Yarrowia lipolytica strain for synthesizing delta-tocotrienol and application of Yarrowia lipolytica strain

Abstract

The invention discloses a Yarrowia lipolytica strain for synthesizing delta-tocotrienol and application of the Yarrowia lipolytica strain, and belongs to the technical field of genetic engineering and bioengineering. According to the invention, a metabolic pathway of the delta-tocotrienol is constructed in the Yarrowia lipolytica, a mutant of a key enzyme, i.e., the uromelanic acid phytase, is screened, and the yield of the delta-tocotrienol is further increased by strengthening a shikimic acid pathway. The yield of delta-tocotrienol of the constructed engineering strain can reach 466.8 mg / L, a foundation is laid for subsequent biosynthesis of vitamin E compounds, and potential value and significance are achieved for development of synthetic biology.

Description

Technical Field

[0001] The invention relates to a Yarrowia lipolytica strain for synthesizing delta-tocotrienol and application thereof, belonging to the technical field of genetic engineering and bioengineering. Background Art

[0002] Vitamin E is the sum of tocopherol and tocotrienol, which is divided into four isomers: α, β, γ and δ. It has anti-aging, antioxidant, and anti-tumor effects, and is widely used in food, medicine, and health care. Previous studies have focused on α-tocopherol because it has the highest activity, the widest distribution, and the most representative. However, recent studies have found that tocotrienol has better functions than α-tocopherol in some aspects. Because tocotrienol contains unsaturated side chains, it can more effectively penetrate into tissues containing saturated fatty acid layers, thereby playing a good antioxidant and free radical scavenging effect. Different forms of tocotrienol differ in physiological activity, chemical activity, and bioavailability, and therefore have different functions. α-Tocotrienol can lower cholesterol by inhibiting 3-hydroxy-3-methylglutaryl coenzyme A reductase. γ-Tocotrienol and δ-tocotrienol are considered to be potential drugs for the treatment of cancer and have a good inhibitory effect on cancer cells. Compared with the other three tocotrienols, δ-tocotrienol has many unique biological functions, such as stronger free radical scavenging activity and anti-inflammatory effects. In addition, δ-tocotrienol has been shown to be able to induce and inhibit the proliferation of pancreatic cancer cells and is a potential effective ingredient for the treatment of pancreatic cancer. Therefore, it is of great significance to construct an efficient synthesis pathway for δ-tocotrienol.

[0003] The traditional chemical method is difficult to prepare δ-tocotrienol, and usually produces side reactions, resulting in reduced selectivity of the target compound. Therefore, in view of the limitations of natural product extraction and chemical synthesis of δ-tocotrienol in terms of product stability, quality safety and price, the rapidly developing metabolic engineering and synthetic biology can provide ideas for the synthesis of δ-tocotrienol. Summary of the invention

[0004] The present invention provides a homogentisate phytyltransferase SyHPT mutant, wherein one or more sites of the 61st, 77th and 146th positions are mutated on the basis of the parent.

[0005] In one embodiment, the mutation comprises mutating isoleucine at position 61, lysine at position 77, and / or isoleucine at position 146 to alanine.

[0006] In one embodiment, the mutation comprises mutating lysine at position 77 to aspartic acid, glutamic acid, tryptophan, tyrosine or phenylalanine.

[0007] The present invention also provides a gene encoding the mutant.

[0008] The present invention also provides a recombinant microorganism expressing the mutant.

[0009] The invention provides an engineered strain of Yarrowia lipolytica for efficiently synthesizing delta-tocotrienol. The strain is Yarrowia lipolytica Δku70, which is used as a starting strain and expresses 4-hydroxyphenylpyruvatedioxygenase (HPD), tocopherol cyclase, and homogentisate phytyltransferase or a mutant of the homogentisate phytyltransferase.

[0010] In one embodiment, the 4-hydroxyphenylpyruvate dioxygenase is 4-hydroxyphenylpyruvate dioxygenase Y1HPD from Yarrowia lipolytica or 4-hydroxyphenylpyruvate dioxygenase PaHPD from Pseudomonas putida.

[0011] In one embodiment, the homogentisate phytyltransferase is the homogentisate phytyltransferase TrHPT derived from wheat, or the homogentisate phytyltransferase SyHPT derived from Synechocystis, or a mutant obtained by mutating one or more of positions 61, 77, and 146 based on the homogentisate phytyltransferase SyHPT from Synechocystis.

[0012] In one embodiment, the mutation includes but is not limited to mutating isoleucine at position 61, lysine at position 77, and / or isoleucine at position 146 to alanine; or mutating lysine at position 77 to aspartic acid, glutamic acid, tryptophan, tyrosine or phenylalanine.

[0013] In one embodiment, the tocopherol cyclase is tocopherol cyclase AtVTE1 from Arabidopsis thaliana.

