A strain of yarrowia lipolytica for synthesizing delta-tocotrienol and application thereof

Abstract

The application discloses a Yarrowia lipolytica strain for synthesizing delta-tocotrienol and an application thereof, and belongs to the technical field of genetic engineering and bioengineering. A delta-tocotrienol metabolic pathway is constructed in Yarrowia lipolytica, a mutant of a key enzyme, benzoate phytyltransferase, is screened, and the production of delta-tocotrienol is further improved by strengthening the shikimic acid pathway. The production of delta-tocotrienol of the constructed engineering strain can reach 466.8 mg / L, which lays a foundation for subsequent biosynthesis of vitamin E compounds, and has potential value and significance for the development of synthetic biology.

Description

TECHNICAL FIELD

[0001] The present application relates to a Yarrowia lipolytica strain for synthesizing δ-tocotrienol and application thereof, and belongs to the technical field of genetic engineering and bioengineering. BACKGROUND

[0002] Vitamin E is the sum of tocopherols and tocotrienols, which are divided into four isomers: alpha, beta, gamma and delta. Vitamin E has the functions of anti-aging, anti-oxidation and anti-tumor, and is widely used in food, medicine and health care. Previous researches mainly focused on alpha-tocopherol because of its highest activity, widest distribution and most representative. However, recent studies have found that tocotrienols have better functions than alpha-tocopherol in some aspects. Because tocotrienols contain unsaturated side chains, they can more effectively penetrate into tissues containing saturated fatty acids, thereby playing a good role in antioxidant and free radical scavenging. Different forms of tocotrienols have differences in physiological activity, chemical activity and bioavailability, and thus have different functions. Alpha-tocotrienol can reduce cholesterol by inhibiting 3-hydroxy-3-methylglutaryl coenzyme A reductase. Gamma-tocotrienol and delta-tocotrienol are considered as potential drugs for treating cancer and have good inhibitory effect on cancer cells. Compared with other three tocotrienols, delta-tocotrienol has many unique biological functions, such as stronger free radical scavenging activity and anti-inflammatory effect. In addition, delta-tocotrienol is proved to be able to induce inhibition of pancreatic cancer cell proliferation and is a potential effective component for treating pancreatic cancer. Therefore, it is of great significance to construct an efficient synthesis pathway of delta-tocotrienol.

[0003] It is difficult to prepare delta-tocotrienol by traditional chemical method, and side reactions often occur, resulting in reduced selectivity of target compounds. Therefore, in view of the limitations of natural product extraction and chemical synthesis of delta-tocotrienol in product stability, quality safety and price, metabolic engineering and synthetic biology, which are rapidly developing, can provide ideas for the synthesis of delta-tocotrienol. SUMMARY

[0004] The present application provides a SyHPT mutant of homogentisate phytyltransferase, which is mutated at one or more of positions 61, 77 and 146 based on the parent.

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

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

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

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

[0009] The application provides a Lipomyces starkeyi engineering strain for efficiently synthesizing delta-tocotrienol, which is based on Lipomyces starkeyi Δku70 as a starting strain, and expresses 4-hydroxyphenylpyruvate dioxygenase (HPD), tocopherol cyclase, and homogentisate phytyltransferase or a mutant of the homogentisate phytyltransferase.

[0010] In an embodiment, the 4-hydroxyphenylpyruvate dioxygenase is 4-hydroxyphenylpyruvate dioxygenase YlHPD derived from Lipomyces starkeyi or 4-hydroxyphenylpyruvate dioxygenase PaHPD derived from Pseudomonas putida.

[0011] In an embodiment, the homogentisate phytyltransferase is homogentisate phytyltransferase TrHPT derived from Triticum aestivum, or homogentisate phytyltransferase SyHPT derived from Synechocystis sp., or a mutant of homogentisate phytyltransferase SyHPT derived from Synechocystis sp. by mutating one or more of positions 61, 77, and 146.

