water (35 mL) was ultrasonically treated for 10 min at room temperature. The mixture was then sealed in a 100 mL hydrothermal synthesis reactor lined with polytetrafluoroethylene and maintained at 140 °C for 12 h. After cooling to room temperature, the resulting white crystalline solid was washed several times with distilled water and anhydrous ethanol and dried at 60 °C for 12 h. The resulting solid (3.0 g) was calcined in air at 600 °C for 2 h in a tube furnace at a heating rate of 0.5 °C / min, and finally naturally cooled to room temperature to obtain oxygen-doped g-C3N4 powder (denoted as Og-C3N4).

[0042] Preparation of Og-C3N4 / PEI: First, a diluted PEI solution was prepared by adding 0.4 g of PEI stock solution to 10 mL of ultrapure water. 0.1 g of the prepared Og-C3N4 powder was weighed and added to 200 mL of ultrapure water and sonicated for 30 min. The diluted PEI solution was then added dropwise to the Og-C3N4 solution while stirring until the mixed suspension was formed, followed by stirring for 12 h. The suspension was then filtered through a 0.2 μm cellulose acetate membrane, and unabsorbed PEI was washed with pure water until the pH of the filtrate reached approximately 7.0. After drying at 60 °C for 18 h in a drying oven, the sample was ground into a fine powder, ultimately preparing Og-C3N4 / PEI.

[0043] In this embodiment, the extraction and quantitative analysis steps of astaxanthin are as follows: 1. Extraction of astaxanthin (1) Take 1 mL of the mixed fermentation broth and centrifuge at 12000 rpm for 5 min (wash twice with pure water).

[0044] (2) After draining the water, resuspend the contents in 2 mL of dimethyl sulfoxide (DMSO) (preheated at 60℃) and shake evenly on a vortex mixer. Then place the container in a 55℃ water bath for 15 min.

[0045] (3) Add 4 mL of anhydrous ethanol.

[0046] (4) Centrifuge the sample at 12,000 rpm for 5 min. Transfer the supernatant to a new centrifuge tube and store it away from light.

[0047] 2. Quantitative analysis of astaxanthin: High performance liquid chromatography was used to detect the concentration of astaxanthin.

[0048] The liquid chromatograph used in this study was an Agilent Technologies 1200 Infinity series; the column was an Acclaim™ 120 C30 column; the UV absorption wavelength was 450 nm; the mobile phase was methanol and methyl tert-butyl ether; the flow rate was controlled at 1.0 mL / min; and the column temperature was 25℃.

[0049] Example 1: Amplification of Gene Elements and Preparation of Target Plasmids According to information provided by NCBI X.denrorhous Geraniol geraniol diphosphate synthase encoding gene crtE The nucleotide sequence of the gene encoding phytoene synthase / lycopene cyclase. crtYB The nucleotide sequence of phytoene desaturase and its encoding gene crtI The nucleotide sequence was optimized by codons of Yersinia lipolytica to obtain gene sequences as shown in SEQ ID No: 1, SEQ ID No: 2, and SEQ ID No: 3. The optimized gene codons made the exogenous gene more compatible with the Yersinia lipolytica chassis.

[0050] Based on the coding gene sequence of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase tHMGR from *Saccharomyces cerevisiae* provided on NCBI, PCR amplification was performed using the *Saccharomyces cerevisiae* genome as a template. The gene sequence of tHMGR is shown in SEQ ID No: 4. H. pluvialis The nucleotide sequences of β-carotene ketolase CrtW and β-carotene hydroxylase CrtZ were obtained by codon optimization, resulting in gene sequences as shown in SEQ ID No: 5 and SEQ ID No: 6. The optimized gene codons improved the compatibility of the exogenous gene with the chassis of Yersinia lipolytica.

[0051] Based on the encoding gene sequences of ATP-citrate lyase and acetyl-CoA synthase from *Saccharomyces lipolyticus* provided on NCBI, PCR amplification was performed using the *Saccharomyces cerevisiae* genome as a template. The gene sequences are shown in SEQ ID No: 7-8.