[0014] In one embodiment, the engineered Yarrowia lipolytica also has one or more of the following improvements:

[0015] (a) Overexpression of shikimate pathway genes ARO1, ARO4, ARO7 and the mutant ARO4 with tyrosine feedback inhibition K221L and ARO7 G139S One or more genes in

[0016] (b) overexpressing key genes of the MVA pathway, including: one or more of the following: GGPP synthase gene (Geranylgeranyldiphosphate synthase, GGPS) YlGGPS, mevalonate kinase gene (Mevalonate kinase, ERG12) ERG12, truncated hydroxymevalonate CoA reductase gene tHMG1, isopentenyldiphosphate delta-isomerase gene (Isopentenyldiphosphate delta-isomerase, IDI 1) IDI 1, farnesyl diphosphate synthase gene (Farnesylpyrophosphate synthetase, ERG20) YlERG20, GGPP synthase gene SaGGPS from Sulfolobus acidocaldarius or GGPP synthase gene XdGGPS from Xanthophyllomyces dendrorhous;

[0017] (c) SyHPT and AtVTE1 were fused using a linker peptide and expressed in Y. lipolytica.

[0018] In one embodiment, the rigid linker peptide comprises TPTP, (TPTP) 2 , EAAAK, or (EAAAK) 2 ; The flexible connecting peptide includes GSG, (GSG) 2 , GGGGS and / or (GGGGS) 2 .

[0019] In one embodiment, the engineered Yarrowia lipolytica strain expresses 4-hydroxyphenylpyruvate dioxygenase PaHPD from Pseudomonas putida, homogentisate phytyltransferase TrHPT from wheat, and tocopherol cyclase AtVTE from Arabidopsis thaliana.

[0020] In one embodiment, the engineered Yarrowia lipolytica strain expresses 4-hydroxyphenylpyruvate dioxygenase PaHPD from Pseudomonas putida, homogentisate phytyltransferase SyHPT from Synechocystis sp., and tocopherol cyclase AtVTE from Arabidopsis thaliana.

[0021] In one embodiment, the engineered Y. lipolytica strain expresses 4-hydroxyphenylpyruvate dioxygenase YlHPD derived from Y. lipolytica, homogentisate phytyltransferase TrHPT derived from wheat, and tocopherol cyclase AtVTE derived from Arabidopsis thaliana.

[0022] In one embodiment, the engineered Y. lipolytica strain expresses 4-hydroxyphenylpyruvate dioxygenase YlHPD from Y. lipolytica, homogentisate phytyltransferase SyHPT from Synechocystis, and tocopherol cyclase AtVTE from Arabidopsis thaliana.

[0023] In one embodiment, the engineered Yarrowia lipolytica strain also overexpresses the shikimate pathway gene ARO1.

[0024] In one embodiment, the engineered Yarrowia lipolytica strain also overexpresses shikimate pathway genes ARO1 and ARO4.

[0025] In one embodiment, the engineered Yarrowia lipolytica strain also overexpresses shikimate pathway genes ARO1 and ARO7.

[0026] In one embodiment, the engineered Yarrowia lipolytica also overexpresses the shikimic acid pathway gene ARO4.

[0027] In one embodiment, the engineered Yarrowia lipolytica strain also overexpresses shikimate pathway genes ARO4 and ARO7.

[0028] In one embodiment, the engineered Yarrowia lipolytica also overexpresses the shikimate pathway gene ARO7.

[0029] In one embodiment, the engineered Yarrowia lipolytica strain also overexpresses shikimate pathway genes ARO1, ARO4, and ARO7.

[0030] In one embodiment, the engineered yeast Yarrowia lipolytica also overexpresses the shikimate pathway gene ARO1 and a mutant ARO4 that is feedback inhibited by tyrosine. K221L and ARO7 G139S .

[0031] In one embodiment, the engineered Yarrowia lipolytica also overexpresses the mevalonate kinase gene ERG12 and the GGPP synthase gene YlGGPS.

[0032] In one embodiment, the engineered Yarrowia lipolytica strain also overexpresses the mevalonate kinase gene ERG12, the GGPP synthase gene YlGGPS, the truncated hydroxymevalonate CoA reductase gene tHMG1, and the isoprene diphosphate isomerase gene IDI 1.

[0033] In one embodiment, the engineered Y. lipolytica strain also overexpresses the farnesyl diphosphate synthase gene YlERG20 and the GGPP synthase gene SaGGPS derived from Sulfolobus acidocaldarius.

[0034] In one embodiment, the engineered Y. lipolytica strain also overexpresses the farnesyl diphosphate synthase gene YlERG20 and the GGPP synthase gene XdGGPS from Xanthophyllomyces dendrorhous.

[0035] In one embodiment, the engineered yeast Yarrowia lipolytica also expresses a fusion of homogentisate phytyltransferase SyHPT and tocopherol cyclase AtVTE from Arabidopsis thaliana; the amino acid sequence of the homogentisate phytyltransferase SyHPT is as shown in SEQ ID NO.6, or has a K77Y mutation based on the amino acid sequence shown in SEQ ID NO.6.

[0036] In one embodiment, the nucleotide sequence of the gene 4-hydroxyphenylpyruvate dioxygenase Y1HPD from Yarrowia lipolytica is shown as SEQ ID NO.1.

[0037] In one embodiment, the nucleotide sequence of the gene 4-hydroxyphenylpyruvate dioxygenase PaHPD from Pseudomonas putida is shown as SEQ ID NO.2.

[0038] In one embodiment, the gene YlHPD or gene PaHPD is integrated at the D17 site.