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

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

[0014] In an embodiment, the Lipomyces starkeyi engineering strain further has one or more of the following improvements:

[0015] (a) overexpressing one or more of the genes ARO1, ARO4, ARO7, and a mutant of ARO4 K221L and ARO7 G139S that is feedback-inhibited by tyrosine; and

[0016] (b) overexpressing one or more of the key genes of the MVA pathway, including: a geranylgeranyl diphosphate synthase gene (GGPS) YlGGPS, a mevalonate kinase gene (ERG12) ERG12, a truncated hydroxymethylglutaryl coenzyme A reductase gene tHMG1, an isopentenyl diphosphate delta-isomerase gene (IDI 1) IDI 1, a farnesyl pyrophosphate synthetase gene (ERG20) YlERG20, a GGPP synthase gene derived from Sulfolobus acidocaldarius SaGGPS, or a GGPP synthase gene derived from Xanthophyllomyces dendrorhous XdGGPS;

[0017] (c) fusing SyHPT and AtVTE using a linker peptide and expressing in Y. lipolytica.

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

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

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

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

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

[0023] In one embodiment, the Y. lipolytica engineered strain further overexpresses the shikimic acid pathway gene ARO1.

[0024] In one embodiment, the Y. lipolytica engineered strain further overexpresses the shikimic acid pathway genes ARO1 and ARO4.

[0025] In one embodiment, the Y. lipolytica engineered strain further overexpresses the shikimic acid pathway genes ARO1 and ARO7.

[0026] In one embodiment, the Y. lipolytica engineered strain further overexpresses the shikimic acid pathway gene ARO4.

[0027] In one embodiment, the Y. lipolytica engineered strain further overexpresses the shikimic acid pathway genes ARO4 and ARO7.

[0028] In one embodiment, the Y. lipolytica engineered strain further overexpresses the shikimic acid pathway gene ARO7.

[0029] In one embodiment, the Y. lipolytica engineered strain further overexpresses the shikimic acid pathway genes ARO1, ARO4, ARO7.

[0030] In one embodiment, the Y. lipolytica engineered strain further overexpresses the shikimic acid pathway genes ARO1 and the mutant ARO4 against feedback inhibition of tyrosine K221L and ARO7 G139S .

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

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

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

[0034] In an embodiment, the Yarrowia lipolytica engineering strain further overexpresses a farnesyl diphosphate synthase gene YlERG20 and a GGPP synthase gene XdGGPS derived from Xanthophyllomyces dendrorhous.

[0035] In an embodiment, the Yarrowia lipolytica engineering strain further expresses a homologous fusion of a homogentisate phytyltransferase SyHPT and a tocopherol cyclase AtVTE derived from Arabidopsis thaliana; the amino acid sequence of the homogentisate phytyltransferase SyHPT is shown in SEQ ID NO. 6, or has a mutation of K77Y based on the amino acid sequence shown in SEQ ID NO. 6.

[0036] In an embodiment, the nucleotide sequence of the gene 4-hydroxyphenylpyruvate dioxygenase YlHPD derived from Yarrowia lipolytica is shown in SEQ ID NO. 1.

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

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

[0039] In an embodiment, the nucleotide sequence of the gene homogentisate phytyltransferase TrHPT derived from wheat is shown in SEQ ID NO. 3.

[0040] In an embodiment, the nucleotide sequence of the gene homogentisate phytyltransferase SyHPT derived from Synechocystis is shown in SEQ ID NO. 4.

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

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

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

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

[0045] The application also provides the use of the engineered Yarrowia lipolytica in the production of δ-tocotrienol.

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

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

[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 fed during the fermentation process.

[0050] The application also claims the use of the engineered Yarrowia lipolytica, or the method, in the production of products containing δ-tocotrienol in the fields of food, medicine and chemical industry.

[0051] In one embodiment, the use is for the preparation of vaccines or medicines containing δ-tocotrienol, or the preparation of cosmetics containing δ-tocotrienol.

[0052] Advantages:

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

[0054] (2) The application strengthens the supply of precursors of δ-tocotrienol, Homogentisic acid (HGA) and Geranylgeranyl diphosphate (GGPP), by overexpressing key genes of shikimic acid pathway and Mevalonic acid (MVA) pathway.

[0055] (3) The application fuses and expresses the key enzymes of δ-tocotrienol, homogentisate phytyltransferase (HPT) and tocopherol cyclase (VTE1) by using rigid / flexible connecting peptides, further improving the yield of δ-tocotrienol.

[0056] (4) The application obtains a new mutant SyHPT by semi-rational design to modify the rate-limiting enzyme HPT K77Y The mutant is integrated into a multi-copy site, and the yield of δ-tocotrienol reaches 189.9 mg / L.

