Lipid-decomposing yeast engineering bacterium for producing DHA (docosahexaenoic acid) as well as construction method and application thereof

Abstract

The invention belongs to the technical field of bioengineering, and particularly relates to a lipid-decomposing yeast engineering bacterium for producing DHA (docosahexaenoic acid) as well as a construction method and application thereof. The lipolytic yeast engineering bacterium is obtained by taking lipolytic yeast as a chassis strain, optimizing T-pfaA, T-pfaB, T-pfaC and T-pfaE genes through codons and then performing heterologous expression in the lipolytic yeast. The method comprises the following steps: transferring a transformed strain seed solution into an SD-Leu C / N 80 nitrogen-limited fermentation culture medium, collecting thalli after fermentation, carrying out freeze-drying, carrying out fatty acid detection, and carrying out quantitative analysis on DHA by adopting GC-MS (Gas Chromatography-Mass Spectrometer) to obtain a DHA standard curve: y = 1.39 * 10 <-8 > x <-0.0007 >, R2 = 0.9991, x is the peak area, y is the DHA yield, the unit is g / L, the DHA yield of the transformed strain is 0.19 mg / g, and a basis is provided for industrial directional synthesis of DHA.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, and specifically relates to an engineered yeast strain that produces DHA. Background Technology

[0002] Docosahexaenoic acid (DHA) is an omega-3 polyunsaturated fatty acid and an essential fatty acid for the human body, playing an irreplaceable physiological role in growth, development, and health maintenance. In infancy, DHA is a key nutrient for brain and retinal development, directly influencing nerve cell proliferation, differentiation, and synapse formation, and is crucial for cognitive and visual function development. Therefore, it is widely used in infant formula. For adults, DHA can regulate lipid metabolism, inhibit inflammatory responses, and protect cardiovascular health. It also plays a positive role in improving memory and delaying the onset of neurodegenerative diseases, leading to continuously increasing demand in functional foods, health foods, and pharmaceuticals.

[0003] With the increasing demand for DHA, its large-scale production technology has become a research hotspot. Currently, the commercial production of DHA mainly relies on marine fish oil extraction and microbial fermentation. Among these methods, fish oil extraction dominates the traditional market due to its high technological maturity, but it faces fundamental bottlenecks: First, the supply of raw materials exhibits significant resource dependence and cyclical fluctuations. Overfishing has led to a decline in deep-sea fish reserves, and ocean acidification and rising temperatures further exacerbate the risk to the supply of raw materials. Second, DHA content in fish oil accounts for only 10%-30% of total fatty acids, and it is highly mixed with PUFAs such as eicosapentaenoic acid (EPA), requiring separation and purification through processes such as molecular distillation and urea encapsulation, which are energy-intensive and costly. Third, the bioaccumulation effect in the food chain makes fish oil prone to carrying persistent organic pollutants such as methylmercury and polychlorinated biphenyls (PCBs), failing to meet the purity requirements of pharmaceutical-grade products.

[0004] Microbial fermentation, with its wide availability of raw materials, short production cycle, high product purity, environmental friendliness, and ability to be industrially scaled and controlled, has become an alternative method for the industrial production of DHA. *Cypripedium* ( Thraustochytrids As a type of eukaryotic microorganism widely found in marine and freshwater environments, *DHA* possesses unique advantages in DHA production. Patent 202311363079.X discloses recombinant engineered bacteria for producing polyunsaturated fatty acids. This involves transforming host cells with the Δ4 desaturase gene from *Cyclochytrium*, resulting in engineered bacteria for DHA production, which shows promise for industrial application. Therefore, screening microbial hosts with high lipid accumulation capabilities and reconstructing the DHA synthesis pathway through metabolic engineering is a core strategy to overcome current production bottlenecks.