[0039] In one embodiment, the nucleotide sequence encoding the gene homogentisate phytyltransferase TrHPT derived from wheat is shown as SEQ ID NO.3.

[0040] In one embodiment, the nucleotide sequence encoding the gene homogentisate phytyltransferase SyHPT from Synechocystis is shown as SEQ ID NO.4.

[0041] In one embodiment, the nucleotide sequence encoding the gene tocopherol cyclase AtVTE1 derived from Arabidopsis thaliana is shown as SEQ ID NO.5.

[0042] In one embodiment, the gene TrHPT or the gene SyHPT and AtVTE1 are integrated at the E4 site.

[0043] In one embodiment, the nucleotide sequences of genes ARO1, ARO4, and ARO7 are shown in SEQ ID NO.7, SEQ ID NO.8, and SEQ ID NO.9, respectively; K221L and ARO7 G139S The nucleotide sequences are shown in SEQ ID NO.17 and SEQ ID NO.18 respectively.

[0044] In one embodiment, the nucleotide sequences of genes YlGGPS, ERG12, tHMG1, IDI 1, ERG20, SaGGPS and XdGGPS are shown as SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15 and SEQ ID NO.16, respectively.

[0045] The present invention also provides the use of the Yarrowia lipolytica engineered bacteria in producing delta-tocotrienol.

[0046] In one embodiment, the engineered Yarrowia lipolytica is fermented in a fermentation medium at 28-30° C. for at least 72 hours.

[0047] In one embodiment, the fermentation is performed for 72 to 120 hours, or 96 to 120 hours.

[0048] In one embodiment, the fermentation medium is YPD medium or a medium containing the following components: peptone, yeast extract, metal ions, vitamins, ferrous sulfate and glucose.

[0049] In one embodiment, glucose is also fed during the fermentation process.

[0050] The present invention also claims to protect the application of the engineered Yarrowia lipolytica or the method in producing products containing delta-tocotrienol in the fields of food, medicine and chemical industry.

[0051] In one embodiment, the use is for preparing a vaccine or a drug containing delta-tocotrienol, or preparing a cosmetic containing delta-tocotrienol.

[0052] Beneficial effects:

[0053] (1) The present invention uses the engineered bacteria of Yarrowia lipolytica PO1f-Δku70 (MatA, Δku70::leu2-270, ura3-302, xpr2-322, axp1-2) as a host, and constructs the biosynthetic pathway of δ-tocotrienol for the first time by screening and expressing genes of the δ-tocotrienol synthesis pathway from different sources.

[0054] (2) The present invention strengthens the supply of homogentisic acid (HGA) and geranylgeranyl diphosphate (GGPP), the precursors of delta-tocotrienol, by overexpressing key genes of the shikimate pathway and the mevalonic acid (MVA) pathway.

[0055] (3) The present invention utilizes a rigid / flexible connecting peptide to fuse and express the key enzymes of δ-tocotrienol, homogentisate phytyltransferase (HPT) and tocopherol cyclase (VTE1), thereby further increasing the yield of δ-tocotrienol.

[0056] (4) The present invention transforms the rate-limiting enzyme HPT through semi-rational design to obtain a new mutant SyHPT K77Y , the mutant was integrated into the multi-copy site, so that the δ-tocotrienol production reached 189.9 mg / L.

[0057] (5) The present invention also achieved the expanded production of δ-tocotrienol in a 5L bioreactor, with the yield reaching 466.8 mg / L, laying the foundation for the subsequent synthesis of vitamin E compounds. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 Schematic diagram of the heterologous synthesis of δ-tocotrienol in Yarrowia lipolytica; the biosynthetic pathway of δ-tocotrienol can be divided into three metabolic modules: the shikimic acid pathway, the MVA pathway, and the δ-tocotrienol biosynthetic pathway. The shikimic acid pathway is shown in the pink box, the MVA pathway is shown in the flesh-colored box, and the δ-tocotrienol biosynthetic pathway is shown in the blue box.

[0059] Figure 2 The results of HPLC comparison between the fermentation broth of △Ku70-4 strain and the δ-tocotrienol standard.

[0060] Figure 3 The LC-MS test comparison results of the fermentation broth of the △Ku70-4 strain and the δ-tocotrienol standard; wherein A is the LC-MS test result of the δ-tocotrienol standard; B is the LC-MS test result of the fermentation broth of the △Ku70-4 strain.

[0061] Figure 4 This is a graph showing the δ-tocotrienol production of the engineered strains Δku70-1 to Δku70-4 under YPD culture.

[0062] Figure 5 This is a graph showing the δ-tocotrienol production of the engineered strains Δku70-4, VE-1 to VE-8 under YPD culture.

[0063] Figure 6 Figure 3. Delta-tocotrienol production of engineered strains with enhanced MVA pathway in YPD culture.

[0064] Figure 7This is a graph showing the δ-tocotrienol production of engineered strains VE-12 to VE-20 under YPD culture.

[0065] Figure 8 The protein structure and mutation results of SyHPT; A is the spatial structure of the protein, B to D are the effects of expressing different variants; the blue line in E and F represents the wild-type SyHPT (WT), and the red line represents the mutant K77Y.