[0057] (5) The application also realizes the large-scale production of δ-tocotrienol in a 5L bioreactor, with a yield of 466.8 mg / L, laying a foundation for the subsequent synthesis of vitamin E compounds. BRIEF DESCRIPTION OF DRAWINGS

[0058] Figure 1 It is a metabolic schematic diagram of heterologous synthesis of δ-tocotrienol in Yarrowia lipolytica. The biosynthesis pathway of δ-tocotrienol can be divided into three metabolic modules: shikimic acid pathway, MVA pathway and δ-tocotrienol biosynthesis pathway. The shikimic acid pathway is shown in the pink box, the MVA pathway is shown in the meat box, and the δ-tocotrienol biosynthesis pathway is shown in the blue box.

[0059] Figure 2 It is the HPLC detection comparison result of the fermentation broth of △Ku70-4 strain and δ-tocotrienol standard.

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

[0061] Figure 4 It is the δ-tocotrienol yield graph of the engineering strains Δku70-1 to Δku70-4 under YPD culture.

[0062] Figure 5 It is the δ-tocotrienol yield graph of the engineering strains Δku70-4, VE-1 to VE-8 under YPD culture.

[0063] Figure 6 It is the δ-tocotrienol yield graph of the engineering strains with enhanced MVA pathway under YPD culture.

[0064] Figure 7Figure 1 shows the production of δ-tocotrienol by the engineered strains VE-12 to VE-20 in YPD culture.

[0065] Figure 8 Figure 6 shows the protein structure of SyHPT and the results of mutations; wherein A is the protein spatial structure, 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] Figure 9 Figure 4 shows the production of δ-tocotrienol by the engineered strain VE-23 in a 5L fermenter in fed-batch fermentation. DETAILED DESCRIPTION

[0067] (I) Culture medium

[0068] LB medium: 10 g / L of proteose peptone, 5 g / L of yeast powder, 10 g / L of sodium chloride. Add 20 g / L of 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 of uracil, 10 g / L of tryptophan, 10 g / L of leucine, 10 g / L of histidine, and appropriate deletion of corresponding amino acids as needed).

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

[0071] (II) Preparation of Y. lipolytica competence: Frozen-EZ Yeast Transformation II reagent kit was used to prepare Y. lipolytica competence, 30°C, and 5 mL of YPD medium was used to culture Y. lipolytica to the middle order (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 aspirate the supernatant;

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

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

[0075] (III) Transformation of Y. lipolytica:

[0076] 1. Scrape the yeast colonies from the YPD plate and inoculate them in YPD liquid medium, and place them in a constant temperature incubator at 30°C for 16-22 h;

[0077] 2. Prepare yeast transformation buffer solution (if multiple transformations, scale up x n): 50% PEG 3350, add 90 μL; 2 M lithium acetate (M CHCOOLi 65.99), 5 μL; ssDNA, 5 μL, which is boiled in water for 3 min before adding to the transformation buffer, then cooled on ice;

[0078] 3. Take 500 μL of the bacterial solution with a pipette and add to a centrifuge tube, centrifuge the tube at an appropriate speed, and discard the supernatant to obtain the yeast cells. Transfer the yeast cells to a centrifuge tube containing 100 μL of the transformation buffer, mix slowly and evenly, add 0.25-0.5 μg of plasmid DNA or linear DNA, mix thoroughly and shake for 2 min (at least 0.25-0.5 μg of DNA is added for each plasmid);

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

[0080] 5. Transfer the centrifuge tube to a centrifuge and centrifuge at 8000 rpm for 2 min. Discard the supernatant in a sterile clean bench, resuspend the cells with an appropriate amount of sterile water, and add to the YPD solid medium. Uniformly spread on the YPD solid medium with a sterile spreader, and then place the medium in a constant temperature incubator at 30°C for culture.

[0081] (Four) HPLC determination of δ-tocotrienols: Shimadzu high performance liquid chromatography was used for determination, which was equipped with a variable wavelength detector and an Agilent ZORBAX Nikpase XDB-C18 column. HPLC conditions: column: InertSustain C18 250 mm x 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 for 26 min; column temperature: 40°C; injection volume: 10 μL; detector wavelength: 280 nm.

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

[0083] (vi) Strain information is shown in Table 1.

