Recombinant yarrowia lipolytica for co-production of microbial oil and astaxanthin as well as construction method and application of recombinant yarrowia lipolytica
By constructing an astaxanthin synthesis pathway and expressing acetyl-CoA carboxylase in *Yersinia lipolytica*, and optimizing fermentation conditions, the problem of low astaxanthin yield in recombinant *Yersinia lipolytica* fermentation was solved, achieving efficient co-production of microbial oil and astaxanthin, laying the foundation for industrial application.
Patent Information
- Application Number
- CN202511731805.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-11-24
- Publication Date
- 2026-01-16
AI Technical Summary
The yield of astaxanthin produced by recombinant Yersinia lipolyticis fermentation in existing technologies is low and far from industrial application.
Recombinant Yersinia lipolyticis was constructed to express geraniol-geraniol synthase CrtE, phytoene synthase/lycopene cyclase CrtYB, and phytoene desaturase CrtI from Paffirula rubra, β-carotene ketolase CrtW and β-carotene hydroxylase CrtZ from Haematococcus pluvialis, and acetyl-CoA carboxylase from Saccharomyces cerevisiae, Pichia pastoris, or Cryptococcus neoformans. Fermentation conditions such as nitrogen source, metal ions, and temperature were optimized, and molasses, an inexpensive raw material, was used for fermentation.
The yield of microbial oil and astaxanthin was significantly increased. In 50 mL culture medium, the yield of microbial oil reached 65.92% and the yield of astaxanthin reached 299.31 mg/L. In a 5 L fermenter, the yield of microbial oil reached 80.14% and the yield of astaxanthin reached 3.09 g/L, laying the foundation for industrialization.
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Abstract
Description
Technical Field
[0001] This invention relates to a recombinant lipophilic yeast that co-produces microbial oil and astaxanthin, its construction method, and its application, belonging to the field of genetic engineering technology. Background Technology
[0002] Yarrowia lipolytica ( Yarrowialipolytica Yersinia lipolytica possesses advantages such as broad substrate utilization, high lipid synthesis efficiency, strong environmental tolerance, and comprehensive genetic modification tools. It can directly metabolize fatty acids, vegetable oils, and even industrial waste oils, converting them into lipids without complex pretreatment, thus realizing an economic model of "oil production from waste." Besides conventional sugars like glucose and sucrose, it can also utilize pentose sugars such as xylose and arabinose, as well as waste glycerol, demonstrating high substrate flexibility and independence from grain-based carbon sources. Furthermore, under optimized conditions, the lipid content of Yersinia lipolytica can reach 30%-70% of its cell dry weight, with some engineered strains exceeding 80%. Natural Yersinia lipolytica primarily accumulates oleic acid, an ideal component for biodiesel; through metabolic engineering, it can also directionally synthesize functional unsaturated fatty acids such as DHA, EPA, and arachidonic acid. In addition, Yersinia lipolytica exhibits strong environmental adaptability, tolerating the high osmotic pressure, high product concentration, and extreme pH conditions encountered during industrial fermentation, reducing the risk of fermentation failure and ensuring continuous production. Currently, the whole genome of Yersinia lipophila has been sequenced and mature genetic manipulation tools are available, enabling precise regulation of metabolic networks through metabolic engineering.
[0003] Astaxanthin is a fat-soluble pigment, and *Yersinia lipolytica*'s high lipid accumulation capacity serves as its natural "storage unit." Utilizing *Yersinia lipolytica*'s comprehensive genetic manipulation tools, metabolic engineering strategies can achieve the synergistic accumulation of lipids and astaxanthin. Furthermore, lipid synthesis and astaxanthin synthesis share the same precursor, acetyl-CoA, and metabolic regulation can simultaneously increase the production of both. In addition, compared to other naturally synthesized astaxanthin microorganisms (such as *Haematococcus pluvialis*), *Yersinia lipolytica* has advantages such as rapid growth, no need for light, and tolerance to lower pH levels.
[0004] However, the yield of astaxanthin produced by recombinant Yersinia lipolyticis fermentation in existing technologies is still low, and there is a significant gap compared to industrialization. Summary of the Invention
[0005] To address the shortcomings of existing technologies, this invention provides a recombinant Yersinia lipolytica yeast that co-produces microbial oil and astaxanthin, its construction method, and its application, thereby increasing the yield of microbial oil and astaxanthin from the recombinant Yersinia lipolytica yeast.
[0006] To solve the above-mentioned technical problems, the present invention adopts the following technical solution: A recombinant Yersinia lipolytica that co-produces microbial oil and astaxanthin, wherein the recombinant Yersinia lipolytica expresses geraniol-geraniol diphosphate synthase CrtE, phytoene synthase / lycopene cyclase CrtYB, and phytoene desaturase CrtI derived from Pharfia redis; 3-hydroxy-3-methylglutaryl-CoA reductase tHMGR derived from Saccharomyces cerevisiae; and β-carotene ketolase CrtW and β-carotene hydroxylase CrtZ derived from Haematococcus pluvialis.