[0052] Based on the coding gene sequence of choline kinase from Saccharomyces cerevisiae provided on NCBI, PCR amplification was performed using the Saccharomyces cerevisiae genome as a template. The gene sequence is shown in SEQ ID No: 9. Inositol polyphosphokinase from Arabidopsis thaliana was synthesized by Genscript Biotech Co., Ltd., and codon optimization was performed. The gene sequence is shown in SEQ ID No: 10.

[0053] Based on the coding gene sequences of farnesyl diphosphate synthase and isopentenyl pyrophosphate isomerase from Yersinia lipolytica provided on NCBI, PCR amplification was performed using the Yersinia lipolytica genome as a template. The gene sequences are shown in SEQ ID No: 11-12.

[0054] (II) Construction of recombinant plasmids 1. Using BB1-23 (Plasmid #98496) as the vector plasmid, construct recombinant plasmids containing CrtI, CrtE, CrtYB, tHMGR, CrtW, CrtZ, ACL, ACS, IPK, CK, ERG20, and IDI.

[0055] The gene sequences of CrtI, CrtE, CrtYB, CrtW, CrtZ, and IPK were synthesized by GenScript Biotech Co., Ltd. Recombinant plasmids BB1-23-ACL and BB1-23-ACS were amplified by PCR using the *Yersinia lipolytica* genome as a template to obtain the ACL and ACS gene sequences. BB1-23-tHMGE and BB1-23-CK were amplified by PCR using the *Saccharomyces cerevisiae* genome as a template to obtain the tHMGE and CK gene sequences. BB1-23-ERG20 and BB1-23-IDI were amplified by PCR using the *Yersinia lipolytica* genome as a template to obtain the ERG20 and IDI gene sequences. The PCR amplification system is shown in Table 1, and the primers are shown in Table 2.

[0056] Table 1

[0057] PCR amplification conditions:

[0058] Table 2:

[0059] 2. Construct expression cassettes for CrtI, CrtE, CrtYB, tHMGR, CrtW, CrtZ, ACL, ACS, IPK, CK, ERG20, and IDI. The recombinant plasmid BB2-AB-pGPM1-CrtI-ScCYC1tt was created by inserting plasmid BB1-23-CrtI, plasmid BB1-12-pGPM1, and plasmid BB1-34-ScCYC1tt into plasmid BB2-AB using the GoldenGate method with Bpi1 enzyme and T4 ligase. The recombinant plasmid BB2-BC-pPDC1-CrtE-RPP1Btt was created by inserting plasmid BB1-23-CrtE, plasmid BB1-12-pPDC1, and plasmid BB1-34-R into plasmid BB2-AB. PP1Btt was inserted into plasmid BB2-BC using the Goldengate method with Bpi1 enzyme and T4 ligase to obtain plasmid BB2-BC-pPDC1-CrtE-RPP1Btt; the recombinant plasmid BB2-CD-pMDH3-CrtYB-RPS2tt was obtained by inserting plasmid BB1-23-CrtYB, plasmid BB1-12-pMDH3, and plasmid BB1-34-RPS2tt into plasmid BB2-CD using the Goldengate method with Bpi1 enzyme and T4 ligase to obtain plasmid BB2-CD-pMDH3-CrtYB-RPS2tt. t; Recombinant plasmid BB2-DE-pADH2-tHMGR-RPL2Att is obtained by inserting plasmid BB1-23-tHMGR, plasmid BB1-12-pADH2, and plasmid BB1-34-RPL2Att into plasmid BB2-DE using the GoldenGate method with Bpi1 enzyme and T4 ligase. Recombinant plasmid BB2-EF-pTEF1-CrtW-IDP1tt is obtained by inserting plasmid BB1-23-CrtW, plasmid BB1-12-pTEF1, and plasmid BB1-34-RPL2Att into plasmid BB2-DE using the GoldenGate method with Bpi1 enzyme and T4 ligase. IDP1tt was inserted into plasmid BB2-EF using the GoldenGate method with Bpi1 enzyme and T4 ligase to obtain plasmid BB2-EF-pTEF1-CrtW-IDP1tt; the recombinant plasmid BB2-FG-pGAP-CrtZ-RPS25Att was obtained by inserting plasmid BB1-23-CrtZ, plasmid BB1-12-pGAP, and plasmid BB1-34-RPS25Att into plasmid BB2-AB using the GoldenGate method with Bpi1 enzyme and T4 ligase.The recombinant plasmid BB2-AB-pGAP-ACL-RPS25Att was obtained by inserting plasmids BB1-23-ACL, BB1-12-pGAP, and BB1-34-RPS25Att into plasmid BB2-AB using the GoldenGate method with Bpi1 enzyme and T4 ligase. The recombinant plasmid BB2-BC-pMDH3-ACS-RPS2tt was obtained by inserting plasmids BB1-23-ACS, BB1-12-pMDH3, and BB1-34-RPS2tt into plasmid BB2-AB. The plasmid BB2-BC-pMDH3-ACS-RPS2tt was obtained by inserting plasmid BB1-23-IPK, plasmid BB1-12-pADH2, and plasmid BB1-34-RPS2tt into plasmid BB2-CD using the GoldenGate method with Bpi1 enzyme and T4 ligase. The recombinant plasmid BB2-CD-pADH2-IPK-RPS2tt was obtained by inserting plasmid BB1-23-IPK, plasmid BB1-12-pADH2, and plasmid BB1-34-RPS2tt into plasmid BB2-CD using the GoldenGate method with Bpi1 enzyme and T4 ligase. 2-DE-pGPM1-CK-RPL2Att is obtained by inserting plasmid BB1-23-CK, plasmid BB1-12-pGPM1, and plasmid BB1-34-RPL2Att into plasmid BB2-DE using the GoldenGate method with Bpi1 enzyme and T4 ligase. The recombinant plasmid BB2-AB-pGPM1-ERG20-ScCYC1tt is obtained by inserting plasmid BB1-23-ERG20, plasmid BB1-12-pGPM1, and plasmid BB1-34-ScCYC1tt into plasmid BB2-DE. Plasmid BB2-AB-pGPM1-ERG20-ScCYC1tt was obtained by inserting plasmid BB2-AB into plasmid BB2-AB using the GoldenGate method with Bpi1 enzyme and T4 ligase. Recombinant plasmid BB2-BC-pPDC1-IDI-RPP1Btt was obtained by inserting plasmid BB1-23-IDI, plasmid BB1-12-pPDC1, and plasmid BB1-34-RPP1Btt into plasmid BB2-BC using the GoldenGate method with Bpi1 enzyme and T4 ligase.

[0060] The construction process of the recombinant plasmid BB2-AB-pGPM1-CrtI-ScCYC1tt is as follows: GoldenGate was assembled using Bpi1 enzyme and T4 ligase from Nanjing Fomax Biotechnology Co., Ltd., and the reaction system is shown in Table 3.

[0061] Table 3 BB2-AB 1 µL BB1-12-pGPM1 1 µL BB1-23-CrtI 1 µL BB1-34-ScCYC1tt 1 µL Bpi1 0.5 µL BSA 1 µL T4 ligase 0.5 µL T4buffer 1 µL distilled water 3 µL The circular recombinant vector was transformed into Escherichia coli DH5α competent cells. The positive recombinant plasmid BB2-AB-pGPM1-CrtI-ScCYC1tt was obtained by screening for ampicillin resistance on plates and verifying by colony PCR and sequencing.

[0062] The construction process for other recombinant plasmids is the same as described above.

[0063] 3. The construction process of recombinant plasmid BB3-A6-intE-IEYBtWZ is as follows: The recombinant plasmid BB3-A6-intE-IEYBtWZ was constructed by inserting plasmids BB2-AB-pGPM1-CrtI-ScCYC1tt, BB2-BC-pPDC1-CrtE-RPP1Btt, BB2-CD-pMDH3-CrtYB-RPS2tt, BB2-DE-pADH2-tHMGR-RPL2Att, BB2-EF-pTEF1-CrtW-IDP1tt, and BB2-FG-pGAP-CrtZ-RPS25Att into plasmid BB3-A6-intB using the GoldenGate method with Bsa1 enzyme and T4 ligase. Figure 1 .

