Yarrowia lipolytica strain for synthesizing δ-tocotrienol, and use

Abstract

The present invention relates to the technical field of genetic engineering and bioengineering. Disclosed are a Yarrowia lipolytica strain for synthesizing δ-Tocotrienol and a use. According to the present invention, a δ-Tocotrienol metabolic pathway is constructed in Yarrowia lipolytica, a mutant of a key enzyme homogentisate phytyltransferase is screened, and the yield of δ-Tocotrienol is further improved by enhancing a shikimate pathway. The engineered strain constructed in the present invention can achieve a δ-tocotrienol yield of 466.8 mg / L.

Description

A *Yamylostella lipolytica* strain for synthesizing δ-tocotrienol and its application Technical Field

[0001] This invention relates to a *Yamylostella lipolytica* strain for synthesizing δ-tocotrienol and its applications, belonging to the fields of genetic engineering and bioengineering technology. Background Technology

[0002] Vitamin E is the sum of tocopherols and tocotrienols, existing in four isomers: α, β, γ, and δ. It possesses anti-aging, antioxidant, and anti-tumor properties and has wide applications in food, pharmaceuticals, and healthcare. Previous research has primarily focused on α-tocopherol due to its highest activity, widest distribution, and most representative characteristics. However, recent studies have discovered that tocotrienols exhibit superior functions compared to α-tocopherol in certain aspects. Because tocotrienols contain unsaturated side chains, they can more effectively penetrate tissues containing saturated fatty acid layers, thus providing excellent antioxidant and free radical scavenging effects. Different forms of tocotrienols differ in physiological activity, chemical activity, and bioavailability, thus possessing different functions. α-Tocotrienols can lower cholesterol by inhibiting 3-hydroxy-3-methylglutaryl-CoA reductase. γ-Tocotrienols and δ-Tocotrienols are considered potential drugs for cancer treatment, showing good inhibitory effects on cancer cells. Compared to the other three tocotrienols, δ-tocotrienol possesses many unique biological functions, such as stronger free radical scavenging activity and anti-inflammatory effects. Furthermore, δ-tocotrienol has been shown to induce and inhibit the proliferation of pancreatic cancer cells, making it a potential effective ingredient for treating pancreatic cancer. Therefore, constructing an efficient synthetic pathway for δ-tocotrienol is of great significance.

[0003] Traditional chemical methods for preparing δ-tocotrienols are challenging and often generate side reactions, leading to reduced selectivity for the target compound. Therefore, the rapidly developing fields of metabolic engineering and synthetic biology offer promising approaches to the synthesis of δ-tocotrienols, addressing limitations in product stability, safety, and price associated with natural product extraction and chemical synthesis. Summary of the Invention

[0004] This invention provides a phytohexyltransferase SyHPT mutant, which, based on the parent, mutates one or more sites at positions 61, 77, and 146.

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

[0006] In one embodiment, the mutation includes mutating the 77th lysine residue 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 recombinant microorganisms expressing the mutants.

[0009] This invention provides a highly efficient engineered *Yamylostella lipolytica* strain for synthesizing δ-tocotrienols. It is based on *Yamylostella lipolytica* Δku70 strain, which expresses 4-hydroxyphenylpyruvate dioxygenase (HPD), tocopherol cyclase, and homogentisic acid phytotransferase or a mutant of the homogentisic acid phytotransferase.

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

[0011] In one embodiment, the homogentisic acid phytotransferase is a homogentisic acid phytotransferase TrHPT derived from wheat, or a homogentisic acid phytotransferase SyHPT derived from Synesthesia agallocha, or a mutant obtained by mutating one or more sites at positions 61, 77, and 146 based on the homogentisic acid phytotransferase SyHPT from Synesthesia agallocha.

[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 AtVTE1, a tocopherol cyclase derived from Arabidopsis thaliana.

[0014] In one embodiment, the engineered *Yamylostella lipolytica* strain further has one or more of the following improvements:

[0015] (a) Overexpression of shikimic acid pathway genes ARO1, ARO4, ARO7, and the tyrosine feedback-inhibiting mutant ARO4 K221L and ARO7 G139S One or more genes in;

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

[0017] (c) Using a linker peptide, SyHPT and AtVTE1 were fused and expressed in Yarrowia lipolytica.

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

[0019] In one embodiment, the engineered *Yarrowia lipophila* strain expresses 4-hydroxyphenylpyruvate dioxygenase PaHPD derived from *Pseudomonas putida*, thymine phytotransferase TrHPT derived from wheat, and tocopherol cyclase AtVTE derived from *Arabidopsis thaliana*.

[0020] In one embodiment, the engineered *Yolopsis lipolyticus* strain expresses 4-hydroxyphenylpyruvate dioxygenase PaHPD derived from *Pseudomonas putida*, urea homogentisate phytotransferase SyHPT derived from *Synthia spp.*, and tocopherol cyclase AtVTE derived from *Arabidopsis thaliana*.

[0021] In one embodiment, the engineered *Yolopsis lipolytica* strain expresses 4-hydroxyphenylpyruvate dioxygenase YlHPD derived from *Yolopsis lipolytica*, ursolic acid phytotransferase TrHPT derived from wheat, and tocopherol cyclase AtVTE derived from *Arabidopsis thaliana*.