[0084] Table 1 Strains involved in the present application

[0085]

[0086]

[0087] Example 1: Construction of the pathway for synthesis of δ-tocotrienols in Yarrowia lipolytica

[0088] For the synthesis of δ-tocotrienol, TrHPT from wheat and SyHPT from Synechocystis sp. PCC6803 were introduced to catalyze the formation of 2-methyl-6-geranylgeranyl-benzoquinone (MGGBQ) from hydroguinanic acid (HGA) and geranylgeranyl pyrophosphate (GGPP); further, AtVTE1 from Arabidopsis thaliana was introduced to catalyze the formation of δ-tocotrienol from MGGBQ. All the heterologous genes were commissioned to a company for synthesis, and plasmid templates containing the target genes were obtained. First, we designed primers for the heterologous genes using Snapgene software, and the endogenous gene YlHPD was amplified from the genome of Yarrowia lipolytica PO1f-Δku70 (published in the paper “Combining 26s rDNA and the Cre-loxP System for Iterative Gene Integration and Efficient Marker Curation in Yarrowia lipolytica”) using primers YLHPD-F / YLHPD-R, the gene PaHPD was amplified from the plasmid template containing the target gene commissioned for synthesis using primers PAHPD-F / PAHPD-R, the gene TrHPT was amplified from the plasmid template containing the target gene commissioned for synthesis using primers TrHPT-F / TrHPT-R, the gene SyHPT was amplified from the plasmid template containing the target gene commissioned for synthesis using primers SyHPT-F / SyHPT-R, the gene AtVTE1 was amplified from the plasmid template containing the target gene commissioned for synthesis using primers AtVTE1-F / AtVTE1-R, and the homologous arms of the D17 site and the E4 site (the upstream homologous arm sequence of the D17 site is shown in SEQ ID NO. 19, and the downstream homologous arm sequence is shown in SEQ ID NO. 20; the upstream homologous arm sequence of the E4 site is shown in SEQ ID NO. 21, and the downstream homologous arm sequence is shown in SEQ ID NO. 22) and the fragment formed by the target genes were PCR amplified together, and the PCR amplified fragment and the corresponding Cas9 plasmid of the site were added during the process of yeast transformation. The fragment P TEF -YlHPD-T XPR2 or P TEF -PaHPD-T XPR2 was integrated into the D17 site of the strain, and the fragment P TEF -TrHPT-TXPR2 / P TEF -SyHPT-T XPR2 or P TEF -AtVTE1-T XPR2 The genes of the above δ-tocotrienol biosynthesis pathway were combined by integrating into the E4 site of the strain, and four engineering 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 the optimal gene combination.

[0089] The Δku70-1-Δku70-4 engineering strains obtained were respectively fermented in YPD medium at 30°C, 220 rpm for 96h, and 10% olive oil was added to the culture medium after 24h of fermentation for extracting δ-tocotrienol. After fermentation, the fermentation broth and organic phase were all poured into a 50mL centrifuge tube, and the upper organic phase was all poured into a 5mL centrifuge tube after centrifugation at 12000rpm for 5min. Then, 100μL of organic phase was dissolved in 900μL of dimethyl sulfoxide for 10-fold dilution, filtered into a liquid phase bottle for detection. After LC-MS identification, the results showed that δ-tocotrienol synthesis was achieved in the four strains, as shown in Figures 2-3 . The gene combination YlHPD-SyHPT-AtVTE1 showed the highest δ-tocotrienol yield, reaching 35.1mg / L (as shown in Figure 4 ).

[0090] All primers are listed in Table 2.

[0091] Table 2 Primer sequences

[0092]

[0093] Example 2: Increasing the supply of HGA by strengthening the shikimic acid pathway

[0094] To promote the supply of the precursor hypoacetic acid, 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. Primers ARO1-F / ARO1-R were used to extract the genome of Yarrowia lipolytica PO1f-Δku70 (published in the paper "Combining 26s rDNA and the Cre-loxP System for Iterative Gene Integration and Efficient Marker Curation in Yarrowia") Gene ARO1 was amplified from *Lactobacillus lipolitica* genome, ARO4 was amplified from the genome using primers ARO4-F / ARO4-R, and ARO7 was amplified from the genome using primers ARO7-F / ARO-R. The upstream and downstream homologous arms of the A3 site were amplified using primers A3-armup-F / A3-armdown-R (the upstream homologous arm was 1181 bp long, and the downstream homologous arm was 1200 bp long). Gene ARO4 was synthesized. K221L and ARO7 G139S (The nucleotide sequences are shown in SEQ NO.17 and SEQ NO.18, respectively). Approximately 1 μg of the integrated fragment and approximately 600 ng of sgRNA were transformed into the *Yeast Transformation* strain Δku70-4 constructed in Example 1 using the Frozen-EZ Yeast Transformation II kit. The transformed strain was plated on screening solid medium and incubated 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 220 rpm for 96 h, with 10% olive oil added for extraction of δ-tocotrienols, followed by HPLC analysis. The results showed that ( Figure 5 The recombinant engineered strain VE-7 produced the highest yield of δ-tocotrienol, at 58.2 mg / L.