[0005] Yarrowia lipolytica ( Yarrowia lipolytica As a "generally recognized safe" strain, *Lactobacillus lipolyticus* possesses strong lipid accumulation capabilities, mature gene-editing tools, and high robustness, making it a host for sustainable lipid synthesis. A key characteristic of *Lactobacillus lipolyticus* is its ability to efficiently convert large amounts of carbon into lipids and accumulate them within cells under conditions of abundant carbon but limited nitrogen. Lipolyticus accumulation can reach over 60% of the cell's dry weight, far exceeding that of ordinary microorganisms. Simultaneously, *Lactobacillus lipolyticus* exhibits strong environmental adaptability, utilizing various inexpensive carbon sources such as glucose, sucrose, glycerol, and lignocellulose hydrolysate for growth and metabolism, significantly reducing production costs. Furthermore, the genetic background of *Lactobacillus lipolyticus* is relatively clear, and gene-editing tools (such as the CRISPR-Cas9 system) are becoming increasingly mature, facilitating precise modification and regulation of metabolic pathways. To address the shortcomings of existing DHA production technologies and leverage the unique advantages of lipolytic yeast, this study utilizes genetic engineering to precisely modify the metabolism of lipolytic yeast, constructing engineered strains with high DHA synthesis efficiency, excellent growth performance, and industrial application potential. Simultaneously, it establishes efficient construction methods and fermentation processes. This has significant theoretical and practical value for promoting innovation in DHA production technology, reducing production costs, ensuring product quality, and achieving sustainable industrial development. Summary of the Invention

[0006] To address the above problems, this invention proposes an engineered DHA-producing lipophilic yeast strain, its construction method, and its application.

[0007] The technical solution of this invention is implemented as follows: This invention is the first to use *Cypripedium rupestris*. Thraustochytrium The PKS fatty acid synthesis pathway of sp. ATCC 26185 was heterologously expressed in lipolytic yeast, resulting in a DHA-producing heterologous expression strain. Based on this, the present invention provides a method for constructing a DHA-producing engineered lipolytic yeast strain, comprising the following steps: (1) T-pfaB, T-pfaC and T-pfaE were constructed into vector 1 to obtain plasmid vector 1-T-pfaB, vector 1-T-pfaC and vector 1-T-pfaE respectively; T-pfaA was constructed into vector 2 in three segments, and then linked to vector 3 by cloning and homologous recombination to construct plasmid vector 3-T-pfaA; (2) In step (1), the vectors 3-T-pfaA and 1-T-pfaC were double-digested to obtain pfaA and pfaC fragments, respectively, which served as the backbone. The vectors 1-T-pfaB and 1-T-pfaE were double-digested to obtain pfaB and pfaE fragments, respectively. Then, the pfaA and pfaE fragments, and the pfaB and pfaC fragments were ligated in pairs using ligase to construct plasmid vectors 3-T-pfaA+T-pfaE and 1-T-pfaB+T-pfaC. (3) The vector 3-T-pfaA+T-pfaE in step (2) is double-digested to form the backbone, and the vector 1-T-pfaB+T-pfaC is double-digested to form the fragment. Then the two are ligated by ligase to construct the plasmid vector 3-T-pfaA+T-pfaB+T-pfaC+T-pfaE. (4) Transform the plasmid vector 3-T-pfaA+T-pfaB+T-pfaC+T-pfaE from step (3) into lipophilic yeast to construct the engineered lipophilic yeast.

[0008] Preferably, the accession number of T-pfaA in NCBI is A0A1B3PEI6.1, the accession number of T-pfaB in NCBI is A0A1B3PEI8.1, the accession number of T-pfaC in NCBI is A0A1B3PEI9.1, and the accession number of T-pfaE in NCBI is A0A1B3PEJ0.1.

[0009] Preferably, the synthetic sequence of the above-mentioned codon-optimized T-pfaA from *Saccharomyces lipolyticus* is shown in SEQ ID NO.1; the synthetic sequence of the codon-optimized T-pfaB from *Saccharomyces lipolyticus* is shown in SEQ ID NO.2; the synthetic sequence of the codon-optimized T-pfaC from *Saccharomyces lipolyticus* is shown in SEQ ID NO.3; and the synthetic sequence of the codon-optimized T-pfaE from *Saccharomyces lipolyticus* is shown in SEQ ID NO.4.

[0010] Preferably, the above-mentioned carrier 1 is a PYLXP2 carrier, carrier 2 is a pUC57 carrier, and carrier 3 is a PYLXP carrier; carrier 1-pfaB, carrier 1-pfaC and carrier 1-pfaE also include the promoter pTEF and the terminator XPR2.