[0007] The nucleotide sequence of CrtE is shown in SEQ ID No: 1 in the sequence listing; the nucleotide sequence of CrtYB is shown in SEQ ID No: 2 in the sequence listing; the nucleotide sequence of CrtI is shown in SEQ ID No: 3 in the sequence listing; the nucleotide sequence of tHMGR is shown in SEQ ID No: 4 in the sequence listing; the nucleotide sequence of CrtW is shown in SEQ ID No: 5 in the sequence listing; and the nucleotide sequence of CrtZ is shown in SEQ ID No: 6 in the sequence listing.
[0008] The recombinant Yersinia lipolytica expressed acetyl-CoA carboxylase derived from Saccharomyces cerevisiae, Pichia pastoris, or Cryptococcus neoformans.
[0009] The nucleotide sequence of the acetyl-CoA carboxylase derived from *Saccharomyces cerevisiae* is shown in NCBI Gene ID: 855750; the nucleotide sequence of the acetyl-CoA carboxylase derived from *Pichia pastoris* is shown in NCBI Gene ID: 8196923; and the nucleotide sequence of the acetyl-CoA carboxylase derived from *Cryptococcus cryptococcosus* is shown in SEQ ID No: 7 in the sequence listing.
[0010] The original strain of the recombinant Yersinia lipolytica was Yersinia lipolytica po1f.
[0011] A method for constructing a recombinant *Yersinia lipophila* strain that co-produces microbial oil and astaxanthin includes: using BB3-A6-intB as a vector plasmid, constructing a recombinant plasmid containing CrtI, CrtE, CrtYB, tHMGR, CrtW, and CrtZ; integrating the recombinant plasmid into the intB site of the po1f genome to obtain the astaxanthin-producing *Yersinia lipophila* strain Yl-A; using BB3-intE1 as a vector, constructing recombinant plasmids containing acetyl-CoA carboxylase from *Saccharomyces cerevisiae*, *Pichia pastoris*, and *Cryptococcus cryptococcosus*, respectively, and introducing them into the intE1 site of the Yl-A genome to obtain the recombinant *Yersinia lipophila* strain.
[0012] The nucleotide sequence of CrtE is shown in SEQ ID No: 1 in the sequence listing; the nucleotide sequence of CrtYB is shown in SEQ ID No: 2 in the sequence listing; the nucleotide sequence of CrtI is shown in SEQ ID No: 3 in the sequence listing; the nucleotide sequence of tHMGR is shown in SEQ ID No: 4 in the sequence listing; the nucleotide sequence of CrtW is shown in SEQ ID No: 5 in the sequence listing; and the nucleotide sequence of CrtZ is shown in SEQ ID No: 6 in the sequence listing. The nucleotide sequence of the acetyl-CoA carboxylase of *Saccharomyces cerevisiae* is shown in NCBI Gene ID: 855750; the nucleotide sequence of the acetyl-CoA carboxylase of *Pichia pastoris* is shown in NCBI Gene ID: 8196923; and the nucleotide sequence of the acetyl-CoA carboxylase of *Cryptococcus cryptococcosus* is shown in SEQ ID No: 7 in the sequence listing.
[0013] The application of the recombinant Yersinia lipophila in the co-production of microbial oils and astaxanthin.
[0014] The fermentation medium for recombinant *Yarrowia lipolytica* consisted of: 38-42 g / L glucose, 0.48-0.52 g / L MgSO4·7H2O, 7.4-7.6 g / L compound nitrogen source, 11.8-12.0 g / L KH2PO4 and 2.7-2.9 g / L K2HPO4·3H2O, and 0.8-1.2 mmol / L zinc chloride. The compound nitrogen source was (NH4)2SO4 and yeast extract in a mass ratio of 1:1. The fermentation temperature for recombinant *Yarrowia lipolytica* was controlled as follows: the fermentation temperature was 30℃ for the first 48 hours after the start of fermentation, and 25℃ after 48 hours.
[0015] The fermentation medium for recombinant Yersinia lipolytica was molasses; dissolved oxygen was controlled at 38-42% during fermentation; the fermentation temperature was controlled as follows: 30℃ for the first 48 hours after fermentation began, and 25℃ after 48 hours; molasses was added at a flow rate of 18-22 mL / h after 24 hours of fermentation.
[0016] Compared with the prior art, the present invention achieves the following beneficial effects: This invention transfers optimized gene fragments of CrtE, CrtYB, and CrtI from Pharfia rubra, optimized gene fragments of CrtW and CrtZ from Haematococcus pluvialis, and a gene fragment of tHMGR from Saccharomyces cerevisiae into Yersinia lipolytica to construct a complete astaxanthin synthesis pathway, enabling Yersinia lipolytica to synthesize astaxanthin de novo.