[0095] Table 3 Primer sequences

[0096]

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

[0098] To promote the supply of 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) of Yarrowia lipolytica, GGPP synthase gene SaGGPS derived from Sulfolobus acidocaldarius and GGPP synthase gene XdGGPS derived from Xanthophyllomyces dendrorhous were overexpressed. The genes YlGGPS were amplified from 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”) using primers YlGGPS-F / YlGGPS-R, the genes ERG12 were amplified from Yarrowia lipolytica genome using primers ERG12-F / ERG12-R, the gene tHMG1 was amplified from Yarrowia lipolytica genome using primers tHMG1-F / tHMG1-R, the gene IDI 1 was amplified from Yarrowia lipolytica genome using primers IDI 1-F / IDI 1-R, the gene ERG20 was amplified from Yarrowia lipolytica genome using primers ERG20-F / ERG20-R, the gene SaGGPS was amplified from the plasmid template containing the target gene synthesized by commission using primers SaGGPS-F / SaGGPS-R, the gene XdGGPS was amplified from the plasmid template containing the target gene synthesized by commission using primers XdGGPS-F / XdGGPS-R, the upper and lower homologous arms of C7 site were amplified using primers C7-armup-F / C7-armdown-R (the upper homologous arm was 1000 bp in length and the lower homologous arm was 999 bp in length), the upper and lower homologous arms of E5 site were amplified using primers E5-armup-F / E5-armdown-R (the upper homologous arm was 1183 bp in length and the lower homologous arm was 838 bp in length), and the upper and lower homologous arms of C1 site were amplified using primers C1-armup-F / C1-armdown-R (the upper homologous arm was 1000 bp in length and the lower homologous arm was 1000 bp in length).About 1 μg of the integration fragment and about 600 ng of sgRNA were transformed into the Y. lipolytica engineering strain VE-7 constructed in Example 2 using the Frozen-EZ Yeast Transformation II kit, and plated on a selection solid medium and incubated at 30°C for 3 days until colonies appeared. The engineering strains VE-9 to VE-12 were constructed respectively. The engineering strains VE-9 to VE-12 were fermented in YPD medium at 30°C, 220 rpm for 96 h, and 10% olive oil was added for extraction of δ-tocotrienol, and then analyzed by HPLC. The analysis results showed that the yield of δ-tocotrienol of the recombinant engineering strain VE-12 reached 102.8 mg / L. Figure 6

[0099] Table 4 primer sequences

[0100]

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

[0102] In order to improve the yield of δ-tocotrienol, the key enzymes SyHPT and AtVTE1 of δ-tocotrienol in Y. lipolytica were fused by rigid connection peptide TPTP, (TPTP)2, EAAAK, (EAAAK)2 or flexible connection peptide GGGGS, (GGGGS)2, GSG, (GSG)2. The genes SyHPT and AtVTE1 were amplified by 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, GSG2-F / GSG2-R, and the sequence of the connection peptide was added to the primer sequence for amplifying the genes SyHPT and AtVTE1 in the form of a homologous arm. About 1 μg of the integration fragment and about 600 ng of sgRNA were transformed into the Y. lipolytica engineering strain VE-12 constructed in Example 3 using the Frozen-EZ Yeast Transformation II kit, and plated on a selection solid medium and incubated at 30°C for 3 days until colonies appeared. The obtained engineering strains VE-13 to VE-20 were fermented in YPD medium at 30°C, 220 rpm for 96 h, and 10% olive oil was added for extraction of δ-tocotrienol, and then analyzed by HPLC. The analysis results showed that the yield of δ-tocotrienol of the recombinant engineering strain VE-20 was the highest, reaching 137.9 mg / L. Figure 7

[0103] Table 5 primer sequences​​

[0104]

[0105] Table 6. Nucleotide sequences of linking peptides

[0106]

[0107]