[0011] Preferably, the primers used for constructing the three segments of T-pfaA are T-PFA1-1-F as shown in SEQ ID NO.5, T-PFA1-1-R as shown in SEQ ID NO.6, T-PFA1-2-F as shown in SEQ ID NO.7, T-PFA1-2-R as shown in SEQ ID NO.8, T-PFA1-3-F as shown in SEQ ID NO.9, and T-PFA1-3-R as shown in SEQ ID NO.10.

[0012] Preferably, the restriction enzyme used for the above double digestion is NheI, AvrII, ClaI or SalI; the ligase is T4 ligase; and the lipophilic yeast is Yersinia lipophilia PO1f.

[0013] Secondly, this invention applies to protect the engineered lipophilic yeast strains constructed by the above-described construction method.

[0014] Thirdly, this invention application protects the application of the above-mentioned engineered lipophilic yeast in the fermentation production of DHA.

[0015] Fourthly, this invention claims a method for fermenting and producing DHA, in which the seed culture of the above-mentioned engineered lipophilic yeast is inoculated into SD-Leu C / N 80 nitrogen-limited fermentation medium and cultured, and then freeze-dried to obtain DHA.

[0016] Preferably, the above culture conditions are 28-32℃, 200-250 r for 90-150 h; the OD value of the seed culture is 0.15-0.35. The standard curve for DHA is y=1.39×10⁻⁶. -8 x-0.0007, R 2 =0.9991, where x is the peak area and y is the DHA yield in g / L.

[0017] The Shimadzu GCMS-TQ8050NX triple quadrupole gas chromatography-mass spectrometry system with a flame ionization detector was used for analysis using an HP-INNOWAX capillary column (30 m × 0.25 mm). The GC-MS conditions were as follows: 60℃ (1 min), ramped to 240℃ in 10 min, and held at 240℃ for 8 min.

[0018] The present invention has the following beneficial effects: This invention is the first to heterologously express *Cyclochytrium* in a lipophilic yeast strain. ThraustochytriumThe PKS pathway gene cluster of sp. ATCC26185 was heterologously expressed in *Yersinia lipophila* using *Yersinia lipophila* as the chassis strain after codon optimization, yielding a DHA-producing *Yersinia lipophila* PO1f transformant strain. The seed culture of the transformant strain (OD=0.25) was transferred to SD-LeuC / N 80 nitrogen-limited fermentation medium. After fermentation, the cells were collected, lyophilized, and analyzed for fatty acid content. Gas chromatography-mass spectrometry (GC-MS) was used for quantitative analysis of DHA, resulting in a DHA standard curve: y=1.39×10⁻⁶. -8 x-0.0007, R 2 =0.9991, where x is the peak area and y is the DHA yield in g / L, and the DHA yield of the transformed strain is 0.19 mg / g; this application realizes the production of DHA in lipolytic yeast, which provides a foundation for the industrial-scale targeted synthesis of DHA. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 This is a diagram of the PYLXP-T-pfaA Snapgene.

[0021] Figure 2 Image of T-pfaA fragment amplification and PYLXP digestion gel.

[0022] Figure 3 Verification using PYLXP-T-pfaA enzyme digestion.

[0023] Figure 4 Snapgene diagrams for PYLXP-T-pfaA+ T-pfaE and PYLXP2-T-pfaB+T-pfaC.

[0024] Figure 5 Gel images showing the digestion results of PYLXP-T-pfaA, PYLXP2-T-pfaB, PYLXP2-T-pfaC, and PYLXP2-T-pfaE.

[0025] Figure 6 Verification was performed by digesting PYLXP-T-pfaA+T-pfaE and PYLXP2-T-pfaB+T-pfaC.

[0026] Figure 7 The PYLXP-T-pfaA+T-pfaB+T-pfaC+T-pfaE snapgene diagram.

[0027] Figure 8 Gel images of PYLXP-T-pfaA+T-pfaE and PYLXP2-T-pfaB+T-pfaC enzyme digestion.

[0028] Figure 9 This is a gel image for PYLXP-T-pfaA+T-pfaB+T-pfaC+T-pfaE enzyme digestion verification.