[0064] 4. The construction process of recombinant plasmid BB3rN-AC-ACS-ACL is as follows: The recombinant plasmids BB2-AB-pGAP-ACL-RPS25Att and BB2-BC-pMDH3-ACS-RPS2tt were inserted into plasmid BB3rN-AC using the GoldenGate method with Bsa1 enzyme and T4 ligase to obtain the recombinant plasmid BB3rN-AC-ACL-ACS. Figure 2 .

[0065] 5. The construction process of recombinant plasmid BB3rN-AE-ACS-ACL-IPK-CK is as follows: The recombinant plasmids BB2-AB-pGAP-ACL-RPS25Att, BB2-BC-pMDH3-ACS-RPS2tt, BB2-CD-pADH2-IPK-RPS2tt, and BB2-DE-pGPM1-CK-RPL2Att were inserted into plasmid BB3rN-AE using the GoldenGate method with Bsa1 enzyme and T4 ligase to obtain the recombinant plasmid BB3rN-AE-ACS-ACL-IPK-CK. The structure of the recombinant plasmid BB3rN-AE-ACS-ACL-IPK-CK is shown below. Figure 3 .

[0066] 6. The construction process of recombinant BB3rN-AH-ACS-ACL-IPK-CK-ERG20-IDI is as follows: The recombinant plasmids BB2-AB-pGAP-ACL-RPS25Att, BB2-BC-pMDH3-ACS-RPS2tt, BB2-CD-pADH2-IPK-RPS2tt, BB2-DE-pGPM1-CK-RPL2Att, BB2-AB-pGPM1-ERG20-ScCYC1tt, and BB2-BC-pPDC1-IDI-RPP1Btt were inserted into plasmid BB3rN-AH using the GoldenGate method with Bsa1 enzyme and T4 ligase to obtain the recombinant plasmid BB3rN-AE-ACS-ACL-IPK-CK-ERG20-IDI. The structure of the recombinant plasmid BB3rN-AH-ACS-ACL-IPK-CK-ERG20-IDI is shown below. Figure 4 .

[0067] Example 2 Construction of recombinant bacteria 1. Construction of recombinant strain PA The plasmid BB3-A6-intE-IEYBtWZ containing the CrtI-CrtE-CrtYB-tHMGR-CrtW-CrtZ gene expression cassette was introduced into Yersinia lipolyticis po1f. The CrtI-CrtE-CrtYB-tHMGR-CrtW-CrtZ expression cassette was integrated into the intE site of the genome, resulting in recombinant strain PA.

[0068] The specific method is as follows: ① Competent cells were prepared by overnight culture of the original Yersinia lipolytica in YPD liquid medium (containing 2% peptone, 1% yeast extract and 2% glucose).

[0069] ② Linearized BB3-A6-intE-IEYBtWZ was introduced into competent cells of Yeast lipolyticis using the Zymogen Frozen EZYeast Transformation Kit II from Zymo Research Corporation for homologous recombination.

[0070] ③ Screening was performed using antibiotic plates on a selection medium. Single colonies grew in 3-4 days. Positive clones that were correctly identified by PCR were named recombinant bacteria PA.

[0071] 2. Construction of recombinant bacteria P-AC, P-ACI, and P-ACIE Plasmids containing the gene expression cassettes ACS, ACL, ACS, ACL, IPK, CK, ACS, ACL, IPK, CK, ERG20, and IDI, such as BB3rN-AE-ACS-ACL, BB3rN-AE-ACS-ACL-IPK-CK, or BB3rN-AH-ACS-ACL-IPK-CK-ERG20-IDI, were introduced into the intB site of the genome of *Yarrowia lipolyticis* PA ​​to obtain recombinant strains P-AC, P-ACI, and P-ACIE. The specific construction method is the same as that for PA.