[0017] This invention expresses acetyl-CoA carboxylases from different sources in Yersinia lipolytica, promoting the conversion of acetyl-CoA to malonyl-CoA. On the one hand, this increases the accumulation of lipids in Yersinia lipolytica, and on the other hand, the accumulation of lipids provides a natural storage space for the synthesis of astaxanthin, thus providing a natural "storage carrier" for the synthesis of astaxanthin and increasing the yield of astaxanthin.
[0018] This invention optimizes the fermentation conditions of recombinant *Yarrowia lipolytica* by employing specific nitrogen sources, metal ions, and a specific temperature-variable strategy. This improves the yield of microbial oil and astaxanthin in the recombinant *Yarrowia lipolytica*. In 50 mL of culture medium, fermentation for 120 h yielded a microbial oil yield of 65.92%. The astaxanthin yield reached 299.31 mg / L.
[0019] This invention utilizes inexpensive molasses in a 5L fermenter to co-produce microbial oil and astaxanthin from Yeast Extract. After 168 hours of fermentation, the yield of microbial oil reached 80.14%, and the yield of astaxanthin reached 3.09 g / L, laying the foundation for further industrialization. Attached Figure Description
[0020] Figure 1 This is a bar chart showing the production of astaxanthin and oils by engineered strains Yl-A, Yl-AC1, Yl-AC2, and Yl-AC3 in Example 3.
[0021] Figure 2 A bar chart showing the production of astaxanthin and microbial oils by the engineered strain Yl-AC3 in different nitrogen sources.
[0022] Figure 3 A bar chart showing the production of astaxanthin and microbial oil by engineered strain Yl-AC3 in the presence of different metal ions.
[0023] Figure 4 A bar chart showing the production of astaxanthin and microbial oils by engineered strain Yl-AC3 under different temperature conditions.
[0024] Figure 5 Bar and line graphs of astaxanthin and microbial oil produced by engineered strain Yl-AC3 using molasses as raw material in a 5 L fermenter. Detailed Implementation
[0025] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.
[0026] The Latin names of the microorganisms in the examples are as follows: Red Pfaff yeast: Xanthophyllomycesdendrorhous Saccharomyces cerevisiae Haematococcus pluvialis: Haematococcuspluvialis Pichia pastoris: Komagataellaphaffii Cryptococcus pluvialis: Apiotrichumporosum The original strain used in the examples was Yersinia lipolytica po1f, which can be purchased from commercial sources.
[0027] In this embodiment, the steps for oil extraction and quantitative analysis are as follows: 1. Extraction of microbial oil: After fermentation, the bacterial broth was centrifuged to obtain bacterial sludge. The sludge was washed several times with sterile water and then freeze-dried for 48 hours. 20 mg of the freeze-dried sludge was weighed and placed in a 15 mL sealed glass tube. 1.5 mL of n-hexane, 0.5 mL of methyl benzoate, and 2 mL of a solution containing 15 vol% H₂SO₄ (prepared with methanol) were added sequentially. After sealing the glass tube, the tube was incubated in a metal bath at 100°C until the freeze-dried sludge powder was completely dissolved. Then, 1 mL of sterile water was added, and the tube was centrifuged at 2500 rpm for 5 minutes at 4°C until the water and oil phases separated. The supernatant was then analyzed by gas chromatography.
[0028] 2. Determination of microbial lipids in the product The content of various fatty acid methyl esters was analyzed using a gas chromatograph (Agilent Technologies, 6890 N Network GC-System). Instrument accessories included a DBWax capillary column (length: 30 m, diameter: 0.25 mm, membrane: 0.25 μm) and a flame ionization detector (FID, Agilent 6890 GC). The operating pressure was 1.083 bar. Nitrogen was used as the carrier gas at a flow rate of 1 mL / min. The GC temperature program was as follows: injection port temperature 250 °C, column temperature increased from 40 °C to 250 °C at a rate of 8 °C / min, held at 250 °C for 10 min, and then decreased back to 40 °C. The fatty acid methyl ester standard was RM3 FAME Mix (Sigma Aldrich, Taufkirchen, Germany; 07256-1AMP). The standard was prepared as a 10 mg / L GC standard solution using n-hexane and analyzed using the same program. Fatty acids with a content less than 1% were classified as trace fatty acids.
[0029] 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 liquid, centrifuge at 12000 rpm for 5 min, and wash the bacterial sludge twice with pure water after centrifugation. Then proceed with the following steps (2). (2) After draining the water, resuspend in 2 mL of dimethyl sulfoxide (DMSO) (preheated at 60℃), shake evenly on a vortex mixer, and then place in a 55℃ water bath for 15 min. (3) Add 4 mL of anhydrous ethanol; (4) Centrifuge the sample at 12,000 rpm for 5 min, and transfer the supernatant to a new centrifuge tube for storage in the dark.