[0029] Figure 10 The image shows the results of GC-MS fatty acid detection; black represents the control transfected with empty PYLXP, purple represents the DHA standard, blue represents the transfected plasmid PYLXP-T-pfaA+T-pfaB+T-pfaC+T-pfaE, and brown represents the original strain PO1f. Detailed Implementation

[0030] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0031] Unless otherwise specified, the experimental methods used in the following experimental examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0032] This invention utilizes high-DHA-producing *Cypripedium* from a superior source. Thraustochytrium The PKS gene cluster of sp. ATCC26185 yielded a novel source of heterologous expression transformation strain of lipolytic yeast, different from previous reports, of the DHA-to-PKS pathway gene cluster.

[0033] Example: Construction of DHA-producing lipolytic yeast engineered strain The strain Yersinia lipophila PO1f used in this embodiment was provided by the Oil and Fat Center of Muyuan Laboratory.

[0034] The vectors used in this embodiment, PYLXP2, pUC57, and PYLXP, are all commonly used vectors, sourced from Wong L, Engel J, Jin E, et al. YaliBricks, a versatile genetic toolkit forstreamlined and rapidpathway engineering in Yarrowia lipolytica . Metab EngCommun. 2017 Oct 1;5:68-77.

[0035] The protein sequences of four genes were obtained from NCBI: T-pfaA (A0A1B3PEI6.1), T-pfaB (A0A1B3PEI8.1), T-pfaC (A0A1B3PEI9.1), and T-pfaE (A0A1B3PEI0.1), abbreviated as T-pfaA, T-pfaB, T-pfaC, and T-pfaE, respectively. After codon optimization for *Saccharomyces lipolyticus* (gene sequences as shown in SEQ ID NO. 1-4), gene synthesis was performed. T-pfaB, T-pfaC, and T-pfaE were constructed into the PYLXP2 vector using the pTEF promoter and XPR2 terminator, respectively. T-pfaA was too long, so it was first constructed into the pUC57 vector in three segments, and then cloning and homologous recombination were performed to transfer it into the PYLXP vector.

[0036] The specific construction steps are as follows: (1) First, the three fragments of T-pfaA were integrated into the PYLXP vector via homologous recombination to construct PYLXP-T-pfaA. The three T-pfaA gene fragments were amplified from the pUC57 vector using the three pairs of primers with homologous arms shown in Table 1. The PCR amplification system is shown in Table 2. The PYLXP vector was digested with SnaBI and KpnI enzymes, and the fragments were recovered by gel digestion. The plasmid digestion system is shown in Table 3. PYLXP-T-pfaA was constructed using a one-step cloning method via homologous recombination. The one-step cloning system is shown in Table 4. The Snapgene diagram of PYLXP-T-pfaA is shown below. Figure 1 As shown.

[0037] Table 1 Primer Table Table 2 PCR amplification system Table 3 Plasmid digestion system Table 4 One-step cloning system X = [0.02 × number of base pairs per fragment] ng (0.03 pmol).

[0038] Y = [0.02 × number of base pairs per cloning vector] ng (0.03 pmol).

[0039] Figure 2 The image shows a gel image of T-pfaA fragment amplification and PYLXP digestion. Figure 2 It can be seen that, Thraustochytrium The T-pfaA gene of strain sp. ATCC26185 was amplified in three segments: lanes 2-3 were T-pfaA-1 (3014 bp); lanes 5-6 were T-pfaA-2 (2492 bp); lanes 8-9 were T-pfaA-3 (3018 bp); and lanes 12-15 were 7425 bp digested with PYLXP, SnaBI, and KpnI.

[0040] Figure 3 For PYLXP-T-pfaA vector restriction enzyme digestion verification, EcoRI and XhoI double digestion were used. Lane 1 of PYLXP in the left image corresponds to lane 1 in the right image; lane 2 of PYLXP-T-pfaA corresponds to lanes 2, 3, and 4 of the three parallel transformant plasmids in the right image; the right image shows PYLXP, PYLXP-T-pfaA-1, PYLXP-T-pfaA-2, and PYLXP-T-pfaA-3, respectively. Figure 3 The left image shows that the simulated enzyme digestion results of Snapgene are consistent with the right image shows that the vector was constructed correctly.