[0066] Fig. 9 This is a graph showing the production of δ-tocotrienol by engineered strain VE-23 in a 5L fermenter by fed-batch fermentation. DETAILED DESCRIPTION

[0067] (I) Culture medium

[0068] LB medium: peptone 10g / L, yeast powder 5g / L, sodium chloride 10g / L. Add 20g / L agar powder to prepare LB solid medium.

[0069] YNB medium: Yeast Nutrition Base 67.4 g / L, glucose 20 g / L, amino acids (5 g / L uracil, 10 g / L tryptophan, 10 g / L leucine, 10 g / L histidine, and appropriate deletion of corresponding amino acids as needed).

[0070] YPD medium: peptone 20 g / L, yeast powder 10 g / L, glucose 20 g / L.

[0071] (II) Preparation of competent cells of Y. lipolytica: The competent cells of Y. lipolytica were prepared using the Frozen-EZYeast Transformation II kit and cultured with 5 mL YPD medium at 30°C until the OD 600 =0.8-1.0). The following steps were carried out at room temperature.

[0072] 1. Centrifuge the cells at 3500 rpm for 5 min and remove the supernatant;

[0073] 2. Add 10 mL of EZ1 solution to wash the precipitate, centrifuge the precipitated cells again, and aspirate the supernatant;

[0074] 3. Add 1 mL of EZ2 solution to resuspend the pelleted cells.

[0075] (III) Transformation of Yarrowia lipolytica:

[0076] 1. Scrape the yeast colony from the YPD plate and inoculate it into YPD liquid medium, then place it in a constant temperature incubator at 30°C and grow for 16-22 hours;

[0077] 2. Prepare yeast transformation buffer solution (if multiple transformations are required, magnification × n): 50% PEG3350, added in a volume of 90 μL; 2M lithium acetate (M CHCOOLi 65.99), volume 5 μL; ssDNA, volume 5 μL, ssDNA needs to be boiled in water for 3 min before adding to the transformation buffer, and then put on ice for cooling;

[0078] 3. Use a pipette to draw 500 μL of bacterial solution into a centrifuge tube, centrifuge the tube at an appropriate speed, and discard the supernatant to obtain yeast cells. Transfer the yeast to a centrifuge tube containing 100 μL of transformation buffer, slowly blow and mix, add 0.25-0.5 μg of plasmid DNA or linear DNA, mix thoroughly and oscillate for 2 minutes (add at least 0.25-0.5 μg of DNA to each plasmid);

[0079] 4. Incubate the centrifuge tube containing the transformation mixture in a metal bath at 30°C for 30-45 min, oscillate the mixture for 15 s every 10 min, and then heat shock at 39°C for an additional 10 min to improve transformation efficiency;

[0080] 5. Transfer the centrifuge tube to a centrifuge and centrifuge at 8000 rpm for 2 minutes. Pour out the supernatant in a sterile clean bench, add an appropriate amount of sterile water to resuspend the bacteria, and add it to the YPD solid medium. Use a sterile coating rod to evenly spread on the YPD solid medium, and then place the medium in a constant temperature incubator at 30°C for cultivation.

[0081] (IV) HPLC determination of δ-tocotrienol: The determination was performed using a Shimadzu high performance liquid chromatograph equipped with a variable wavelength detector and an Agilent ZORBAX Nikpase XDB-C18 column. HPLC conditions: Column: InertSustain C18 250 mm × 4.6 mm column (particle size 5 μm); Mobile phase A, ultrapure water containing 1‰ trifluoroacetic acid; Mobile phase B, acetonitrile containing 1‰ trifluoroacetic acid; Pump A flow rate 0.02 mL / min, Pump B flow rate 0.98 mL / min, isocratic elution 26 min; Column temperature: 40°C; Injection volume: 10 μL; Detector wavelength: 280 nm.

[0082] (V) HPLC determination of homogentisate: The analysis was performed using a Shimadzu LC-20AT high performance liquid chromatography system equipped with a variable wavelength detector and an Agilent ZORBAX Nikpase XDB-C18 column. The mobile phase was water and acetonitrile with 0.1% trifluoroacetic acid added, with a total flow rate of 1 mL / min, an injection volume of 10 μL, binary high pressure gradient elution for 25 min, and a detection wavelength of 292 nm.

[0083] (VI) The strain information is shown in Table 1.

[0084] Table 1 Strains involved in the present invention

[0085]

[0086]

[0087] Example 1: Construction of the synthesis pathway of delta-tocotrienol in Yarrowia lipolytica