[0030] 2. Quantitative analysis of astaxanthin: High performance liquid chromatography (HPLC) was used to detect the concentration of astaxanthin. The liquid chromatograph used in this invention is an Agilent Technologies 1200 Infinity series; the column is an Acclaim™ 120 C30 column; the ultraviolet absorption wavelength is 450 nm; methanol and methyl tert-butyl ether are used as the mobile phase, and are introduced into the analytical column at a volume ratio of 95:5; the flow rate is controlled at 1.0 mL / min; and the column temperature is 25 °C.
[0031] Example 1: Amplification of Gene Elements and Preparation of Target Plasmids (a) Determination and optimization of gene element sequences Based on the nucleotide sequences of the geranylgeranyl diphosphate synthase CrtE gene, the nucleotide sequences of the phytoene synthase / lycopene cyclase CrtYB gene, and the nucleotide sequences of the phytoene desaturase CrtI gene from *Phaeophyton flavus* provided on NCBI, the nucleotide sequences shown in SEQ ID No: 1, SEQ ID No: 2, and SEQ ID No: 3 were obtained through codon optimization.
[0032] 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 nucleotide sequence of tHMGR is shown in SEQ ID No: 4.
[0033] Based on the nucleotide sequences of β-carotene ketolase CrtW and β-carotene hydroxylase CrtZ from Haematococcus pluvialis provided on NCBI, the nucleotide sequences shown in SEQ ID No: 5 and SEQ ID No: 6 were obtained through codon optimization.
[0034] Based on the coding gene sequences of acetyl-CoA carboxylase from *Saccharomyces cerevisiae* and *Pichia pastoris* provided on NCBI, PCR amplification was performed using the genomes of *Saccharomyces cerevisiae* and *Pichia pastoris* as templates, respectively. The coding gene sequences are shown in NCBI Gene IDs: 855750 and 8196923, respectively. Codon optimization was performed based on the coding gene sequence of acetyl-CoA carboxylase from *Cryptococcus cryptococcosus* provided on NCBI, and the optimized nucleotide sequence is shown in SEQ ID No: 7.
[0035] (II) Construction of recombinant plasmids 1. Using BB1-23 (Plasmid #98496) as the vector plasmid, construct recombinant plasmids containing CrtI, CrtE, CrtYB, tHMGR, CrtW, and CrtZ.
[0036] The gene fragments CrtI, CrtE, CrtYB, CrtW, and CrtZ were synthesized by GenScript Biotech Inc.; PCR amplification was performed using the Saccharomyces cerevisiae genome (BY4741) as a template (primers are shown in Table 1) to obtain the tHMGR gene fragment.
[0037] Gene fragments of CrtI, CrtE, CrtYB, tHMGR, CrtW, and CrtZ were inserted into BB1-23 (Plasmid #98496) to construct recombinant plasmids containing CrtI, CrtE, CrtYB, tHMGR, CrtW, and CrtZ, respectively, named BB1-23-CrtI, BB1-23-CrtE, BB1-23-CrtYB, BB1-23-tHMGR, BB1-23-CrtW, and BB1-23-CrtZ.
[0038] 2. Construct expression cassettes for CrtI, CrtE, CrtYB, tHMGR, CrtW, and CrtZ. Plasmids BB1-23-CrtI, BB1-12-pGPM1, and BB1-34-ScCYC1tt were inserted into plasmid BB2-AB using the GoldenGate method with Bpi1 enzyme and T4 ligase to obtain plasmid BB2-AB-pGPM1-CrtI-ScCYC1tt. Plasmids BB1-23-CrtE, BB1-12-pPDC1, and BB1-34-RPP1Btt were inserted into plasmid BB2-BC using the GoldenGate method with Bpi1 enzyme and T4 ligase to obtain plasmid BB2-BC-pPDC1-CrtE-RPP1Btt.
[0039] Plasmids BB1-23-CrtYB, BB1-12-pMDH3, and BB1-34-RPS2tt were inserted into plasmid BB2-CD using the GoldenGate method with Bpi1 enzyme and T4 ligase to obtain plasmid BB2-CD-pMDH3-CrtYB-RPS2tt. Plasmids BB1-23-tHMGR, BB1-12-pADH2, and BB1-34-RPL2Att were inserted into plasmid BB2-DE using the GoldenGate method with Bpi1 enzyme and T4 ligase to obtain plasmid BB2-DE-pADH2-tHMGR-RPL2Att.
[0040] Plasmids BB1-23-CrtW, BB1-12-pTEF1, and BB1-34-IDP1tt were inserted into plasmid BB2-EF using the GoldenGate method with Bpi1 enzyme and T4 ligase to obtain plasmid BB2-EF-pTEF1-CrtW-IDP1tt.