[0022] In one embodiment, the engineered *Yolopsis lipolyticus* strain expresses 4-hydroxyphenylpyruvate dioxygenase YlHPD from *Yolopsis lipolyticus*, urea homogentisate phytotransferase SyHPT from *Synostemma pentaphyllum*, and tocopherol cyclase AtVTE from *Arabidopsis thaliana*.

[0023] In one embodiment, the engineered *Yamylostella lipolytica* strain also overexpresses the shikimic acid pathway gene ARO1.

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

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

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

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

[0028] In one embodiment, the engineered *Yamylostella lipolytica* strain also overexpresses the shikimic acid pathway gene ARO7.

[0029] In one embodiment, the engineered *Yamylostella lipolytica* strain also overexpresses the shikimic acid pathway genes ARO1, ARO4, and ARO7.

[0030] In one embodiment, the engineered *Aylocereus lipophilus* strain also overexpresses the shikimic acid pathway gene ARO1 and the tyrosine feedback-inhibiting mutant ARO4. K221L and ARO7 G139S .

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

[0032] In one embodiment, the engineered *Yolopsis lipolytica* strain also overexpresses the mevalonate kinase gene ERG12, the GGPP synthase gene YlGGPS, the truncated mevalonate-coenzyme A reductase gene tHMG1, and the isoprene diphosphate isomerase gene IDI1.

[0033] In one embodiment, the engineered *Yolostomium lipophilum* 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 *Yamylostella lipolytica* strain also overexpresses the farnesyl diphosphate synthase gene YlERG20 and the GGPP synthase gene XdGGPS derived from *Xanthophyllomyces dendrorhous*.

[0035] In one embodiment, the engineered *Aylocereus lipophilus* strain further expresses homogentisic acid phytosyltransferase SyHPT and tocopherol cyclase AtVTE from *Arabidopsis thaliana*; the amino acid sequence of homogentisic acid phytosyltransferase 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 YlHPD derived from Ylolycoside is shown in SEQ ID NO.1.

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

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

[0039] In one embodiment, the nucleotide sequence encoding the wheat-derived gene hyoscyl phytotransferase TrHPT is shown in SEQ ID NO.3.

[0040] In one embodiment, the nucleotide sequence encoding the gene ursolic acid phytotransferase SyHPT derived from Synesthesia is shown in SEQ ID NO.4.

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

[0042] In one embodiment, the gene TrHPT or the gene SyHPT, along with AtVTE1, is 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; gene ARO4 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 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 and SEQ ID NO.16, respectively.

[0045] The present invention also provides the application of the engineered *Yamylostella lipolytica* strain in the production of δ-tocotrienols.

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

[0047] In one implementation, fermentation lasts 72–120 hours, or 96–120 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 implementation, glucose is also added during fermentation.

[0050] This invention also claims protection for the engineered strain of *Yamylostella lipolytica*, or for the application of the method in the production of products containing δ-tocotrienols in the food, pharmaceutical, and chemical industries.

[0051] In one embodiment, the application is for preparing vaccines or drugs containing δ-tocotrienol, or for preparing cosmetics containing δ-tocotrienol. Beneficial effects:

[0052] (1) This invention uses the engineered strain of Yeast lipophila PO1f-Δku70 (MatA,Δku70::leu2-270,ura3-302,xpr2-322,axp1-2) as the host, and constructs the biosynthetic pathway of δ-tocotrienol for the first time by screening and expressing genes from different sources of δ-tocotrienol synthesis pathway.

[0053] (2) This invention enhances the supply of δ-tocotrienol precursors homogentisic acid (HGA) and geranylgeranyl diphosphate (GGPP) by overexpressing key genes in the shikimic acid and mevalonic acid (MVA) pathways.

[0054] (3) This invention utilizes rigid / flexible linker peptides 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.

[0055] (4) This invention modifies the rate-limiting enzyme HPT through semi-rational design to obtain a new mutant, SyHPT. K77Y The mutant was integrated into a multicopy site, resulting in a δ-tocotrienol yield of 189.9 mg / L.

[0056] (5) The present invention also achieved the scaled production of δ-tocotrienol in a 5L bioreactor, with a yield of 466.8 mg / L, laying the foundation for the subsequent synthesis of vitamin E compounds. Attached Figure Description

[0057] Figure 1 is a metabolic schematic diagram of heterologous synthesis of δ-tocotrienols in *Agropyron lipolytica*. The biosynthetic pathway of δ-tocotrienols 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 in the flesh-colored box, and the δ-tocotrienol biosynthetic pathway in the blue box.

[0058] Figure 2 shows the HPLC detection comparison results of the fermentation broth of strain △Ku70-4 and the δ-tocotrienol standard.

[0059] Figure 3 shows the comparison results of LC-MS detection of fermentation broth of strain △Ku70-4 and δ-tocotrienol standard; where A is the LC-MS detection result of δ-tocotrienol standard and B is the LC-MS detection result of fermentation broth of strain △Ku70-4.