[0088] In order to synthesize δ-tocotrienol, TrHPT from wheat and SyHPT from Synechocystis were introduced to catalyze the formation of 2-methyl-6-geranylgeranyl-benzoquinone (MGGBQ) from homogentisic acid (HGA) and geranylgeranyl pyrophosphate (GGPP); further, AtVTE1 from Arabidopsis thaliana was introduced to catalyze MGGBQ to form δ-tocotrienol. All heterologous genes were commissioned to be synthesized by the company to obtain the plasmid template containing the target gene. First, we used Snapgene software to design primers for heterologous genes, and used primers YLHPD-F / YLHPD-R from the genome of Yarrowia lipolytica PO1f-Δku70 (published in the paper "Combining 26s rDNAand the Cre-loxP System for Iterative Gene Integration and Efficient Marker Curation in Yarrowia lipolitica》) to amplify the endogenous gene YlHPD, the gene PaHPD was amplified from a commissioned synthetic plasmid template containing the target gene using primers PAHPD-F / PAHPD-R, the gene TrHPT was amplified from a commissioned synthetic plasmid template containing the target gene using primers TrHPT-F / TrHPT-R, the gene SyHPT was amplified from a commissioned synthetic plasmid template containing the target gene using primers SyHPT-F / SyHPT-R, the gene AtVTE1 was amplified from a commissioned synthetic plasmid template containing the target gene using primers AtVTE1-F / AtVTE1-R, the homology arms of the D17 site and the E4 site (published in the paper "Remodelling metabolism for high-level resveratrol production in Yarrowialipolytica") were linked using primers D17-armup-F / D17-armup-R and E4-armup-F / E4-armdown-R (the upstream homology arm sequence of the D17 site is shown in SEQ ID NO.19, the length of the downstream homology arm is shown in SEQ ID NO.20; the upstream homology arm sequence of the E4 site is shown in SEQ ID NO.21, and the downstream homology arm sequence is shown in SEQ ID NO.22) and the fragment formed by the target gene are PCR amplified together, and the PCR amplified fragment and the Cas9 plasmid of the corresponding site are added during the yeast transformation process. The fragment containing the promoter P TEF -YlHPD-T XPR2 or P TEF -PaHPD-T XPR2 Integrate into the D17 site of the strain and insert the promoter fragment P TEF -TrHPT-TXPR2 / P TEF -SyHPT-T XPR2 or P TEF -AtVTE1-T XPR2 The genes of the above-mentioned δ-tocotrienol biosynthesis pathway were integrated into the E4 site of the strain, and four engineered strains were obtained: Δku70-1 (PaHPD-TrHPT-AtVTE1), Δku70-2 (PaHPD-SyHPT-AtVTE1), Δku70-3 (YlHPD-TrHPT-AtVTE1) and Δku70-4 (YlHPD-SyHPT-AtVTE1) to screen out the optimal gene combination.

[0089] The constructed Δku70-1-Δku70-4 engineered strains were fermented in YPD medium at 30°C and 220rpm for 96h, and 10% olive oil was added to the medium after 24h of fermentation to extract δ-tocotrienol. After the fermentation was completed, the fermentation broth and the organic phase were poured into a 50mL centrifuge tube and centrifuged at 12000rpm for 5min in a centrifuge. The upper organic phase was poured into a 5mL centrifuge tube and centrifuged at 12000rpm for 5min. Subsequently, 100μL of the organic phase was dissolved in 900μL of dimethyl sulfoxide and diluted 10 times, and filtered through a filter membrane into a liquid phase bottle for detection. After LC-MS identification, the results showed that all four strains achieved the synthesis of δ-tocotrienol, such as Figures 2-3 The gene combination YlHPD-SyHPT-AtVTE1 showed the highest δ-tocotrienol production, reaching 35.1 mg / L ( Figure 4 ).

[0090] All primers are listed in Table 2.

[0091] Table 2 Primer sequences

[0092]

[0093] Example 2: Increasing HGA supply by strengthening the shikimate pathway

[0094] To promote the supply of the precursor homogentisate, the endogenous genes ARO1 (nucleotide sequence as shown in SEQ ID NO.7), ARO4 (nucleotide sequence as shown in SEQ ID NO.8), and ARO7 (nucleotide sequence as shown in SEQ ID NO.9) of Yarrowia lipolytica were overexpressed, and primers ARO1-F / ARO1-R were used to extract the nucleotide sequence of Yarrowia lipolytica PO1f-Δku70 genome (published in the paper "Combining 26s rDNA and the Cre-loxP System for Iterative Gene Integration and Efficient Marker Curation in Yarrowia lipolytica"). lipolitica》) were used to amplify the gene ARO1, the gene ARO4 was amplified from the Yarrowia lipolytica genome with primers ARO4-F / ARO4-R, the gene ARO7 was amplified from the Yarrowia lipolytica genome with primers ARO7-F / ARO-R, and the upstream and downstream homology arms of the A3 site were amplified with primers A3-armup-F / A3-armdown-R (the upstream homology arm length was 1181 bp, and the downstream homology arm length was 1200 bp). Synthetic gene ARO4 K221L and ARO7 G139S (The nucleotide sequences are shown in SEQ NO.17 and SEQ NO.18, respectively). About 1 μg of the integration fragment and about 600 ng of sgRNA were transformed into the Yeast lipolytica engineered strain Δku70-4 constructed in Example 1 using the yeast transformation kit Frozen-EZ Yeast Transformation II, spread on the screening solid culture medium, and cultured at 30°C for 3 days until colonies appeared. The obtained VE-1-VE-8 engineered strains were fermented in YPD medium at 30°C and 220rpm for 96h, and 10% olive oil was added to extract δ-tocotrienol for HPLC analysis. The analysis results showed that ( Figure 5 ), the production of δ-tocotrienol of the recombinant engineering bacteria VE-7 was the highest, which was 58.2 mg / L.

