Yarrowia lipolytica with high yield of beta-carotene as well as construction method and application of yarrowia lipolytica

Abstract

The invention discloses yarrowia lipolytica with high yield of beta-carotene as well as a construction method and application of the yarrowia lipolytica. According to the yarrowia lipolytica engineering bacterium, geranyl diphosphate synthase gene xdGPS, phytoene dehydrogenase gene CarB, phytoene cyclization / phytoene synthesis bifunctional enzyme mutant gene GarRPY27R, acetyl-coenzyme A synthetase mutant gene ACSL641P and pyruvate ferredoxin oxidoreductase gene nifJ are integrated and expressed on a chromosome, and the yarrowia lipolytica engineering bacterium is obtained. Meanwhile, a 3-hydroxy-3-methylglutaryl CoA reductase gene HMGR (3-hydroxy-3-methylglutaryl CoA reductase) derived from the yarrowia lipolytica strain is subjected to overexpression; the yield of the beta-carotene is increased to 13.186 g / L, and the production efficiency is 0.14 g / L / h.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, specifically to a high-β-carotene-producing Yersinia lipolytica strain, its construction method, and its applications. Background Technology

[0002] Carotenoids are tetraterpenoid pigments naturally produced by some plants, algae, fungi, and bacteria. More than a thousand carotenoids are known, including β-carotene, lycopene, astaxanthin, and lutein. β-carotene possesses various physiological activities such as antioxidation, anti-aging, anti-tumor, immune enhancement, osteoporosis prevention, and arteriosclerosis prevention, showing broad application prospects in the food and pharmaceutical health fields. The carotenoid market was estimated at $1.5 billion in 2017 and is projected to grow at a CAGR of 5.7%. Currently, β-carotene is mainly synthesized chemically. Other methods include extraction from natural plant raw materials such as palm oil or carrots, and fermentation using natural producers (such as Dunaliella salina). Chemical synthesis processes are complex, highly polluting, and the products contain potentially harmful isomers. Plant extraction methods require large amounts of plant resources and are time-consuming and cumbersome. In contrast, microbial fermentation is not limited by raw materials or seasons and features short cycles, sustainability, and high efficiency. While natural microorganisms such as Dunaliella salina and Blakelyella trispora can be used for the fermentation production of β-carotene, the yield is low, limiting its industrial application. Therefore, it is necessary to construct recombinant strains that efficiently synthesize β-carotene using metabolic engineering and synthetic biology techniques. Yersinia lipolytica, a food-safe, unconventional yeast, is one of the ideal hosts for β-carotene synthesis.

[0003] A recombinant *Yersinia lipolytica* strain for β-carotene production, such as the high-β-carotene-producing *Yersinia lipolytica* GVD-A disclosed in patent publication number CN112831427A, involves using *Yersinia lipolytica* T1, a low-β-carotene-producing strain, as the starting strain. Endogenous key enzymes HMGs, HMGR, ERG20, GGS1, ERG8, ERG12, ERG19, and IDI are randomly integrated in multiple rounds. Exogenous GGPP synthase GPS (which can directly catalyze IPP and DMAPP into GGPP) and hemoglobin VHb are introduced. Finally, the key gene AAL8, which reduces liposome degradation, is knocked out to obtain *Yersinia lipolytica* engineered strain GVD-A with a β-carotene yield of 7.8 g / L. For example, the patent publication number CN114806914A discloses a method for constructing Yersinia lipolytica-IV, which produces high levels of β-carotene. This method involves heterologously introducing multiple copies of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), geranyl-geranyl diphosphate synthase (GPS), phytoene dehydrogenase (CarB), and phytoene synthase / phytoene cyclase (CarRP), and overexpressing acetyl-CoA carboxylase (ACC) and citrate lyase (ACL) to obtain Yersinia lipolytica-IV, which produces 2.7 g / L of β-carotene. However, existing technologies suffer from low yields, long fermentation cycles, and low production efficiency in the synthesis of β-carotene by Yersinia lipolytica. For example, the engineered strain GVD-A produces 7.8 g / L of β-carotene after 216 hours of fermentation, while the engineered strain Yli-IV produces 2.7 g / L of β-carotene after 144 hours of fermentation, which is still insufficient to meet the needs of industrial production.

[0004] Therefore, there is an urgent need to develop a more efficient β-carotene-producing Yersinia lipolytica to promote the large-scale industrial production of β-carotene. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a high-β-carotene-producing *Yersinia lipolytica* strain, its construction method, and its applications. This invention utilizes genetic engineering methods to heterologously express geranyl diphosphate synthase (GPS), phytoene dehydrogenase (CarB), a bifunctional enzyme mutant of phytoene cyclization / phytoene synthesis [GarRP(Y27R)], an acetyl-CoA synthase mutant ACS(L641P), and pyruvate ferroreductase (PFO) in the *Yersinia lipolytica* strain Po1f, as well as overexpressing 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) derived from the *Yersinia lipolytica* strain itself, thereby obtaining a high-β-carotene-producing recombinant *Yersinia lipolytica* strain ZPY-B7.