[0041] (2) Using the YaliBricks gene editing tool from lipolytic yeast, ligation was performed via enzyme digestion. First, T-pfaA and T-pfaC were digested with NheI and SalI, respectively, as the backbone. T-pfaB and T-pfaE were digested with AvrII and SalI, respectively, as fragments. T-pfaA and T-pfaE, and T-pfaB and T-pfaC, were then ligated pairwise via T4 (Table 7) to construct the vectors PYLXP-T-pfaA+T-pfaE and PYLXP2-T-pfaB+T-pfaC. The Snapgene diagrams of the two vectors are shown below. Figure 4 As shown, PYLXP-T-pfaA+T-pfaE was digested with NheI and SalI to form a vector, and PYLXP2-T-pfaB+T-pfaC was digested with AvrII and SalI to form a fragment. The digestion system is shown in Tables 5-6. The fragments were then ligated at T4 (Table 7) to construct an expression vector integrating the four genes into the plasmid PYLXP-T-pfaA+T-pfaB+T-pfaC+T-pfaE. The Snapgene diagram is shown below. Figure 7 As shown.

[0042] Depend on Figure 5 It can be seen that lanes 2-3 are PYLXP-T-pfaA NheI SalI 15870 bp; lanes 5-6 are PYLXP2-T-pfaB AvrII SalI 7168 bp; lanes 8-9 are PYLXP2-T-pfaC NheI SalI 10851 bp; and lanes 11-12 are PYLXP2-T-pfaEAvrII SalI 1885 bp.

[0043] Verification was performed using double digestion with XhoI and SalI. Figure 6 In the left image, lane 1 of PYLXP-T-pfaA corresponds to lane 1 in the right image; lane 2 of PYLXP2-T-pfaB corresponds to lane 2 in the right image; lane 3 of PYLXP2-T-pfaC corresponds to lane 3 in the right image; lane 4 of PYLXP2-T-pfaE corresponds to lane 4 in the right image; lane 5 of PYLXP-T-pfaA+T-pfaE corresponds to lanes 5, 6, and 7 of the right image, representing the three parallel transformant plasmids; lane 6 of PYLXP2-T-pfaB+T-pfaC corresponds to lanes 8, 9, and 10 of the right image, representing the three parallel transformant plasmids. Figure 6 The left image shows that the simulated enzyme digestion results of Snapgene are consistent with the right image shows that the vector was constructed correctly.

[0044] Figure 8 Lanes 2-3 were digested with PYLXP-T-pfaA+S-pfaE NheI SalI, yielding 17732 bp; lanes 5-6 were digested with PYLXP2-T-pfaB+S-pfaC AvrII SalI, yielding 12657 bp.

[0045] Figure 9 Lane 1 of PYLXP-T-pfaA in the left image corresponds to lane 1 in the right image; lane 2 of PYLXP-T-pfaA+T-pfaE corresponds to lane 2 in the right image; lane 3 of PYLXP2-T-pfaB+T-pfaC corresponds to lane 3 in the right image; lane 4 of PYLXP-T-pfaA+T-pfaB+T-pfaC+T-pfaE corresponds to the three parallel transformant plasmids in lanes 4, 5, and 6 in the right image; Figure 9 The left image shows that the simulated enzyme digestion results of Snapgene are consistent with the right image shows that the vector was constructed correctly.

[0046] Table 5 Plasmid digestion system Table 6 Plasmid digestion system Table 7 T4 Connection System (3) Transform the constructed plasmid into lipophilic yeast PO1f. Transformation method: streak lipophilic yeast PO1f on YPD solid plate and culture at 30℃ for 48 h. Pick a single colony and inoculate it into a shaker tube containing about 5 mL of YPD liquid medium. Culture overnight at 30℃ and 220 r. Measure the OD. Inoculate into a 250 mL Erlenmeyer flask containing 100 mL of the initial OD=0.15. Culture at 30℃ and 220 r for about 3.5 h until the OD=0.5. Prepare competent cells. Collect all cells at 4℃ and 6000 r. Resuspend in 1 mL of 1×TE / 0.1 M LiOAc aqueous solution and transfer to a 1.5 mL centrifuge tube for washing twice. Finally, resuspend in 0.5 mL of 1×TE / 0.1 M LiOAc to complete the preparation of competent cells. After preparing the transformation system, incubate at 30℃ for 30 min (twisting and mixing once every 10 min), then incubate at 42℃ for 15 min (twisting and mixing once every 5 min). After centrifugation, wash once with 1 mL of 1×TE / 0.1 M LiOAc aqueous solution, spread on Leu-deficient plates, and verify the PCR level of the transformed molecule (Table 8).