[0095] Table 3 Primer sequences

[0096]

[0097] Example 3: Enhancing MVA pathway flux to increase GGPP supply

[0098] In order to promote the supply of the precursor GGPP, the endogenous genes YlGGPS (nucleotide sequence as shown in SEQ ID NO.10), ERG12 (nucleotide sequence as shown in SEQ ID NO.11), tHMG1 (nucleotide sequence as shown in SEQ ID NO.12), IDI 1 (nucleotide sequence as shown in SEQ ID NO.13), ERG20 (nucleotide sequence as shown in SEQ ID NO.14), the GGPP synthase gene SaGGPS from Sulfolobus acidocaldarius and the GGPP synthase gene XdGGPS from Xanthophyllomyces dendrorhous were overexpressed. The YlGGPS gene was amplified from the Yarrowia lipolitica PO1f-Δku70 genome (published in the paper "Combining 26srDNA and the Cre-loxP System for Iterative Gene Integration and EfficientMarker Curation in Yarrowia lipolitica") using primers YlGGPS-F / YlGGPS-R, the ERG12 gene was amplified from the Yarrowia lipolitica genome using primers ERG12-F / ERG12-R, the tHMG1 gene was amplified from the Yarrowia lipolitica genome using primers tHMG1-F / tHMG1-R, and the IDI1 gene was amplified from the Yarrowia lipolitica genome using primers IDI1-F / IDI1-R. 1. Gene ERG20 was amplified from the Yarrowia lipolytica genome using primers ERG20-F / ERG20-R, gene SaGGPS was amplified from a synthetic plasmid template containing the target gene using primers SaGGPS-F / SaGGPS-R, gene XdGGPS was amplified from a synthetic plasmid template containing the target gene using primers XdGGPS-F / XdGGPS-R, and the upstream and downstream homologous residues of the C7 locus were amplified using primers C7-armup-F / C7-armdown-R. The upstream homology arm was amplified with primers E5-armup-F / E5-armdown-R (the upstream homology arm was 1183 bp in length, and the downstream homology arm was 838 bp in length), and the upstream and downstream homology arms of the C1 site were amplified with primers C1-armup-F / C1-armdown-R (the upstream homology arm was 1000 bp in length, and the downstream homology arm was 1000 bp in length).About 1 μg of the integration fragment and about 600 ng of sgRNA were transformed into the Yeast lipolytica engineered strain VE-7 constructed in Example 2 using the yeast transformation kit Frozen-EZ Yeast Transformation II, spread on the screening solid culture medium, and cultured at 30°C for 3 days until colonies appeared. Engineered strains VE-9 to VE-12 were constructed separately. The engineered strains VE-9 to VE-12 were fermented in YPD medium at 30°C and 220rpm for 96 hours, and 10% olive oil was added to extract δ-tocotrienol for HPLC analysis. The analysis results showed that (. Figure 6 ), the production of δ-tocotrienol of the recombinant engineered bacteria VE-12 can reach 102.8 mg / L.

[0099] Table 4 Primer sequences

[0100]

[0101] Example 4: Fusion and expression of SyHPT and AtVTE1

[0102] In order to improve the production of δ-tocotrienol, the key enzymes SyHPT and AtVTE1 for δ-tocotrienol in Yarrowia lipolytica were cleaved by rigidly linking the peptides TPTP, (TPTP 2 ,EAAAK,(EAAAK) 2 or flexible linker peptide GGGGS, (GGGGS) 2 、GSG、(GSG) 2 The genes SyHPT and AtVTE1 were amplified using primers TPTP-F / TPTP-R, TPTP2-F / TPTP2-R, EAAAK-F / EAAAK-R, EAAAK2-F / EAAAK2-R, GGGGS-F / GGGGS-R, GGGGS2-F / GGGGS2-R, GSG-F / GSG-R, and GSG2-F / GSG2-R. The sequence of the connecting peptide was added to the primer sequence for amplifying the SyHPT and AtVTE1 genes in the form of homology arms. About 1 μg of the integration fragment and about 600 ng of sgRNA were transformed into the Yeast lipolytica engineered strain VE-12 constructed in Example 3 using the yeast transformation kit Frozen-EZ Yeast Transformation II, spread on the screening solid culture medium, and cultured at 30°C for 3 days until colonies appeared. The obtained VE-13-VE-20 engineered strain was fermented in YPD medium at 30°C and 220rpm for 96h, and 10% olive oil was added to extract δ-tocotrienol, and then analyzed by HPLC. The analysis results showed that ( Figure 7), the production of δ-tocotrienol of the recombinant engineering bacteria VE-20 was the highest, which was 137.9 mg / L.