[0006] The technical solution of the present invention is as follows:

[0007] A high-β-carotene-producing *Yersinia lipolytica* engineered strain, wherein the chromosome of the engineered *Yersinia lipolytica* strain integrates and expresses the germyl diphosphate synthase gene xdGPS, the phytoene dehydrogenase gene CarB, and the phytoene cyclization / phytoene synthesis bifunctional enzyme mutant gene GarRP. Y27R And acetyl-CoA synthase mutant gene ACS L641P The pyruvate ferroreductase gene nifJ was used, and the 3-hydroxy-3-methylglutaryl-CoA reductase gene HMGR, derived from the *Yersinia lipolytica* strain, was overexpressed. The preservation number of the engineered *Yersinia lipolytica* strain is CCTCC NO: M 20251523.

[0008] Furthermore, the engineered Yeast lipolytica strain uses Yeast lipolytica Po1f as its host.

[0009] Further, the gene sequence of the gerany diphosphate synthase gene xdGPS is shown in SEQ ID NO.1; the gene sequence of the phytopene dehydrogenase gene CarB is shown in SEQ ID NO.2; and the gene sequence of the phytopene cyclization / phytopene synthesis bifunctional enzyme mutant gene GarRP is shown in SEQ ID NO.2. Y27R The gene sequence is shown in SEQ ID NO.3; the acetyl-CoA synthase mutant gene ACS L641PR The gene sequence is shown in SEQ ID NO.4; the pyruvate ferric reductase gene nifJ R The gene sequence is shown in SEQ ID NO.5; the gene sequence of the 3-hydroxy-3-methylglutaryl-CoA reductase gene HMGR is shown in SEQ ID NO.6.

[0010] A method for constructing the engineered *Yarrowia lipophila* strain, the method comprising the following steps:

[0011] (1) Construction of zpURA plasmid: Using pET28a plasmid as a template, the Kan-Ori fragment was amplified by PCR. At the same time, the 16S rDNA fragment, 28S rDNA fragment, Loxp-URA3-Loxp fragment, pTEF fragment and tLIP2 fragment were amplified by PCR from the genome of Yeastia lipolytica. Then, the Kan-Ori fragment, 16S rDNA fragment, 28S rDNA fragment, Loxp-URA3-Loxp fragment, pTEF fragment and tLIP2 fragment were homologously ligated by Gibson assembly to obtain zpURA plasmid.

[0012] (2) Construction of zpLEU plasmid: Using pET28a plasmid as a template, the Kan-Ori fragment was amplified by PCR. At the same time, the 16S rDNA fragment, 28S rDNA fragment, Loxp-LEU2-Loxp fragment, pTEF fragment and tLIP2 fragment were amplified by PCR from the genome of Yeastia lipolytica. Then, the Kan-Ori fragment, 16S rDNA fragment, 28S rDNA fragment, Loxp-LEU2-Loxp fragment, pTEF fragment and tLIP2 fragment were homologously ligated by Gibson assembly to obtain zpLEU plasmid.

[0013] (3) Construction of zpURA-xdGPS-CarB-GarRPY27R plasmid: xdGPS, CarB, and GarRP were digested with BamHI and AvrII restriction enzymes. Y27R Genes and zpURA plasmids were then ligated using T4 ligase to combine xdGPS, CarB, and GarRP. Y27R The genes were ligated into the zpURA plasmid to obtain the zpURA-xdGPS expression vector, CarB-zpURA expression vector, and GarRP expression vector, respectively. Y27R -zpURA expression vector, and then expressed using zpURA-xdGPS, CarB-zpURA expression vector, and GarRP respectively. Y27R Using the zpURA expression vector as a template, PCR amplification yielded pTEF-xdGPS-tLIP2 expression cassette, pTEF-CarB-tLIP expression cassette, and pTEF-GarRP expression cassette. Y27R -tLIP2 expression cassette; finally, using zpURA plasmid as a template, the pTEF-xdGPS-tLIP2 expression cassette, pTEF-CarB-tLIP expression cassette, and pTEF-GarRP expression cassette were assembled using the Gibson method. Y27R The -tLIP2 expression cassette is ligated to the zpURA plasmid to obtain zpURA-xdGPS-CarB-GarRP. Y27R plasmids;

[0014] (4) Construct zpLEU-HMGR-ACS L641P -nifJ plasmid: The HMGR fragment was amplified from the genome of *Yarrowia lipolyticis* by PCR. Using zpLEU plasmid as a template, the HMGR fragment was ligated to zpLEU plasmid using the Gibson assembly method to obtain zpLEU-HMGR. ACS was digested with BamHI and AvrII restriction enzymes. L641P The nifJ gene and zpLEU plasmid were then ligated using T4 ligase to connect the ACS gene. L641PThe nifJ gene was ligated into the zpLEU plasmid to obtain zpLEU-ACS. L641P Expression vectors, zpLEU-nifJ expression vector, and then zpLEU-HMGR and zpLEU-ACS expression vectors respectively. L641P Using expression vectors zpLEU-nifJ as templates, PCR amplification was performed to obtain pTEF-HMGR-tLIP2 expression cassettes and pTEF-ACS. L641P -tLIP expression cassette and pTEF-nifJ-tLIP2 expression cassette; finally, using zpLEU plasmid as a template, the pTEF-HMGR-tLIP2 expression cassette and pTEF-ACS expression cassette were assembled using the Gibson method. L641P The -tLIP expression cassette and pTEF-nifJ-tLIP2 expression cassette were ligated with the zpLEU plasmid to obtain zpLEU-HMGR-ACS. L641P -nifJ plasmid;

[0015] (5) Construction of recombinant strain ZPY-A: zpURA-xdGPS-CarB-GarRP was digested with Not I restriction endonuclease. Y27R The plasmid was linearized and then transformed into competent cells of Yeast lipophila po1f. After screening and PCR identification, a positive clone recombinant strain ZPY-A with the target gene integrated was obtained.