[0041] Plasmids BB1-23-CrtZ, BB1-12-pGAP, and BB1-34-RPS25Att were inserted into plasmid BB2-FG using the GoldenGate method with Bpi1 enzyme and T4 ligase to obtain plasmid BB2-FG-pGAP-CrtZ-RPS25Att.
[0042] The construction process of the recombinant plasmid BB2-AB-pGPM1-CrtI-ScCYC1tt is as follows: GoldenGate assembly was performed using Bpi1 enzyme and T4 ligase from Nanjing Fomax Biotechnology Co., Ltd.
[0043] 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.
[0044] The construction process for other recombinant plasmids is the same as described above.
[0045] 3. The construction process of recombinant plasmid BB3-A6-intB-IEYBtWZ is as follows: The 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 were inserted into plasmid BB3-A6-intB using the GoldenGate method with Bsa1 enzyme and T4 ligase to obtain the recombinant plasmid BB3-A6-intB-IEYBtWZ.
[0046] 4. The construction process of recombinant plasmids BB3-intE1-ScACC, BB3-intE1-KpACC, and BB3-intE1-CpACC is as follows: After PCR amplification using ScACC-F and ScACC-R, KpACC-F and KpACC-R, and CpACC-F and CpACC-R respectively, ScACC, KpACC, and CpACC were obtained (primers are shown in Table 1). Using plasmid BB3-intE1 as a vector, the vector was digested with Asis1 and cloned with the above fragments in one step to obtain circular vectors.
[0047] The method for constructing BB3-intE1 is as follows: using BB3-A6-intB as the backbone, replacing intB with the insertion site intE1 of Yersinia lipolyticis, and replacing the kanamycin sulfate selection marker with hygromycin B selection marker.
[0048] ScACC used Saccharomyces cerevisiae BY4741 as a template for amplification, KpACC used Pichia pastoris GS115 as a template for amplification, and CpACC used Cryptococcus cryptococcus Y3 as a template for amplification.
[0049] The circular vector was transformed into Escherichia coli DH5α competent cells, and positive recombinant plasmids BB3-intE1-ScACC, BB3-intE1-KpACC, and BB3-intE1-CpACC were obtained by screening with antibiotic plates and verification by colony PCR and sequencing.
[0050] Table 1
[0051] Example 2 Construction of recombinant bacteria 1. Construction of recombinant strain Yl-A The plasmid BB3-A6-intB-IEYBtWZ, containing the gene expression cassette of CrtI-CrtE-CrtYB-tHMGR-CrtW-CrtZ, was introduced into Yersinia lipolyticis po1f and integrated into the intB site of the genome to obtain the recombinant strain Yl-A.
[0052] 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).
[0053] ② Linearized BB3-A6-intB-IEYBtWZ was introduced into competent cells of Yeast lipolyticis using the Zymogen Frozen EZYeast Transformation Kit II from Zymo Research Corporation for homologous recombination.
[0054] ③ Screening was performed using antibiotic plates on the selection medium. Single colonies grew in 3-4 days. The positive clones that were correctly identified by PCR were named recombinant bacteria Yl-A.
[0055] 2. Construction of recombinant bacteria Yl-AC1, Yl-AC2, and Yl-AC3 The recombinant plasmids BB3-intE1-ScACC, BB3-intE1-KpACC, and BB3-intE1-CpACC containing the ScACC / KpACC / CpACC gene expression cassettes were introduced into the intE1 region of the genome of Yersinia lipolytica Yl-A, respectively, to obtain recombinant bacteria Yl-AC1, Yl-AC2, and Yl-AC3.
[0056] 3. Construction of two contrasting recombinant strains (1) Comparison of recombinant strain Yl-A1 The gene sequences of CrtS and CrtR from *Phaeff's red yeast* were codon-optimized to obtain the nucleotide sequences shown in SEQ ID No: 16 and SEQ ID No: 17. These sequences replaced the nucleotide sequences of β-carotene ketolase CrtW and β-carotene hydroxylase CrtZ from *Haematococcus pluvialis*. A plasmid containing the gene expression cassette of CrtI-CrtE-CrtYB-tHMGR-CrtS-CrtR was constructed according to the method in Example 1. The plasmid was introduced into *Yersinia lipophila* po1f according to the method in Example 2 to obtain the comparative recombinant strain Yl-A1. (2) Comparison of recombinant strain Yl-A2 The nucleotide sequences of β-carotene ketolase CrtW and β-carotene hydroxylase CrtZ from Haematococcus pluvialis provided on NCBI were codon-optimized to obtain the nucleotide sequences shown in SEQ ID No: 18 and SEQ ID No: 19. A plasmid containing the gene expression cassette of CrtI-CrtE-CrtYB-tHMGR-CrtS-CrtR was constructed according to the method of Example 1. The plasmid was introduced into Yersinia lipophila po1f according to the method of Example 2 to obtain the comparative recombinant strain Yl-A2.