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

[0109] In the δ-tocotrienol biosynthetic pathway, HPT and VTE1 are two rate-limiting enzymes, restricting the synthesis and conversion of MGGBQ. The protein structure of SyHPT was predicted using AlphaFold 3, and HGA and GGPP were subsequently successfully docked into the substrate-binding pocket using Discovery Studio. To determine the key amino acid residue sites of SyHPT, we selected SyHPT substrates. For residues within the specified range, alanine scanning was used to screen residue sites. Based on the strain Δku70-4 constructed in Example 1, 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 using SyHPT as a template, and single-point mutations were performed through circular amplification. Fermentation was carried out according to the method in Example 1. The results showed that, compared with the expression WT type (strain Δku70-4), mutating valine and lysine at positions 61, 77, and 146 of SyHPT to alanine increased the yield of δ-tocotrienols in the constructed recombinant strain. The efficiency at position 77 reached 36%, therefore, lysine at position 77 was selected for the next mutation. Subsequently, lysine at position 77 was mutated to acidic amino acids (aspartic acid, glutamic acid) and amino acids with benzene rings on the side chain (tryptophan, tyrosine, and phenylalanine) to enhance the interaction with the substrate molecule. The results showed that mutating lysine to tyrosine further increased the yield of δ-tocotrienols compared to SyHPT. K77A The efficiency was increased by 27%, and by 67% compared to WT. To elucidate the catalytic mechanism of the mutant, we performed molecular dynamics simulations. Based on the experimental results, the mutant exhibits a more stable protein structure than WT. Furthermore, the mutant SyHPT… K77YThe mutant SyHPT has more hydrogen bonds than WT, and the binding free energy is -73.4 kcal / mol, while the binding free energy of WT is -71.7 kcal / mol. This means that the mutant SyHPT K77Y has higher stability and stronger binding ability to the substrate. Subsequently, the mutant and the δ-tocotrienol synthesis pathway genes were integrated into the multi-copy site ZETA of Y. lipolytica (disclosed in the paper "YaliCMulti and YaliHMulti: Stable, efficient multi-copy integration tools for engineering Yarrowia lipolytica"), while the LEU2 and URA3 markers were complemented, further improving the yield of δ-tocotrienol. About 1 μg of the integration fragment and about 600 ng of sgRNA were transformed into the Y. lipolytica engineering strain VE-20 constructed in Example 4 using the Frozen-EZ Yeast Transformation II kit, and plated on the selection solid medium and cultured at 30°C for 3 days until colonies appeared. The obtained engineering strains VE-21-VE-26 were fermented in YPD medium at 30°C, 220 rpm for 96 h, and 10% olive oil was added for extraction of δ-tocotrienol, and HPLC analysis, and 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: Scale-up culture of recombinant bacteria in a 5L fermenter

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

[0115] Fermentation medium: 40 g / L of proteose peptone, 20 g / L of yeast extract, 5 mL / L of metal ion solution, 3 mL / L of vitamin solution, 75 mg / L of ferrous sulfate, and 40 g / L of 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 copper 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 nicotinic acid, 1 g / L calcium pantothenate, 1 g / L pyridoxal phosphate, 1 g / L thiamine hydrochloride, and 25 g / L myo-inositol.

[0118] Feeding medium: 200 g / L proteose peptone, 100 g / L yeast extract.

[0119] Preparation of primary seed liquid: a colony was picked from a YPD plate and inoculated in a 10 mL YPD-containing shake flask, which was incubated at 28°C, 220 rpm for 24 hours to obtain a primary seed liquid;

[0120] Preparation of secondary seed liquid: the primary seed liquid was transferred into a 200 mL YPD-containing shake flask, which was incubated at 28°C, 220 rpm for 24 hours.

[0121] The secondary seed liquid was transferred into a 2.2 L fermenter containing 2.2 L fermentation medium. The pH was set to 5.0, the dissolved oxygen was set to 20%, and the fermentation was carried out at 28°C. In order to capture δ-tocotrienol, 10% (v / v) olive oil was added to the medium to extract δ-tocotrienol at 24 h of fermentation. When the initial glucose was consumed, 800 g / L glucose and the feeding medium were added, and the glucose flow rate was controlled to keep the glucose concentration between 0.3-0.8 g / L. The feeding medium flow rate was 12-15 mL / h. Ammonia water with a concentration of 50% was added to control the pH at 5.0. The stirring rate was adjusted according to the DO to maintain the DO at 20%.