[0016] (6) Construction of recombinant strain ZPY-B: zpLEU-HMGR-ACS was digested with Not I restriction endonuclease. L641P The -nifJ plasmid was linearized and then transformed into competent cells of recombinant bacteria ZPY-A. After screening and PCR identification, a positive clone of recombinant bacteria ZPY-B with the target gene integrated was obtained.

[0017] (7) Screening for high β-carotene-producing strains: The PCR-verified positive recombinant strain ZPY-B was inoculated into YNB screening medium and cultured. After screening, the high β-carotene-producing Yersinia lipolytica engineered strain ZPY-B7 was obtained.

[0018] The application of the *Yersinia lipolytica* engineered strain or the *Yersinia lipolytica* engineered strain constructed by the construction method in the production of β-carotene.

[0019] Further, after activating the engineered Yersinia lipolytica, the seed culture was prepared by inoculating it into YPD liquid medium, and then the seed culture was inoculated into fermentation medium for continuous fed-batch fermentation to obtain β-carotene.

[0020] Furthermore, the seed culture solution is cultured at a temperature of 28–30°C for 24 hours.

[0021] 8. The application according to claim 6, wherein the fermentation medium is YPD liquid medium.

[0022] Furthermore, the temperature of the continuously fed fermentation is 30℃, the aeration rate is 3vvm, the stirring speed is 400-1000rpm, the dissolved oxygen content is 20%, and the pH is 6.8. Specifically, the continuously fed fermentation is as follows: during fermentation of 36-60h, the stirring speed is set to 600rpm, the aeration rate is 1vvm, and 400mL of 600g / L glycerol is added at a flow rate of 16mL / h; during fermentation of 60-120h, 500mL of 600g / L glucose is added at a flow rate of 8mL / h.

[0023] Furthermore, the yield of β-carotene is 13.186 g / L, and the production efficiency is 0.14 g / L / h.

[0024] The beneficial technical effects of this invention are as follows:

[0025] The MVA pathway is a key metabolic pathway for β-carotene synthesis, with acetyl-CoA being its core substrate. This patent, based on the expression of an exogenous β-carotene synthesis module, introduces heterologous pyruvate ferroreductase (PFO) and acetyl-CoA synthase to enhance the synthesis efficiency of acetyl-CoA. Furthermore, by overexpressing HMGR, the MVA pathway of *Yarrowia lipolytica* is strengthened, increasing the metabolic flux and biomass of β-carotene synthesis. A 5L fed-batch fermentation process is employed, resulting in a simple and efficient process. Ultimately, the recombinant strain achieved a β-carotene yield exceeding 13 g / L, reaching the highest reported level to date and demonstrating promising prospects for industrial application. Attached Figure Description

[0026] Figure 1 This is a schematic diagram of the zpURA plasmid structure.

[0027] Figure 2 This is a schematic diagram of the zpLEU plasmid structure.

[0028] Figure 3 zpURA-xdGPS-CarB-GarRP Y27R Schematic diagram of plasmid structure.

[0029] Figure 4 zpLEU-HMGR-ACS L641P Schematic diagram of the structure of the -nifJ plasmid.

[0030] Figure 5 This is a preliminary screening image of the recombinant bacteria in Example 4.

[0031] Figure 6The graph shows the β-carotene production of the original strain polf, recombinant strains ZPY-A, ZPY-B1 to ZPY-B10 in Example 4.

[0032] Figure 7 The graph shows the yield of β-carotene produced by the ZPY-B7 strain in Example 5 through continuous fed-batch fermentation.

[0033] Figure 8 This is a photograph of the ZPY-B7 strain used in Example 5 for continuous fed-batch fermentation to produce β-carotene. Detailed Implementation

[0034] The present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0035] The gene synthesis, primer synthesis, and sequencing in this invention were all outsourced to Wuxi Tianlin Biotechnology Co., Ltd.

[0036] Unless otherwise specified, the experimental methods used in the following embodiments of the present invention, including plasmid construction, enzyme digestion, preparation of competent cells, and transformation, are all conventional methods. Specific experimental conditions can be determined through simple experiments if necessary.

[0037] Unless otherwise specified, all materials and reagents used in the following embodiments of the present invention are commercially available.

[0038] The high-β-carotene-producing Yarrowia lipolytica strain ZPY-B7 described in the following embodiments of the present invention is deposited at the China Center for Type Culture Collection (CCTCC), with the strain name Yarrowia lipolyticaβ-s4, deposit address: Wuhan University, Bayi Road, Hongshan District, Wuhan City, Hubei Province, accession number: CCTCC NO: M 20251523, and deposit date: July 4, 2025.

[0039] The pET28a plasmid of this invention was purchased from Merck Life Sciences.

[0040] The genes involved in this invention include the geranylide diphosphate synthase gene xdGPS from Xanthophyllomyces dendrorhous, the phytoene dehydrogenase gene CarB from Mucor lusitanicus, and the mutant gene GarRP from Mucor lusitanicus, which is a bifunctional enzyme for phytoene cyclization / phytoene synthesis. Y27R ACS, a mutant gene of acetyl-CoA synthase from Salmonella enterica L641P The gene nifJ, a pyruvate ferroreductase from Trichormus variabilis, was synthesized after codon optimization.