[0057] Microbial oil and astaxanthin were produced using the recombinant bacteria from Example 2, respectively.
[0058] The specific method is as follows: Take the strain from the seed preservation tube, inoculate it into the YPD test tube at an inoculation rate of 1% (volume ratio), and incubate at 30℃ for 24 hours to obtain the seed liquid; The seed culture was inoculated at a rate of 1% (v / v) into 50 mL of minimal culture medium (40 g / L glucose, 0.5 g / L MgSO4·7H2O, 7.5 g / L (NH4)2SO4, 11.93 g / L KH2PO4, and 2.8 g / L K2HPO4·3H2O). The culture was incubated at 25℃ and 220 rpm with shaking for 5 days, with 4 mL of 40 g / L glucose added every 24 hours. After 120 hours of fermentation, the microbial oil content of the recombinant strains Yl-A, Yl-AC1, Yl-AC2, and Yl-AC3 were 47.15%, 54.63%, 50.13%, and 58.99%, respectively, and the astaxanthin content was 169.91 mg / L, 221.30 mg / L, 196.90 mg / L, and 242.79 mg / L, respectively. Figure 1 This indicates that expressing acetyl-CoA carboxylase in Yersinia lipolytica not only promotes lipid accumulation, but also that microbial lipids, as natural reservoirs of lipid-soluble compounds, can further promote astaxanthin accumulation.
[0059] The comparative recombinant strains Yl-A1 and Yl-A2 from Example 2 were fermented and cultured according to the above method.
[0060] After 120 hours, the astaxanthin content of the recombinant strain Yl-A1, which did not ferment, was compared to that of the recombinant strain Yl-A2, which was 33.76 mg / L.
[0061] The results showed that CrtS and CrtR from *Phaefflera rubrum*, even with codon optimization, could not be successfully expressed in *Yarrowia lipolytica*, possibly because these two enzymes could not fold correctly in *Yarrowia lipolytica*, making it difficult to catalyze the conversion of β-carotene to astaxanthin. In contrast, CrtW and CrtZ from *Haematococcus pluvialis* exhibited good enzymatic activity and successfully catalyzed the conversion of β-carotene to astaxanthin. However, the astaxanthin yield of the recombinant strain Yl-A2 (33.76 mg / L) was significantly lower than that of the recombinant strain Yl-A (169.91 mg / L). Although both recombinant strains Yl-A2 and Yl-A contained optimized CrtW and CrtZ, the difference in nucleotide sequences affected the enzyme expression level, thus affecting the astaxanthin yield.
[0062] Example 4: Effects of different nitrogen sources on the production of microbial oils and astaxanthin by engineered strains Nitrogen sources are essential nutrients for microbial growth. Organic nitrogen sources can provide proteins and amino acids, which are beneficial for the accumulation of biomass by the microorganisms; inorganic nitrogen sources are more conducive to the synthesis of microbial products. Therefore, a suitable nitrogen source is extremely important for the growth of microorganisms and the accumulation of fermentation products.
[0063] Based on Example 3, only the type of nitrogen source was changed. Yeast powder, peptone, corn steep liquor powder, and urea were combined with (NH4)2SO4 to form a mixed nitrogen source (mass ratio of 1:1). Yl-AC3 was used as the fermentation strain, and all other operations were the same as in Example 3. The results of fermentation for 120 hours are shown in Table 2 and [Table data missing]. Figure 2 .
[0064] The results showed that, compared with a single nitrogen source, the mixed nitrogen source of (NH4)2SO4 combined with yeast extract yielded the highest oil content and astaxanthin production, at 64.19% and 264.71 mg / L, respectively. Peptone, corn steep liquor powder, and urea, however, were detrimental to the production of oil and astaxanthin.
[0065] Table 2
[0066] Example 5: Effects of different types of metal ions on the production of microbial oils and astaxanthin by engineered strains Metal ions play a very important role in the growth of microorganisms, such as maintaining the stability of enzyme molecular structure and maintaining the stability of cell osmotic pressure.
[0067] Based on Example 3, the nitrogen source was (NH4)2SO4 and yeast powder in a mass ratio of 1:1. Different types of metal chlorides were added to investigate Mn. 2+ Cu 2+ Ca 2+ Zn2+ The effects of *Yarrowia lipolytica* on the production of microbial oils and astaxanthin were investigated. The final concentration of metal ions added to the culture medium was 1 mmol / L. Yl-AC3 was used as the fermentation strain, and other operations were the same as in Example 3. The results after 120 h of fermentation are shown in Table 3. Figure 3 .