[0047] Table 8 Yeast Conversion System (4) Fermentation: Transformant single colonies were inoculated into shake tubes containing about 5 mL of SD-Leu seed medium and cultured at 30℃ and 220 r for 36 h. They were then transferred to 250 mL Erlenmeyer flasks containing 30 mL of SD-Leu C / N 80 nitrogen-limiting fermentation medium at an initial OD of 0.25 and cultured at 30℃ and 220 r for 120 h. After fermentation, all cells were collected, washed once with PBS, and stored at -80℃ overnight before being lyophilized for fatty acid detection and analysis.

[0048] The culture medium formula used is as follows: SD-Leu seed culture medium: glucose 20 g / L, SC-Leu Broth 8 g / L; SD-Leu C / N 80 nitrogen-limited fermentation medium: glucose 40 g / L, Do Supplement-Leu 1.29 g / L, Yeast Nitrogen Base (without amino acids and ammonium sulfate) (YNB) 1.7 g / L, ((NH4)SO4) 1.1 g / L.

[0049] Implementation Results Example Fatty acid analysis method: Weigh approximately 40 mg of lyophilized bacterial powder into a 1.5 mL centrifuge tube, add 100 μL of 2 g / L glycerol-C17 (triglyceride-heptadecanoate) internal standard, then add 500 μL of 0.5 N sodium hydroxide solution, and shake the sample at 1200 rpm for 2 hours. Prepare a blank solution (without cell precipitate) by adding only 100 μL of internal standard and 500 μL of 0.5 N sodium hydroxide. After the reaction, add 40 μL of 98% sulfuric acid to neutralize the sample. Immerse the tip of a pipette into the sample surface, and then slowly add sulfuric acid; add 400 μL of n-hexane to each sample to extract fatty acid methyl esters. Shake at 1200 rpm for 10 minutes. Centrifuge at 14000 rpm for 2 minutes, carefully remove 250 μL of the n-hexane phase into a gas chromatography-mass spectrometry (GC-MS) bottle for analysis. The analysis was performed using a Shimadzu GCMS-TQ8050NX triple quadrupole gas chromatography-mass spectrometry system with a flame ionization detector and an HP-INNOWAX capillary column (30 m × 0.25 mm). The GC-MS conditions were as follows: 60℃ (1 min), ramped to 240℃ after 10 min, and held at 240℃ for 8 min.

[0050] Product detection data: The standard curve was prepared using hexane to prepare DHA methyl ester standards at concentrations of 0.0013281 g / L, 0.0026563 g / L, 0.0053125 g / L, 0.010625 g / L, 0.02125 g / L, 0.0425 g / L, and 0.085 g / L, respectively. GC-MS analysis was performed, and the standard curve was plotted based on peak area and concentration. DHA standard curve plotted: y = 1.39 * 10 -8 *x-0.0007, R 2 =0.9991 (x is the peak area, y is in g / L).

[0051] Depend on Figure 10 It can be seen that, according to calculations, the DHA yield of the transformed strain is 0.19 mg / g. Neither the starting strain Yersinia lipophila PO1f nor the transformed empty vector plasmid PYLXP produces DHA. However, the transformed PYLXP-T-pfaA+T-pfaB+T-pfaC+T-pfaE plasmid produces DHA.