[0041] The culture media used in this invention include LB solid medium, LB liquid medium, YPD liquid medium, and YPD solid medium, and the specific formulations of each medium are as follows:

[0042] LB solid medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, 20 g / L agar powder;

[0043] LB liquid medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride;

[0044] YPD liquid medium: 20 g / L tryptone, 10 g / L yeast extract, 20 g / L glucose;

[0045] YPD solid medium: 20 g / L tryptone, 10 g / L yeast extract, 20 g / L glucose, 15 g / L agar powder;

[0046] YNB selection medium: 1.7 g / L YNB, 10 g / L glucose, 5 g / L NH4Cl, 50 mM phosphate buffer pH 6.8 (pH 6.8), 2 g / L casein amino acids, 20 g / L agar powder.

[0047] The primers used in this invention are shown in Table 1.

[0048] Table 1 Primer Table

[0049]

[0050] Note: Homologous arms at both ends of the primer sequence are indicated by a single underscore, restriction enzyme sites are indicated by a single underscore in italics, and Loxp sites are indicated by a double underscore.

[0051] The xdGPS gene sequence in this invention is shown in SEQ ID NO.1; the CarB gene sequence is shown in SEQ ID NO.2; GarRP Y27R The gene sequence is shown in SEQ ID NO.3; ACS L641P The gene sequences are shown in SEQ ID NO.4; the nifJ gene sequence is shown in SEQ ID NO.5; and the HMGR gene sequence is shown in SEQ ID NO.6.

[0052] xdGPS, SEQ ID NO.1:

[0053]

[0054] CarB,SEQ ID NO.2:

[0055]

[0056] GarRP Y27R ,SEQ ID NO.3:

[0057]

[0058] ACS L641P ,SEQ ID NO.4:

[0059]

[0060] nifJ,SEQ ID NO.5:

[0061]

[0062] HMGR,SEQ ID NO.6:

[0063]

[0064] Example 1: Obtaining the target gene

[0065] (1) xdGPS: Based on the nucleotide sequence of the xdGPS gene (GenBank: DQ016502.1) from Xanthophyllomyces dendrorhous provided on NCBI, after codon optimization, the optimized xdGPS gene was synthesized by Wuxi Tianlin Biotechnology Co., Ltd. in accordance with the xdGPS gene sequence SEQ ID NO:1.

[0066] (2) CarB: Based on the nucleotide sequence of the phytoene dehydrogenase gene CarB (GenBank: AJ238028.1) from Mucor lusitanicus provided on NCBI, after codon optimization, the optimized CarB gene was synthesized by Wuxi Tianlin Biotechnology Co., Ltd. according to the CarB gene sequence SEQ ID NO:2.

[0067] (3) GarRP Y27R Based on the nucleotide sequence of the bifunctional enzyme gene GarRP (GenBank: AJ250827.1) from *Mucor lusitanicus*, provided on NCBI, a mutation site was introduced to change the amino acid at position 27 from tyrosine to arginine. After codon optimization, the sequence was then processed according to GarRP... Y27R GarRP, with gene sequence SEQ ID NO:3, was synthesized and optimized by Wuxi Tianlin Biotechnology Co., Ltd. Y27R Gene.

[0068] (4)ACS L641P Based on the nucleotide sequence of the acetyl-CoA synthase gene ACS (GenBank: CP074626.1) from Salmonella enterica provided by NCBI, a mutation site was introduced, changing the amino acid at position 641 from leucine to proline. After codon optimization, the sequence was then processed according to the ACS sequence. L641P Gene sequence SEQ ID NO:4 was synthesized and optimized by Wuxi Tianlin Biotechnology Co., Ltd. L641P Gene.

[0069] (5) nifJ: Based on the nucleotide sequence of the pyruvate ferroreductase gene nifJ (GenBank: CP000117.1) from Trichormus variabilis provided on NCBI, after codon optimization, the optimized nifJ gene was synthesized by Wuxi Tianlin Biotechnology Co., Ltd. in accordance with the nifJ gene sequence SEQ ID NO:5.

[0070] (6) HMGR: Based on the gene sequence (SEQ ID NO: 6) of the 3-hydroxy-3-methylglutaryl-CoA reductase gene HMGR (GenBank: XM_503558.1) of Yersinia lipolytica provided on NCBI, it was obtained from the Yersinia lipolytica Po1f genome by PCR using the primers in Table 1.

[0071] Example 2: Construction of plasmids

[0072] (1) Construction of zpURA plasmid

[0073] According to the 16S rDNA (GenBank: MW281683.1) and 28S rDNA (GenBank: MW281683.1) of Yersinia lipolytica Po1f provided by NCBI, GQ458023.1 ), URA3 (GenBank: XP_065950430.1), TEF promoter (GenBank: AF054508.1 The nucleotide sequence of the LIP2 terminator (GenBank: AJ012632.1) was used to obtain fragments 16S rDNA, 28S rDNA, Loxp-URA3-Loxp, pTEF, and tLIP2 from the genome of *Yarrowia lipolytica* using primers in Table 1 via PCR (PCR reaction system shown in Table 2, PCR amplification conditions shown in Table 3). Using pET28a plasmid as a template, the fragment Kan-Ori was obtained via PCR using primers in Table 1. The above fragments were then homologously recombined using the standard Gibson assembly method to prepare the zpURA plasmid. The structure of the zpURA plasmid is shown in [Table 1]. Figure 1 .