[0068] The results show that Mn 2+ Cu 2+ It can promote the accumulation of oils, but it is detrimental to the accumulation of astaxanthin; Ca 2+ It has a negative impact on both; Zn 2+ The addition of [a specific ingredient] can increase the yield of microbial oil and astaxanthin to 65.15% and 277.09 mg / L, respectively.
[0069] Table 3
[0070] Example 6: Effects of different fermentation temperatures on the production of microbial oils and astaxanthin by engineered strains Fermentation temperature significantly affects the growth of microorganisms and the efficiency of product synthesis. Therefore, this application investigated the production of microbial oils and astaxanthin by Yersinia lipolytica under different conditions of 18-37℃.
[0071] Based on Example 3, the nitrogen source was (NH4)2SO4 and yeast extract in a mass ratio of 1:1. Zinc chloride was added to the culture medium at a final concentration of 1 mmol / L. Yl-AC3 was used as the fermentation strain. Other operations were the same as in Example 3. The results after 120 h of fermentation are shown in Table 4. Figure 4 .
[0072] The results showed that low temperatures were beneficial for astaxanthin accumulation but reduced oil accumulation; relatively higher temperatures were more conducive to oil accumulation. Therefore, this application selected a variable fermentation temperature. For the first 48 hours of fermentation, microorganisms were kept at 30℃, which was beneficial for microbial growth and product accumulation; after 48 hours, they were placed at 25℃, which was beneficial for astaxanthin synthesis. Using this variable temperature strategy, the yields of microbial oil and astaxanthin were 65.92% and 299.31 mg / L, respectively. This indicates that astaxanthin yield is more sensitive to temperature changes, and the variable temperature strategy is more suitable for the co-production of microbial oil and astaxanthin.
[0073] Table 4
[0074] Example 7: Co-production of microbial oil and astaxanthin from inexpensive raw material molasses in a 5 L fermentation tank. To reduce fermentation costs and achieve more efficient synergistic production of microbial oils and astaxanthin, this application selects inexpensive molasses as the fermentation substrate. Molasses is a byproduct of the sugar industry, mainly composed of sucrose, and also contains nutrients such as vitamins and proteins, making it highly suitable for microbial growth.
[0075] ① Seed culture: a. Primary seed culture: Take 1% of the recombinant strain Yl-AC3 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.
[0076] ② Batch feeding fermentation Seed culture solution obtained from seed culture was inoculated (10% inoculum) into a 5L fermenter containing fermentation medium (40 g / L molasses). Recombinant strain Yl-AC3 underwent fed-batch fermentation in the 5L fermenter at 30℃, 500 rpm, and dissolved oxygen maintained at 40%. After 24 h of fermentation, molasses was added at a flow rate of 20 mL / h. After 48 h, the fermentation temperature was reduced to 25℃ to accumulate astaxanthin. Finally, after 168 h of fermentation, the OD of the strain... 600 The concentration reached 294.13, the cell dry weight was 140.3 g / L, the microbial lipid accumulation reached 80.14%, and the astaxanthin production reached 3.09 g / L. Figure 5 ).
[0077] In the 5L fermenter, the cell density was significantly increased, which is crucial for the production of the intracellular product astaxanthin; a constant pH value was maintained in the fermenter, which is beneficial for cell growth; and the residual sugar concentration was continuously monitored during fermentation to ensure that the strain did not experience substrate deficiency during growth.
[0078] This invention constructs a complete astaxanthin synthesis pathway in *Yersinia lipolytica*, enabling the de novo synthesis of astaxanthin. To expand the astaxanthin storage space, acetyl-CoA carboxylases from different sources were expressed, successfully increasing the synthesis of lipids in *Yersinia lipolytica* and providing a natural "storage" for astaxanthin synthesis. Subsequently, by varying the nitrogen source, metal ion addition, and fermentation temperature, different fermentation strategies were employed, significantly increasing the yield of microbial lipids and astaxanthin. Finally, using inexpensive molasses as a raw material, continuous fed-batch fermentation was carried out in a 5L fermenter, achieving a cell dry weight of 140.3 g / L, a microbial lipid yield of over 80%, and an astaxanthin content of over 3%. This successfully achieved the co-production of microbial lipids and astaxanthin by recombinant strains in a fermenter, and holds promise for further increasing the yield of microbial lipids and astaxanthin in larger fermenters, laying the foundation for subsequent industrialization.
Claims
1. A recombinant Yarrowia lipolytica co-producing microbial lipid and astaxanthin, characterized in that: The recombinant Yarrowia lipolytica expresses geranylgeranyl diphosphate synthase CrtE derived from Phaffia rhodozyma, phytoene synthase / lycopene cyclase CrtYB, phytoene desaturase CrtI, 3-hydroxy-3-methylglutaryl CoA reductase tHMGR derived from Saccharomyces cerevisiae, beta-carotene ketolase CrtW and beta-carotene hydroxylase CrtZ derived from Haematococcus pluvialis.