[0103] Table 5 Primer sequences

[0104]

[0105] Table 6 Connector peptide nucleotide sequence

[0106]

[0107]

[0108] Example 5: Semi-rational design of SyHPT

[0109] In the biosynthetic pathway of δ-tocotrienol, HPT and VTE1 are two rate-limiting enzymes that limit the synthesis and conversion of MGGBQ. The protein structure of SyHPT was predicted using AlphaFold 3, and then HGA and GGPP were successfully docked into the substrate binding pocket using Discovery Studio. In order to determine the key amino acid residue sites of SyHPT, we selected the SyHPT substrate For residues within the range, the alanine scanning method was used to screen the residue sites. Based on the strain Δku70-4 constructed in Example 1, using SyHPT shown in SEQ ID NO.4 as a template, specific mutation primers (SY61-F / SY61-R, SY65-F / SY65-R, SY77-F / SY77-R, SY128-F / SY128-R, SY136-F / SY136-R, SY146-F / SY146-R, SY150-F / SY150-R, SY194-F / SY194-R, SY280-F / SY280-R) were designed, and single-point mutation was performed by circular amplification. Fermentation was performed according to the method of Example 1. The results showed that compared with the expression WT type (strain Δku70-4), after the valine and lysine at positions 61, 77 and 146 of SyHPT were mutated to alanine, the production of δ-tocotrienol in the constructed recombinant bacteria was improved, among which the efficiency at position 77 reached 36%, so lysine at position 77 was selected for the next mutation. Subsequently, we mutated lysine at position 77 to acidic amino acids (aspartic acid, glutamic acid) and amino acids with benzene rings on the side chains (tryptophan, tyrosine and phenylalanine) to enhance the interaction with the substrate molecule. The results showed that after lysine was mutated to tyrosine, the production of δ-tocotrienol was further improved, compared with SyHPT. K77AThe mutant SyHPT increased by 27% and 67% compared to WT. In order to clarify the catalytic mechanism of the mutant, we performed molecular dynamics simulations. According to the experimental results, the mutant exhibited a more stable protein structure than WT. In addition, the mutant SyHPT K77Y The number of hydrogen bonds of mutant SyHPT is more than that of WT, and its binding free energy is -73.4 kcal / mol, while that of WT is -71.7 kcal / mol. K77Y It has higher stability and stronger binding ability to the substrate. Subsequently, the mutant and the δ-tocotrienol synthesis pathway gene were integrated into the multi-copy site ZETA of Yarrowia lipolytica (published in the paper "YaliCMulti andYaliHMulti:Stable, efficient multi-copy integration tools for engineeringYarrowia lipolytica"), and the LEU2 and URA3 tags were supplemented at the same time, further improving the production of δ-tocotrienol. About 1 μg of the integrated fragment and about 600 ng of sgRNA were transformed into the Yarrowia lipolytica engineering strain VE-20 constructed in Example 4 using the yeast transformation kit Frozen-EZ Yeast Transformation II, spread on the screening solid culture medium, and cultured at 30°C for 3 days until colonies appeared. The obtained engineered strains VE-21 to VE-26 were fermented in YPD medium at 30°C and 220 rpm for 96 h, and 10% olive oil was added to extract δ-tocotrienol. After HPLC analysis, the δ-tocotrienol yield of strain VE-23 reached 189.9 mg / L ( Figure 8 ).

[0110] Table 7 Primer sequences

[0111]

[0112]

[0113] Example 6: Scaling up the recombinant bacteria in a 5L fermenter

[0114] In order to further increase the production of δ-tocotrienol, strain VE-23 was used for fed-batch fermentation in a 5 L fermenter.

[0115] Fermentation medium: 40 g / L peptone, 20 g / L yeast extract, 5 mL / L metal ion solution, 3 mL / L vitamin solution, 75 mg / L ferrous sulfate and 40 g / L glucose.

[0116] Metal ion solution: 4.5 g / L calcium chloride dihydrate, 4.5 g / L zinc sulfate heptahydrate, 3 g / L ferrous sulfate heptahydrate, 1 g / L cupric chloride dihydrate, 1 g / L boric acid, 0.4 g / L sodium molybdate dihydrate, 0.3 g / L cobalt chloride hexahydrate, 0.1 g / L copper sulfate pentahydrate, 0.1 g / L potassium iodide and 15 g / L EDTA;

[0117] Vitamin solution: 50 mg / L biotin, 200 mg / L p-aminobenzoic acid, 1 g / L niacin, 1 g / L calcium pantothenate, 1 g / L pyridoxine phosphate, 1 g / L thiamine hydrochloride and 25 g / L inositol.

[0118] Feed medium: peptone 200g / L, yeast powder 100g / L.

[0119] Preparation of primary seed solution: Pick colonies from the YPD plate and inoculate them into a shake flask containing 10 mL of YPD. Cultivate at 28°C and 220 rpm for 24 hours to obtain primary seed solution.

[0120] Preparation of secondary seed solution: Transfer all the primary seed solution to a shake flask containing 200 mL of YPD and culture at 28°C and 220 rpm for 24 h.

[0121] Transfer all the secondary seed liquid to a fermentation tank containing 2.2L fermentation medium. Set the pH to 5.0, the dissolved oxygen to 20%, and ferment at 28°C. In order to capture δ-tocotrienol, 10% (v / v) olive oil was added to the culture medium at 24h of fermentation to extract δ-tocotrienol. When the initial glucose was exhausted during fermentation, 800g / L glucose and feed medium were added. The flow rate of glucose was controlled so that the concentration of glucose was always maintained between 0.3-0.8g / L, and the flow rate of feed medium was 12-15mL / h; 50% ammonia water was added to control the pH at 5.0; the stirring rate was adjusted according to DO so that DO was maintained at 20%.