[0074] Table 2 PCR reaction system

[0075] composition concentration Volume (μL) Template (plasmid, genomic DNA) 20-60 ng / μL 0.5 upstream primer 10μM 0.5 Downstream primer 10μM 0.5 Phanta Flash Master Mix (Novizan) 2× 25 Ultrapure water - 23.5

[0076] Table 3 PCR amplification conditions

[0077]

[0078] (2) Construction of zpLEU plasmid

[0079] According to the 16S rDNA (GenBank: MW281683.1) and 28S rDNA (GenBank: MW281683.1) of Yersinia lipolytica Po1f provided by NCBI, GQ458023.1 ), LEU2 (GenBank: XM_501284.3), TEF promoter (GenBank: AF054508.1 The nucleotide sequence of the LIP2 terminator (GenBank: AJ012632.1) was used to obtain fragments 16S rDNA, 28S rDNA, Loxp-LEU2-Loxp, pTEF, and tLIP2 from the genome of *Yarrowia lipolytica* Po1f using primers in Table 1 (PCR reaction system shown in Table 2, PCR amplification conditions shown in Table 3). Using pET28a plasmid (preserved in the laboratory) as a template, the fragment Kan-Ori was obtained by PCR using primers in Table 1. Homologous recombination of the above fragments was performed using the standard Gibson assembly method to obtain plasmid zpLEU. The structure of the zpLEU plasmid is shown in [Table 1]. Figure 2 .

[0080] (3)zpURA-xdGPS-CarB-GarRP Y27R plasmid construction

[0081] xdGPS, CarB, and GarRP synthesized in Example 1 Y27R The gene was digested with BamHI and AvrII restriction enzymes and then ligated into zpURA plasmid vectors that had been digested with the same enzymes.

[0082] The specific method is as follows: 0.5 μL of restriction endonucleases BamHI and AvrII, 10 μL of xdGPS, CarB or GarRP are added respectively. Y27R Gene fragments, 2.0 μL of 10× buffer, and 7 μL of sterile water were added to an EP tube and incubated at 37°C for 2 hours to obtain xdGPS, CarB, and GarRP, respectively. Y27R Insert fragment;

[0083] 0.5 μL of restriction endonucleases BamHI and AvrII, 1.0 μg of zpURA plasmid expression vector, and 2.0 μL of 10× buffer were added to an EP tube, and sterile water was added to a final volume of 20 μL. The tube was incubated at 37°C for 2 h to obtain the zpURA plasmid vector fragment.

[0084] The xdGPS, CarB, and GarRP were ligated using T4 ligase. Y27RThe inserted fragments were ligated to the vector fragments of the zpURA plasmid; the ligation products were then transformed into E. coli DH5α competent cells and cultured overnight at 37°C. Single colonies were picked the following day and inoculated into LB broth, cultured on a shaker at 37°C for 12 hours, and the cells were collected. Plasmids were extracted and purified to obtain the xdGPS gene expression vector zpURA-xdGPS, the CarB gene expression vector zpURA-CarB, and GarRP. Y27R Gene expression vector zpURA-GarRP Y27R .

[0085] Subsequently, xdGPS gene expression vector zpURA-xdGPS, CarB gene expression vector zpURA-CarB, and GarRP were used respectively. Y27R Gene expression vector zpURA-GarRP Y27R Using the primers in Primer Table 1 as templates, expression cassette fragments pTEF-xdGPS-tLIP2, pTEF-CarB-tLIP, and pTEF-GarRP were obtained by PCR. Y27R -tLIP2; Using zpURA plasmid as a template, the expression vector fragment was obtained by PCR using primers 28S rDNA-f and LEU2-r in Primer Table 1; the fragment was homologously recombined using the conventional Gibson assembly method to obtain zpURA-xdGPS-CarB-GarRP. Y27R Plasmid, zpURA-xdGPS-CarB-GarRP Y27R The structure of the plasmid is shown in Figure 3 .

[0086] (4)zpLEU-HMGR-ACS L641P Construction of -nifJ plasmid

[0087] Based on the HMGR nucleotide sequence of *Yersinia lipolytica* provided on NCBI, the HMGR fragment was obtained from the *Yersinia lipolytica* genome by PCR using primers in Table 1 (PCR reaction system is shown in Table 2, PCR amplification conditions are shown in Table 3). Using zpLEU plasmid as a template, the zpLEU fragment was obtained by PCR using primers in Table 1. The above fragments were homologously recombined using the conventional Gibson assembly method to obtain the HMGR gene expression vector zpLEU-HMGR.

[0088] The ACS synthesized in Example 1 L641P The nifJ gene was digested with BamHI and AvrII restriction endonucleases and then ligated into zpLEU plasmid vectors that had been digested with the same enzymes.

[0089] The specific method is as follows: 0.5 μL of restriction endonuclease BamHI and Avr II, and 10 μL of ACS are added respectively. L641P Alternatively, the nifJ gene fragment, 2.0 μL of 10× buffer and 7 μL of sterile water were added to an EP tube, and the reaction was incubated at 37°C for 2 h to obtain ACS. L641P nifJ inserts fragments;

[0090] 0.5 μL of restriction endonucleases BamHI and AvrII, 1.0 μg of zpLEU plasmid expression vector, and 2.0 μL of 10× buffer were added to an EP tube, followed by the addition of sterile water to a final volume of 20 μL. The tube was incubated at 37°C for 2 h to obtain the zpLEU plasmid vector fragment.