2. The recombinant Yarrowia lipolytica co-producing microbial oil and astaxanthin according to claim 1, characterized in that: The nucleotide sequence of the CrtE is shown in SEQ ID No: 1 in the sequence listing, the nucleotide sequence of the CrtYB is shown in SEQ ID No: 2 in the sequence listing, the nucleotide sequence of the CrtI is shown in SEQ ID No: 3 in the sequence listing, the nucleotide sequence of the tHMGR is shown in SEQ ID No: 4 in the sequence listing, the nucleotide sequence of the CrtW is shown in SEQ ID No: 5 in the sequence listing, and the nucleotide sequence of the CrtZ is shown in SEQ ID No: 6 in the sequence listing.
3. The recombinant Yarrowia lipolytica co-producing microbial oil and astaxanthin according to claim 1, characterized in that: The recombinant Yarrowia lipolytica expresses acetyl-CoA carboxylase derived from Saccharomyces cerevisiae, Pichia pastoris or Cryptococcus.
4. The recombinant Yarrowia lipolytica co-producing microbial oil and astaxanthin according to claim 3, characterized in that: The nucleotide sequence of the acetyl-CoA carboxylase derived from Saccharomyces cerevisiae is shown in NCBI Gene ID: 855750, the nucleotide sequence of the acetyl-CoA carboxylase derived from Pichia pastoris is shown in NCBI Gene ID: 8196923, and the nucleotide sequence of the acetyl-CoA carboxylase derived from Cryptococcus is shown in SEQ ID No: 7 in the sequence listing.
5. The recombinant Yarrowia lipolytica coproducing microbial oil and astaxanthin according to claim 1, characterized in that: The original strain of the recombinant Yarrowia lipolytica is Yarrowia lipolytica po1f.
6. A method of constructing a recombinant Yarrowia lipolytica for co-production of microbial oil and astaxanthin, characterized by: The recombinant plasmid containing CrtI, CrtE, CrtYB, tHMGR, CrtW and CrtZ is constructed by using BB3-A6-intB as a vector plasmid, and the recombinant plasmid is integrated into the intB site of the po1f genome to obtain a Yarrowia lipolytica strain Yl-A for producing astaxanthin; the recombinant plasmid containing acetyl-CoA carboxylase derived from Saccharomyces cerevisiae, Pichia pastoris or Cryptococcus is constructed by using BB3-intE1 as a vector, and is introduced into the intE1 site of Yl-A to obtain a recombinant Yarrowia lipolytica strain.
7. The method of construction of claim 6, wherein: The nucleotide sequence of the CrtE is shown in SEQ ID No: 1 in the sequence listing, the nucleotide sequence of the CrtYB is shown in SEQ ID No: 2 in the sequence listing, the nucleotide sequence of the CrtI is shown in SEQ ID No: 3 in the sequence listing, the nucleotide sequence of the tHMGR is shown in SEQ ID No: 4 in the sequence listing, the nucleotide sequence of the CrtW is shown in SEQ ID No: 5 in the sequence listing, the nucleotide sequence of the CrtZ is shown in SEQ ID No: 6 in the sequence listing; the nucleotide sequence of the acetyl-CoA carboxylase of the Saccharomyces cerevisiae is shown in NCBI Gene ID: 855750; the nucleotide sequence of the acetyl-CoA carboxylase of the Pichia pastoris is shown in NCBI Gene ID: 8196923; the nucleotide sequence of the acetyl-CoA carboxylase of the Cryptococcus is shown in SEQ ID No: 7 in the sequence listing.
8. The recombinant Yarrowia lipolytica of any one of claims 1-5 for use in the production of microbial oil and astaxanthin.
9. Use according to claim 8, characterized in that: The fermentation medium of the recombinant Yarrowia lipolytica is 38-42 g / L glucose, 0.48-0.52 g / L MgSO4·7H2O, 7.4-7.6 g / L of a composite nitrogen source, 11.8-12.0 g / L KH2PO4 and 2.7-2.9 g / L K2HPO4·3H2O, and 0.8-1.2 mmol / L zinc chloride, wherein the composite nitrogen source is (NH4)2SO4 and yeast powder at a mass ratio of 1:1; the fermentation temperature control method of the recombinant Yarrowia lipolytica is as follows: the fermentation temperature is 30℃ within 48 h after the start of fermentation, and the fermentation temperature is 25℃ after 48 h.
10. Use according to claim 8, characterized in that: The fermentation medium of the recombinant Yarrowia lipolytica is molasses; the dissolved oxygen is controlled at 38-42% during the fermentation; the fermentation temperature control method is as follows: the fermentation temperature is 30℃ within 48 h after the start of fermentation, and the fermentation temperature is 25℃ after 48 h; and the molasses is fed at a flow rate of 18-22 mL / h after 24 h of fermentation.