[0122] After 120 h of cultivation, the accumulation of δ-tocotrienol reached 466.8 mg / L Figure 9 , which is the highest yield of δ-tocotrienol production in microorganisms reported to date.

[0123] Although the present application has been disclosed with reference to the preferred embodiments as above, it is not intended to limit the present application, and any person skilled in the art can make various modifications and modifications without departing from the spirit and scope of the present application, and therefore the protection scope of the present application should be defined by the claims.

Claims

1. A phytosanitary phytotransferase mutant, characterized in that, Based on the amino acid sequence shown in SEQ ID NO.6, the isoleucine at position 61 is mutated to alanine, or the lysine at position 77 is mutated to tyrosine.

2. The gene encoding the homogentisic acid phytotransferase mutant of claim 1.

3. A recombinant microorganism expressing the homogentisic acid phytotransferase mutant of claim 1.

4. The engineered strain of *Yamylostella lipolytica*, characterized in that... Expressed 4-hydroxyphenylpyruvate dioxygenase YlHPD The tocopherol cyclase AtVTE1, and the homogentisic acid phytotransferase mutant of claim 1, overexpressing a shikimic acid pathway gene; wherein the shikimic acid pathway gene is (a) or (b): (a) Gene ARO1 , ARO4 and ARO7 ; (b) Genes ARO1 , ARO4 K221L and ARO7 G139S ; Encoding the 4-hydroxyphenylpyruvate dioxygenase YlHPD The nucleotide sequence is shown in SEQ ID NO.1; encoding the tocopherol cyclase. AtVTE1 The nucleotide sequence is shown in SEQ ID NO.5; gene ARO1 , ARO4 , ARO7 The nucleotide sequences are shown in SEQ ID NO.7~SEQ ID NO.9, respectively; gene ARO4 K221L and ARO7 G139S The nucleotide sequences are shown in SEQ ID NO.17 and SEQ ID NO.18, respectively.

5. The engineered *Yamylostella lipolytica* strain according to claim 4, characterized in that, It also has any of the following improvements: Overexpression of mevalonate kinase gene ERG12 and GGPP synthase gene YlGGPS ;or Overexpression of mevalonate kinase gene ERG12 GGPP synthase gene YlGGPS truncated hydroxymethylvalerate coenzyme A reductase gene tHMG1 and isoprene diphosphate isomerase gene IDI 1 ;or Overexpression of mevalonate kinase gene ERG12 GGPP synthase gene YlGGPS truncated hydroxymethylvalerate coenzyme A reductase gene tHMG1 and isoprene diphosphate isomerase gene IDI 1 Farnesyl diphosphate synthase gene YlERG20 GGPP synthase gene SaGGPS ;or Overexpression of mevalonate kinase gene ERG12 GGPP synthase gene YlGGPS truncated hydroxymethylvalerate coenzyme A reductase gene tHMG1 and isoprene diphosphate isomerase gene IDI 1 Farnesyl diphosphate synthase gene YlERG20 GGPP synthase gene XdGGPS ; The gene YlGGPS, ERG12 , tHMG1 , IDI 1 , YlERG20 , SaGGPS and XdGGPS The nucleotide sequences are shown in SEQ ID NO.10~SEQ ID NO.16, respectively.

6. The engineered *Yamylostella lipolytica* strain according to claim 4 or 5, characterized in that, The homogentisic acid phytotransferase mutant linked by a linker peptide and tocopherol cyclase are fused and expressed; the linker peptide is TPTP, EAAAK, GSG, (GSG)2 or GGGGS.

7. A method for preparing δ-tocotrienol, characterized in that, The engineered *Yamylostella lipolytica* strain according to any one of claims 4 to 6 is fermented in a fermentation medium at 28 to 30°C for at least 72 h.

8. The method according to claim 7, characterized in that, Glucose is also added during the fermentation process.

9. The method according to claim 7 or 8, characterized in that, The fermentation medium is YPD medium or a medium containing the following components: peptone, yeast extract, metal ions, vitamins, ferrous sulfate and glucose.

10. The use of the homogentisic acid phytotransferase mutant of claim 1, or the gene of claim 2, or the recombinant microorganism of claim 3, or the engineered *Yamylostella lipolytica* of any one of claims 4-6, or the method of any one of claims 7-9 in the production of products containing δ-tocotrienol.

Images