[0091] The ACS was ligated using T4 ligase. L641P The nifJ insert fragment was ligated to the zpLEU plasmid vector fragment, respectively. The ligation product was transformed into E. coli DH5α competent cells and cultured overnight at 37°C. Single colonies were picked the next day and inoculated into LB broth, cultured on a shaker at 37°C for 12 h, and the cells were collected. The plasmid was extracted and purified to obtain ACS. L641P Gene expression vector zpLEU-ACS L641P , nifJ gene expression vector zpLEU-nifJ.

[0092] Subsequently, the HMGR gene was expressed using the vector zpLEU-HMGR, ACS, and other methods. L641P Gene expression vector zpLEU-ACS L641P Using the nifJ gene expression vector zpLEU-nifJ as a template, the expression cassette fragments pTEF-HMGR-tLIP2 and pTEF-ACS were obtained by PCR according to the primers in Table 1. L641P -tLIP2 and pTEF-nifJ-tLIP2; using plasmid zpLEU as a template, the expression vector fragment was obtained by PCR according to primers 28srDNA-f and LEU2r in Table 1; the above fragment was homologously recombined using the conventional Gibson assembly method to obtain plasmid zpLEU-HMGR-ACS. L641P -nifJ, zpLEU-HMGR-ACS L641P The structure of the -nifJ plasmid is shown in [see...]. Figure 4 .

[0093] Example 3 Construction of recombinant bacteria

[0094] (1) Construction of recombinant strain ZPY-A

[0095] a. Plasmid zpURA-GPS-CarB-GarRP Y27R linearization

[0096] zpURA-xdGPS-CarB-GarRP constructed in Example 2 was digested with Not I restriction endonuclease. Y27R Plasmids, the specific method is as follows:

[0097] 0.5 μL of restriction endonuclease Not I and 5 μL of zpURA-GPS-CarB-GarRP were added respectively. Y27R The plasmid expression vector, 1.0 μL of 10× buffer, and 3.5 μL of sterile water were added to an EP tube and incubated at 37°C for 1 h to prepare zpURA-GPS-CarB-GarRP. Y27R Linearized fragments;

[0098] b. Transformation of Yersinia lipophila po1f strain

[0099] After culturing the original Yersinia lipolyticis po1f strain overnight in YPD liquid medium, competent cells were prepared using the Zymogen Frozen EZ Yeast Transformation Kit II, and zpURA-GPS-CarB-GarRP was used. Y27R The linearized fragment was introduced into competent cells of *Yarrowia lipolyticis* for homologous recombination. Screening was performed using YNB selection medium. After incubation at 28°C for 3 days, single colonies were identified by PCR. Positive clones integrating the target gene were selected and named recombinant strain ZPY-A.

[0100] (2) Construction of recombinant strain ZPY-B

[0101] a. Preparation of zpLEU-HMGR-ACS L641P -nifJ linearized fragment:

[0102] zpLEU-HMGR-ACS constructed in Example 2 was digested with Not I restriction endonuclease. L641P The -nifJ plasmid, the specific method is as follows:

[0103] 0.5 μL of restriction endonuclease Not I and 5 μL of zpLEU-HMGR-ACS were respectively added. L641P The -nifJ expression vector, 1.0 μL of 10× buffer, and 3.5 μL of sterile water were added to an EP tube and incubated at 37°C for 1 h to obtain zpLEU-HMGR-ACS. L641P -nifJ linearizes fragments.

[0104] b. Transformation of Yersinia lipophila strain ZPY-A

[0105] After culturing Yersinia lipolyticis ZPY-A strain overnight in YPD liquid medium, competent cells were prepared using the Zymogen Frozen EZ Yeast Transformation Kit II, and zpLEU-HMGR-ACS were used. L641P The linearized -nifJ fragment was introduced into competent *Yarrowia lipophila* cells for homologous recombination. Screening was performed using YNB selection medium. After incubation at 28°C for 3 days, single colonies were identified by PCR. Positive clones integrating the target gene were selected and named recombinant strain ZPY-B.

[0106] Example 4: Screening of high-β-carotene-producing strains

[0107] Thirty PCR-verified positive recombinant bacteria, ZPY-B, were selected and inoculated onto YNB selection medium for 3 days. The color of the colonies was then compared and analyzed. Figure 5 As shown, 10 colonies exhibiting a deep orange-yellow color were initially screened and numbered ZPY-B1 to ZPY-B10. Subsequently, the recombinant bacteria ZPY-B1 to ZPY-B10 were inoculated into YPD liquid medium and cultured for 5 days to obtain fermentation broth. The β-carotene content in the fermentation broth of each recombinant bacteria was detected, with the original bacteria po1f and the recombinant bacteria ZPY-A serving as controls.

[0108] Extraction and detection method of β-carotene: Take 1 mL of fermentation broth sample and add it to a 2 mL centrifuge tube. Centrifuge at 8000×g for 5 min to collect the cells and discard the supernatant. Then, add 1 mL of 3N HCl to the centrifuge tube to resuspend the cells, vortex thoroughly for 3 min, place in a boiling water bath to lyse the cells for 3 min, immediately place on ice for 5 min, and centrifuge again at 8000×g for 5 min to collect the cells. Add 1 mL of sterile water to the tube to ensure that the cells are thoroughly cleaned, centrifuge at 8000×g for 5 min to collect the cells and discard the supernatant. Add 1 mL of acetone, shake to disrupt the cells for 3 min (with a 60 s interval between each 1 min of shaking), and then sonicate for 10 min until the cells turn white. Centrifuge at 8000×g for 5 min, collect the supernatant (combine the samples from 2-3 extractions), filter through a 0.22 μm organic membrane, and analyze by high performance liquid chromatography.