[0072] Example 3: Application of recombinant bacteria in astaxanthin production The engineered bacteria were cultured using the recombinant bacteria PA, P-AC, P-ACI, and P-ACIE from Example 2 to produce astaxanthin.

[0073] The specific method is as follows: Take the strain from the seed preservation tube, inoculate it into the YPD test tube with a 1% inoculation amount, and culture it at 30℃ for 24 hours to obtain the seed liquid; The seed culture was inoculated at a rate of 1% into 50 mL of fermentation medium (40 g / L glucose, 10 g / L yeast extract, and 20 g / L tryptone). The culture was incubated at 25℃ and 220 rpm with shaking for 5 days. 4 mL of 40 g / L glucose was added every 24 h. After 120 h of fermentation, the astaxanthin yields of recombinant strains PA, P-AC, P-ACI, and P-ACIE were 135.07 mg / L, 166.62 mg / L, 197.39 mg / L, and 227.14 mg / L, respectively, with astaxanthin contents of 8.22 mg / g, 10.51 mg / g, 13.99 mg / g, and 16.08 mg / g, respectively. Figure 5Increasing the supply of acetyl-CoA, simultaneously increasing the supply of acetyl-CoA and IPP, and simultaneously increasing the supply of acetyl-CoA, IPP, and FPP all resulted in astaxanthin yield increases of 23.36%, 46.14%, and 68.16%, respectively, compared to the starting strain. This indicates that increasing precursor supply, whether through increasing acetyl-CoA, artificially constructing new IUP pathways to increase IPP synthesis, or increasing the translocation of IIPP to FPP, are effective methods to increase astaxanthin yield.

[0074] Example 4: Effect of photocatalytic material Og-C3N4 / PEI on the synthesis of astaxanthin by engineered strain Yersinia lipolyticis To verify the effect of different concentrations of the photocatalyst Og-C3N4 / PEI on the production of astaxanthin by the engineered strain P-ACIE, we added Og-C3N4 / PEI at concentrations of 10 mg / L, 20 mg / L, 40 mg / L, 60 mg / L, and 80 mg / L at 20 h of fermentation, and cultured the engineered strain P-ACIE under the following light conditions: LED light strip, light intensity of 1500-2000 Lux, starting from 24 h of fermentation, for 4 days. The astaxanthin yields were 241.40 mg / L, 257.95 mg / L, 258.26 mg / L, 264.96 mg / L, and 245.15 mg / L, respectively. Figure 6 Among them, the 60 mg / L Og-C3N4 / PEI strain achieved the highest astaxanthin yield, which was 16.65% higher than that of the strain without added material. This indicates that adding photocatalytic material to the culture medium under light conditions can promote the level of intracellular NADPH, which can help alleviate the cofactor imbalance caused by astaxanthin production.

[0075] Example 5: Effect of photocatalytic material Og-C3N4 / PEI on the astaxanthin synthesis pathway of engineered strain Yersinia lipolyticis To investigate the effect of adding the photocatalyst Og-C3N4 / PEI on the synthesis of astaxanthin by the engineered strain Yersinia lipolyticis, the gene expression level of the recombinant strain P-ACIE with and without the addition of the photocatalyst Og-C3N4 / PEI was verified by quantitative real-time PCR (qRT-PCR).

[0076] The specific method is as follows: The engineered strain P-ACIE was cultured with 60 mg / L of the photocatalyst Og-C3N4 / PEI and without the photocatalyst Og-C3N4 / PEI for 24 h of fermentation. LED light strips were used for illumination at an intensity of 1500-2000 Lux from the 24 h of fermentation onwards, continuing for 4 days. After 120 h, samples were collected and immediately frozen in liquid nitrogen. mRNA with polyA structures was enriched from total RNA using oligo(dT) magnetic beads, and RNA fragments were broken down to approximately 300 bp. Using RNA as a template, the first strand of cDNA was synthesized using 6-base random primers and reverse transcriptase. Double-stranded cDNA was then synthesized using the first-strand cDNA as a template. After library construction, fragment enrichment was performed by PCR amplification, followed by library screening based on fragment size.