[0052] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for constructing a DHA-producing engineered Yarrowia lipolytica strain, characterized by, The steps are as follows: (1) T-pfaB, T-pfaC and T-pfaE are respectively constructed into vector 1 to obtain plasmid vector 1-T-pfaB, vector 1-T-pfaC and vector 1-T-pfaE; T-pfaA is constructed into vector 2 in three sections, and then connected to vector 3 through cloning and homologous recombination to construct plasmid vector 3-T-pfaA; (2) The vector 3-T-pfaA and vector 1-T-pfaC in step (1) are double enzyme cut to obtain pfaA fragment and pfaC fragment as a skeleton, and the vector 1-T-pfaB and vector 1-T-pfaE are double enzyme cut to obtain pfaB fragment and pfaE fragment, then the pfaA fragment and pfaE fragment, and the pfaB fragment and pfaC fragment are connected by ligase to construct plasmid vector 3-T-pfaA+T-pfaE and vector 1-T-pfaB+T-pfaC; (3) The vector 3-T-pfaA+T-pfaE in step (2) is double enzyme cut as a skeleton, and the vector 1-T-pfaB+T-pfaC is double enzyme cut as a fragment, and then they are connected by ligase to construct plasmid vector 3-T-pfaA+T-pfaB+T-pfaC+T-pfaE; (4) The plasmid vector 3-T-pfaA+T-pfaB+T-pfaC+T-pfaE in step (3) is transformed into Yarrowia lipolytica to construct Yarrowia lipolytica engineering bacteria.

2. The method for constructing DHA-producing Y. lipolytica engineering bacteria according to claim 1, characterized in that: The T-pfaA has the accession number A0A1B3PEI6.1 in NCBI, the T-pfaB has the accession number A0A1B3PEI8.1 in NCBI, the T-pfaC has the accession number A0A1B3PEI9.1 in NCBI, and the T-pfaE has the accession number A0A1B3PEJ0.1 in NCBI.

3. The method for constructing DHA-producing Y. lipolytica engineering bacteria according to claim 2, characterized in that: The T-pfaA codon-optimized by Yarrowia lipolytica has a synthetic sequence as shown in SEQ ID NO. 1; the T-pfaB codon-optimized by Yarrowia lipolytica has a synthetic sequence as shown in SEQ ID NO. 2; The T-pfaC codon-optimized by Yarrowia lipolytica has a synthetic sequence as shown in SEQ ID NO. 3; and the T-pfaE codon-optimized by Yarrowia lipolytica has a synthetic sequence as shown in SEQ ID NO.

4.

4. The method for constructing DHA-producing Y. lipolytica engineering bacteria according to claim 3, characterized in that: The vector 1 is PYLXP2 vector, the vector 2 is pUC57 vector, and the vector 3 is PYLXP vector; the vector 1-pfaB, the vector 1-pfaC and the vector 1-pfaE further include a promoter pTEF and a terminator XPR2.

5. The method for constructing DHA-producing Yarrowia lipolytica engineering bacteria according to claim 3, characterized in that: The primers used for constructing the T-pfaA in three sections are T-PFA1-1-F as shown in SEQ ID NO. 5, T-PFA1-1-R as shown in SEQ ID NO. 6, T-PFA1-2-F as shown in SEQ ID NO. 7, T-PFA1-2-R as shown in SEQ ID NO. 8, T-PFA1-3-F as shown in SEQ ID NO. 9, and T-PFA1-3-R as shown in SEQ ID NO.

10.

6. The method for constructing DHA-producing Y. lipolytica engineering bacteria according to claim 5, characterized in that: The endonuclease used for the double enzyme digestion is NheI, AvrII, ClaI or SalI; the ligase is T4 ligase; and the Yarrowia lipolytica is Yarrowia lipolytica PO1f.

7. The Yarrowia lipolytica engineering strain constructed by the construction method of any one of claims 1-6.

8. The use of the Yarrowia lipolytica engineering strain of claim 7 in the fermentation production of DHA.

9. A method of fermentative production of DHA, characterized by: The seed liquid of the Yarrowia lipolytica engineering strain of claim 7 is inoculated into SD-Leu C / N 80 nitrogen-limited fermentation medium for culture, and DHA is obtained after the culture and freeze-drying.

10. The method of claim 8, wherein: The culture conditions are 28-32°C, 200-250 r for 90-150 h; and the OD value of the seed liquid is 0.15-0.

35. The culture conditions are 28-32°C, 200-250 r for 90-150 h; and the OD value of the seed liquid is 0.15-0.35.

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