[0109] High-performance liquid chromatography (HPLC) detection conditions: Unitary C18 column (4.6 mm × 250 mm, 5 μm), mobile phase: acetonitrile and methanol (85:15, V / V), flow rate: 1.2 mL / min, column temperature: 30 °C, detection wavelength: 450 nm. Three biological replicates were performed on each sample. Quantitative analysis was conducted using β-carotene standards, and the β-carotene content in the sample was calculated using peak area.

[0110] The results are as follows Figure 6 As shown, no β-carotene production was detected in the original strain Po1f. Compared with the recombinant strain ZPY-A, the β-carotene production of recombinant strains ZPY-B1 to ZPY-B10 was significantly increased. Among them, strain ZPY-B7 had the highest β-carotene production, reaching up to 1.3 g / L after 72 h of YPD shake-flask fermentation.

[0111] Example 5: Production of β-carotene by fed-batch fermentation culture of ZPY-B7 strain

[0112] The ZPY-B7 strain obtained in Example 4 was picked up with an inoculation loop and streaked onto YPD solid medium to activate it. It was then cultured at 28–30°C for 36 hours. A single colony was then picked and inoculated into a 0.5L shake flask containing 50mL of YPD liquid medium. The flask was cultured at 30°C and 220rpm for 24 hours to obtain a seed culture. 50mL of the seed culture was then inoculated into a 5L fermenter containing 2L of medium for fed-batch fermentation. The fermenter was set to a temperature of 30°C, an aeration rate of 3 vvm, and a stirring speed of 400–1000rpm to maintain dissolved oxygen at 20% and pH at 6.8. Glucose concentration in the fermenter was periodically sampled and tested. After 36 hours of fermentation, the glucose concentration was below 2.0g / L, and the bacterial OD... 600 Once the temperature reaches 215, begin feeding carbon sources (glycerol and glucose) at a speed of 600 rpm and a flow rate of 1 vvm. From 36 to 60 h, feed 400 mL of 600 g / L glycerol at a flow rate of 16 mL / h. After 60 h, feed 500 mL of 600 g / L glucose at a flow rate of 8 mL / h until 120 h. Figure 7 and Figure 8 The fermentation results are shown, OD 600 The highest value reached 303, and the yield of β-carotene was highest at 96 hours, reaching 13.186 g / L.

[0113] The above description is merely a preferred embodiment of the present invention, and the present invention is not limited to the above embodiments. It is understood that other improvements and variations that are directly derived or conceived by those skilled in the art without departing from the spirit and concept of the present invention should be considered to be included within the protection scope of the present invention.

Claims

1. A high-yield β-carotene-producing engineered Yersinia lipolytica strain, characterized in that, The engineered *Yersinia lipolytica* strain integrates and expresses the following genes on its chromosome: xdGPS (gerany diphosphate synthase), CarB (phytoene dehydrogenase), and GarRP (phytoene cyclization / phytoene synthesis bifunctional enzyme mutant). Y27R And acetyl-CoA synthase mutant gene ACS L641P The pyruvate ferroreductase gene nifJ was used, and the 3-hydroxy-3-methylglutaryl-CoA reductase gene HMGR, derived from the *Yersinia lipolytica* strain, was overexpressed. The preservation number of the engineered *Yersinia lipolytica* strain is CCTCC NO: M 20251523.

2. The engineered *Yarrowia lipophila* strain according to claim 1, characterized in that, The engineered Yersinia lipolytica strain uses Yersinia lipolytica Po1f as its host.

3. The engineered *Yarrowia lipophila* strain according to claim 1, characterized in that, The gene sequence of the gerany diphosphate synthase gene xdGPS is shown in SEQ ID NO.1; the gene sequence of the phytoene dehydrogenase gene CarB is shown in SEQ ID NO.2; the gene sequence of the phytoene cyclization / phytoene synthesis bifunctional enzyme mutant gene GarRP is shown in SEQ ID NO.

1. Y27R The gene sequence is shown in SEQ ID NO.3; the acetyl-CoA synthase mutant gene ACS L641PR The gene sequence is shown in SEQ ID NO.4; the pyruvate ferric reductase gene nifJ R The gene sequence is shown in SEQ ID NO.5; the gene sequence of the 3-hydroxy-3-methylglutaryl-CoA reductase gene HMGR is shown in SEQ ID NO.

6.