[0077] The transcriptome was then validated by qRT-PCR to ensure the reliability of the transcriptome analysis. Key genes in the astaxanthin synthesis pathway of recombinant strains with and without the addition of the photocatalytic material Og-C3N4 / PEI were investigated. crtI , crtE , crtYB , tHMGR , crtW, crtZ, ACS, ACL, IPK, CK, ERG20, IDI Perform qRT-PCR validation ( Figure 7 In addition, the intracellular NADPH content of recombinant Yersinia lipolytica with and without the photocatalyst Og-C3N4 / PEI was also measured. Figure 8 The addition of the photocatalyst Og-C3N4 / PEI significantly increased the transcriptional level of tHMGR, the rate-limiting enzyme in astaxanthin synthesis. We hypothesize that the photocatalyst can increase the supply of the cofactor NADPH under light conditions, and since tHMGR requires one molecule of NADPH for expression, this may be the reason why the photocatalyst promotes the increased transcriptional level of tHMGR. Simultaneously, it increases the intracellular NADPH level, alleviating the NADH / NADPH imbalance caused by astaxanthin synthesis.

[0078] Example 6: High-density fermentation production of astaxanthin using recombinant strain P-ACIE ① Seed culture: a. Primary seed culture: Take 1% of the recombinant strain P-ACIE bacterial culture from the cryopreservation tube and inoculate it into YPD test tubes. Incubate at 30°C and 200 rpm for 24 hours to obtain the primary seed culture. The YPD medium contains 2% peptone, 1% yeast extract and 2% glucose. b. Secondary seed culture: Take the primary seed culture and inoculate it into a new seed culture medium at an inoculation rate of 10%, and culture it at a constant temperature under the same conditions as a to obtain the seed culture for fermentation culture.

[0079] ② Batch feeding fermentation The seed culture obtained from seed culture was inoculated into a 5 L fermenter containing fermentation medium (40 g / L glucose, 20 g / L peptone, 10 g / L yeast extract). Recombinant strain P-ACIE underwent fed-batch fermentation in the 5 L fermenter at 30℃, 500 rpm, and dissolved oxygen maintained at 40%. At 24 h of fermentation, 60 mg / L of Og-C3N4 / PEI was added, and continuous LED illumination was provided at an intensity of 1500-2000 Lux. The initial glucose concentration in the fermenter was 40 g / L. When the glucose was nearly depleted, glucose was added continuously to maintain the glucose concentration below 5 g / L. Samples were taken every 12 h to determine cell dry weight, and astaxanthin was extracted from the fermentation broth to determine its content. After 7 days of fermentation, the cell OD... 600 The concentration reached 284.16, and the astaxanthin yield reached 3.78 g / L, with a content of 22.26 mg / g. Figure 9 ).

[0080] This invention successfully achieved de novo astaxanthin synthesis by constructing a complete astaxanthin synthesis pathway in *Yersinia lipolytica*. Furthermore, by increasing the supply of the precursor acetyl-CoA, constructing an artificial IUP pathway in *Yersinia lipolytica*, and increasing the copy numbers of ERG20 and IDI, as well as increasing the supply of intracellular IPP and FPP, astaxanthin yield was successfully increased by 68.16%. Adding the photocatalyst Og-C3N4 / PEI alleviated the imbalance in intracellular cofactor supply, significantly increasing the expression level of the rate-limiting enzyme tHMGR in the MVA pathway, resulting in a 16.65% increase in astaxanthin yield. Finally, continuous fed-batch fermentation was conducted in a 5 L fermenter, and the production capacity of the strain was comprehensively evaluated. The OD of the recombinant strain in the fermenter was measured. 600 The monitoring of astaxanthin production is expected to further increase astaxanthin yield in larger fermentation tanks, laying the foundation for subsequent industrialization.