[0122] After 120 h of cultivation, the accumulation of δ-tocotrienol reached 466.8 mg / L ( Fig. 9 ), which is the highest yield of delta-tocotrienol production reported so far in microorganisms.

[0123] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A homogentisate phytyltransferase mutant, characterized in that: Based on the parent, one or more sites at positions 61, 77 and 146 are mutated; the parent has an amino acid sequence shown in SEQ ID NO.

6.

2. The homogentisate phytyltransferase mutant according to claim 1, characterized in that The mutations include (a) and / or (b): (a) mutating the isoleucine at position 61, the lysine at position 77, and / or the isoleucine at position 146 to alanine; (b) Mutating the lysine at position 77 to aspartic acid, glutamic acid, tryptophan, tyrosine or phenylalanine.

3. A gene encoding the homogentisate phytyltransferase mutant according to claim 1 or 2.

4. A recombinant microorganism expressing the homogentisate phytyltransferase mutant according to claim 1 or 2.

5. An engineered strain of Yarrowia lipolytica, characterized in that: 4-hydroxyphenylpyruvate dioxygenase, tocopherol cyclase, and homogentisate phytyltransferase or a homogentisate phytyltransferase mutant according to any one of claims 1 to 2 are expressed; The 4-hydroxyphenylpyruvate dioxygenase is 4-hydroxyphenylpyruvate dioxygenase YlHPD derived from Yarrowia lipolytica or 4-hydroxyphenylpyruvate dioxygenase PaHPD derived from Pseudomonas putida; The homogentisate phytyltransferase is homogentisate phytyltransferase TrHPT derived from wheat, or homogentisate phytyltransferase SyHPT derived from Synechocystis; the tocopherol cyclase is tocopherol cyclase AtVTE1 derived from Arabidopsis thaliana.

6. The engineered yeast Yarrowia lipolytica according to claim 5, characterized in that There are also one or more of the following improvements: (a) Overexpression of shikimate pathway genes ARO1, ARO4, ARO7 and the mutant ARO4 with tyrosine feedback inhibition K221L and ARO7 G139S One or more genes in (b) overexpressing key genes of the MVA pathway, including: one or more of the following: GGPP synthase gene, mevalonate kinase gene ERG12, truncated mevalonate-CoA reductase gene tHMG1, isoprene diphosphate isomerase gene IDI 1, farnesyl diphosphate synthase gene YlERG20, GGPP synthase gene SaGGPS from Sulfolobus acidocaldarius, or GGPP synthase gene XdGGPS from Xanthophyllomyces dendrorhous; (c) expressing SyHPT and AtVTE1 connected by a connecting peptide; the connecting peptide includes TPTP, (TPTP)2, EAAAK, (EAAAK)2, GSG, (GSG)2, GGGGS or (GGGGS)2.

7. The engineered yeast Yarrowia lipolytica according to claim 5 or 6, characterized in that The invention also has any of the following improvements (1) to (8): (1) Overexpression of the shikimate pathway gene ARO1; (2) overexpression of shikimate pathway genes ARO1 and ARO4; (3) overexpression of shikimate pathway genes ARO1 and ARO7; (4) overexpression of the shikimate pathway gene ARO4; (5) overexpression of shikimate pathway genes ARO4 and ARO7; (6) overexpression of the shikimate pathway gene ARO7; (7) Overexpression of shikimate pathway genes ARO1, ARO4, and ARO7; (8) Overexpression of the shikimate pathway gene ARO1 and the tyrosine feedback inhibition mutant ARO4 K221L and ARO7 G139S .

8. The engineered strain of Yarrowia lipolytica according to any one of claims 5 to 7, characterized in that Overexpression of the mevalonate kinase gene ERG12 and the GGPP synthase gene YIGGPS; or Overexpression of mevalonate kinase gene ERG12, GGPP synthase gene YlGGPS, truncated hydroxymevalonate CoA reductase gene tHMG1 and isoprene diphosphate isomerase gene IDI 1.

9. The engineered strain of Yarrowia lipolytica according to any one of claims 5 to 7, characterized in that The farnesyl diphosphate synthase gene YIERG20, and the GGPP synthase gene SaGGPS from Sulfolobus acidocaldarius or the GGPP synthase gene XdGGPS from Xanthophyllomyces dendrorhous were also overexpressed.

10. The engineered strain of Yarrowia lipolytica according to claim 5 or 9, characterized in that: Homogentisate phytyltransferase SyHPT and tocopherol cyclase AtVTE from Arabidopsis thaliana were fused and expressed; the homogentisate phytyltransferase SyHPT had a K77Y mutation.

11. A method for preparing delta-tocotrienol, characterized in that: The engineered Yarrowia lipolytica according to any one of claims 5 to 10 is fermented in a fermentation medium at 28 to 30° C. for at least 72 hours.

12. The method according to claim 11, characterized in that Glucose was also fed during the fermentation process.

13. Use of the homogentisate phytyltransferase mutant of claim 1 or 2, or the gene of claim 3, or the recombinant microorganism of claim 4, or the engineered yeast Yarrowia lipolytica of any one of claims 5 to 10, or the method of any one of claims 11 to 12 in the production of products containing δ-tocotrienol in the fields of food, medicine, and chemical industry.

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