4. A method for constructing the engineered *Yarrowia lipophila* strain according to claim 1, characterized in that, The construction method includes the following steps: (1) Construction of zpURA plasmid: Using pET28a plasmid as a template, the Kan-Ori fragment was amplified by PCR. At the same time, the 16S rDNA fragment, 28S rDNA fragment, Loxp-URA3-Loxp fragment, pTEF fragment and tLIP2 fragment were amplified by PCR from the genome of Yeastia lipolytica. Then, the Kan-Ori fragment, 16S rDNA fragment, 28S rDNA fragment, Loxp-URA3-Loxp fragment, pTEF fragment and tLIP2 fragment were homologously ligated by Gibson assembly to obtain zpURA plasmid. (2) Construction of zpLEU plasmid: Using pET28a plasmid as a template, the Kan-Ori fragment was amplified by PCR. At the same time, the 16S rDNA fragment, 28S rDNA fragment, Loxp-LEU2-Loxp fragment, pTEF fragment and tLIP2 fragment were amplified by PCR from the genome of Yeastia lipolytica. Then, the Kan-Ori fragment, 16S rDNA fragment, 28S rDNA fragment, Loxp-LEU2-Loxp fragment, pTEF fragment and tLIP2 fragment were homologously ligated by Gibson assembly to obtain zpLEU plasmid. (3) Construction of zpURA-xdGPS-CarB-GarRPY27R plasmid: xdGPS, CarB, and GarRP were digested with BamHI and AvrII restriction enzymes. Y27R Genes and zpURA plasmids were then ligated using T4 ligase to combine xdGPS, CarB, and GarRP. Y27R The genes were ligated into the zpURA plasmid to obtain the zpURA-xdGPS expression vector, CarB-zpURA expression vector, and GarRP expression vector, respectively. Y27R -zpURA expression vector, and then expressed using zpURA-xdGPS, CarB-zpURA expression vector, and GarRP respectively. Y27R Using the zpURA expression vector as a template, PCR amplification yielded pTEF-xdGPS-tLIP2 expression cassette, pTEF-CarB-tLIP expression cassette, and pTEF-GarRP expression cassette. Y27R -tLIP2 expression cassette; finally, using zpURA plasmid as a template, the pTEF-xdGPS-tLIP2 expression cassette, pTEF-CarB-tLIP expression cassette, and pTEF-GarRP expression cassette were assembled using the Gibson method. Y27R The -tLIP2 expression cassette is ligated to the zpURA plasmid to obtain zpURA-xdGPS-CarB-GarRP. Y27R plasmids; (4) Construct zpLEU-HMGR-ACS L641P -nifJ plasmid: The HMGR fragment was amplified from the genome of *Yarrowia lipolyticis* by PCR. Using zpLEU plasmid as a template, the HMGR fragment was ligated to zpLEU plasmid using the Gibson assembly method to obtain zpLEU-HMGR. ACS was digested with BamHI and Avr II restriction enzymes. L641P The nifJ gene and zpLEU plasmid were then ligated using T4 ligase to connect the ACS gene. L641P The nifJ gene was ligated into the zpLEU plasmid to obtain zpLEU-ACS. L641P Expression vectors, zpLEU-nifJ expression vector, and then zpLEU-HMGR and zpLEU-ACS expression vectors respectively. L641P Using expression vectors zpLEU-nifJ as templates, PCR amplification was performed to obtain pTEF-HMGR-tLIP2 expression cassettes and pTEF-ACS. L641P -tLIP expression cassette and pTEF-nifJ-tLIP2 expression cassette; finally, using zpLEU plasmid as a template, the pTEF-HMGR-tLIP2 expression cassette and pTEF-ACS expression cassette were assembled using the Gibson method. L641P The -tLIP expression cassette and pTEF-nifJ-tLIP2 expression cassette were ligated with the zpLEU plasmid to obtain zpLEU-HMGR-ACS. L641P -nifJ plasmid; (5) Construction of recombinant strain ZPY-A: zpURA-xdGPS-CarB-GarRP was digested with Not I restriction endonuclease. Y27R The plasmid was linearized and then transformed into competent cells of Yeast lipophila po1f. After screening and PCR identification, a positive clone recombinant strain ZPY-A with the target gene integrated was obtained. (6) Construction of recombinant strain ZPY-B: zpLEU-HMGR-ACS was digested with Not I restriction endonuclease. L641P The -nifJ plasmid was linearized and then transformed into competent cells of recombinant bacteria ZPY-A. After screening and PCR identification, a positive clone of recombinant bacteria ZPY-B with the target gene integrated was obtained. (7) Screening for high β-carotene-producing strains: The PCR-verified positive recombinant strain ZPY-B was inoculated into YNB screening medium and cultured. After screening, the high β-carotene-producing Yersinia lipolytica engineered strain ZPY-B7 was obtained.

5. The use of the engineered *Yersinia lipolytica* strain according to claim 1 or the engineered *Yersinia lipolytica* strain constructed by the construction method according to claim 4 in the production of β-carotene.

6. The application according to claim 5, characterized in that, After activating the engineered Yersinia lipolytica, the seed culture was prepared by inoculating it into YPD liquid medium. The seed culture was then inoculated into a fermentation medium for continuous fed-batch fermentation to obtain β-carotene.

7. The application according to claim 6, characterized in that, The seed culture solution was cultured at a temperature of 28–30°C for 24 hours.

8. The application according to claim 6, characterized in that, The fermentation medium is YPD liquid medium.

9. The application according to claim 6, characterized in that, The continuous fed fermentation is carried out at a temperature of 30°C, an aeration rate of 3 vvm, a stirring speed of 400–1000 rpm, a dissolved oxygen content of 20%, and a pH of 6.

8. Specifically, the continuous fed fermentation is carried out as follows: during fermentation of 36–60 h, the stirring speed is set to 600 rpm, the aeration rate is 1 vvm, and 400 mL of 600 g / L glycerol is added at a flow rate of 16 mL / h. During fermentation of 60–120 h, 500 mL of 600 g / L glucose is added at a flow rate of 8 mL / h.

10. The application according to claim 5, characterized in that, The yield of β-carotene was 13.186 g / L, and the production efficiency was 0.14 g / L / h.

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