Claims

1. A recombinant lipophilic yeast strain producing astaxanthin, characterized in that, Yeast lipophila ( Yarrowia lipolytica The expression cassette containing geraniol geraniol diphosphate synthase CrtE, phytopene synthase / lycopene cyclase CrtYB, phytopene desaturase CrtI, 3-hydroxy-3-methylglutaryl-CoA reductase tHMGR, β-carotene ketolase CrtW, β-carotene hydroxylase CrtZ, ATP citrate lyase ACL, acetyl-CoA synthase ACS, choline kinase CK, inositol polyphosphate kinase IPK, farnesyl diphosphate synthase ERG20, and isopentenyl pyrophosphate isomerase IDI was obtained. The *Yersinia lipolytica* strain is *Yersinia lipolytica* po1f. The strain was constructed as follows: using BB3aK-AG as the vector plasmid, a co-expression recombinant plasmid of CrtI, CrtE, CrtYB, tHMGR, CrtW, and CrtZ was constructed, and the recombinant plasmid was integrated into the intE site of the po1f genome to obtain the astaxanthin-producing strain PA. Using BB3rN-AH as the vector plasmid, a co-expression recombinant plasmid of ACL, ACS, IPK, CK, ERG20, and IDI was constructed. The recombinant plasmid was integrated into the intB site of the PA genome to obtain the recombinant Yersinia lipophila strain P-ACIE.

2. The recombinant Yersinia lipolyticis strain according to claim 1, characterized in that, The coding gene sequences of CrtE, CrtYB, CrtI, tHMGR, CrtW, and CrtZ are shown in SEQ ID No: 1-6, respectively; wherein, the genes of CrtE, CrtYB, and CrtI are derived from Pharfia redis yeast (… Xanthophyllomyces denrorhous The tHMGR gene is derived from Saccharomyces cerevisiae (Saccharomyces cerevi Saccharomyces cerevisiae The genes for CrtW and CrtZ are derived from Haematococcus pluvialis (…). Haematococcus pluvialis ).

3. The recombinant Yersinia lipolyticis strain according to claim 1, characterized in that, The recombinant *Yersinia lipolytica* strain enhances the supply of precursor acetyl-CoA by expressing ATP citrate lyase ACL and acetyl-CoA synthase ACS in the host culture. These ATP citrate lyase ACL and acetyl-CoA synthase ACS are derived from *Yersinia lipolytica*. Yarrowia lipolytica The gene sequence is shown in SEQ ID No: 7-8.

4. The recombinant Yersinia lipolyticis strain according to claim 1, characterized in that, The recombinant Yersinia lipolytica strain enhances the isopentenol utilization pathway by expressing choline kinase CK and inositol polyphosphokinase IPK in the host bacteria. Choline kinase CK is derived from Saccharomyces cerevisiae, and the encoding gene is shown in SEQ ID No: 10; inositol polyphosphokinase IPK is derived from Arabidopsis thaliana, and the encoding gene is shown in SEQ ID No:

9.

5. The recombinant Yersinia lipolyticis strain according to claim 1, characterized in that, The recombinant Yersinia lipolytica strain enhances the supply of precursor FPP by expressing farnesyl diphosphate synthase ERG20 and isopentenyl pyrophosphate isomerase IDI in the host strain. Farnesyl diphosphate synthase ERG20 and isopentenyl pyrophosphate isomerase IDI are derived from Yersinia lipolytica, and the encoding genes are shown in SEQ ID No: 11-12.

6. The use of the recombinant Yersinia lipolyticis strain according to any one of claims 1-5 in the fermentation production of astaxanthin.

7. The application according to claim 6, characterized in that, The recombinant Yersinia lipolytica strain was inoculated into a fermentation medium and cultured to synthesize astaxanthin.

8. The application according to claim 7, characterized in that, The fermentation medium contains the photocatalytic material Og-C3N4 / PEI.

9. The application according to claim 8, characterized in that, The amount of the photocatalyst material Og-C3N4 / PEI added is 10~80 mg / L.

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