[0060] Figure 4 shows the yield of δ-tocotrienols of engineered strains Δku70-1 to Δku70-4 under YPD culture.

[0061] Figure 5 shows the yield of δ-tocotrienols of engineered strains Δku70-4, VE-1 to VE-8 under YPD culture.

[0062] Figure 6 shows the yield of δ-tocotrienols by engineered strains that enhance the MVA pathway under YPD culture.

[0063] Figure 7 shows the yield of δ-tocotrienols of engineered strains VE-12 to VE-20 under YPD culture.

[0064] Figure 8 shows the protein structure and mutation results of SyHPT; where A is the protein spatial structure, B to D are the effects of expressing different variants; in E and F, the blue line represents wild-type SyHPT (WT), and the red line represents the mutant K77Y.

[0065] Figure 9 shows the yield of δ-tocotrienol produced by engineered strain VE-23 in a 5L fermenter using a fed-batch fermentation method. Detailed Implementation

[0066] (I) Culture medium

[0067] LB medium: 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride. Add 20 g / L agar powder to prepare LB solid medium.

[0068] 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, with appropriate amino acid deletions as needed).

[0069] YPD medium: peptone 20 g / L, yeast extract 10 g / L, glucose 20 g / L.

[0070] (II) Preparation of competent cells of *Yamylostella lipolytica*: Competent cells of *Yamylostella lipolytica* were prepared using the Frozen-EZ Yeast Transformation II kit. The cells were cultured at 30°C with 5 mL of YPD medium to a medium-order OD of [value missing]. 600 =0.8-1.0). The following steps are performed at room temperature.

[0071] 1. Centrifuge the cells at 3500 rpm for 5 minutes and aspirate the supernatant;

[0072] 2. Add 10 mL of EZ1 solution to wash the precipitate, centrifuge the precipitate cells again, and remove the supernatant;

[0073] 3. Add 1 mL of EZ2 solution to resuspend the precipitated cells.

[0074] (III) Transformation of Yeast Extract:

[0075] 1. Scrape yeast colonies from YPD plates and inoculate them into YPD liquid medium. Place them in a constant temperature incubator at 30℃ and grow for 16-22 hours.

[0076] 2. Prepare the yeast conversion buffer solution (if multiple conversions, multiply by n): 90 μL of 50% PEG3350; 2M lithium acetate (M CHCOOLi65.99), volume 5 μL; ssDNA, volume 5 μL. Before adding ssDNA to the transformation buffer, it needs to be boiled in water for 3 min and then cooled in ice.

[0077] 3. Using a pipette, add 500 μL of bacterial culture to a centrifuge tube. Centrifuge the tube at an appropriate speed, discard the supernatant, and obtain yeast cells. Transfer the yeast cells to a centrifuge tube containing 100 μL of transformation buffer, gently pipette to mix, add 0.25-0.5 μg of plasmid DNA or linear DNA, mix thoroughly, and vortex for 2 min (add at least 0.25-0.5 μg of DNA to each plasmid).

[0078] 4. Incubate the centrifuge tubes containing the conversion mixture in a metal bath at 30°C for 30-45 min, shaking the mixture for 15 s every 10 min, and then subject it to an additional 10 min of heat shock at 39°C to improve the conversion efficiency.

[0079] 5. Transfer the centrifuge tubes to a centrifuge and centrifuge at 8000 rpm for 2 minutes. Pour out the supernatant in a sterile laminar flow hood, add an appropriate amount of sterile water to resuspend the bacteria, and add it to YPD solid medium. Spread the medium evenly on the YPD solid medium using a sterile spreader, and then incubate the medium in a 30°C constant temperature incubator.

[0080] (IV) HPLC Determination of δ-Tocotrienols: The determination was performed using Shimadzu high-performance liquid chromatography (HPLC), equipped with a variable wavelength detector and an Agilent ZORBAX Nikpase XDB-C18 column. HPLC conditions: Column: InertSustain C18 250mm × 4.6mm column (particle size 5μm); Mobile phase A: ultrapure water containing 1‰ trifluoroacetic acid; Mobile phase B: acetonitrile containing 1‰ trifluoroacetic acid; Pump A flow rate: 0.02mL / min, Pump B flow rate: 0.98mL / min, isocratic elution for 26min; Column temperature: 40℃; Injection volume: 10μL; Detector wavelength: 280nm.

[0081] (V) HPLC determination of homogentisic acid: 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 the addition of 0.1% trifluoroacetic acid. The total flow rate was 1 mL / min, the injection volume was 10 μL, and binary high-pressure gradient elution was performed for 25 min. The detection wavelength was 292 nm.

[0082] (vi) The strain information is shown in Table 1.

[0083] Table 1. Strains involved in this invention

[0084] Example 1: Construction of the δ-tocotrienol synthesis pathway in *Yamylostella lipolytica*

[0085] To synthesize δ-tocotrienols, wheat-derived TrHPT and Synthia spp.-derived SyHPT were introduced to catalyze the formation of 2-methyl-6-geranylgeranyl-benzoquinone (MGGBQ) from homogentisic acid (HGA) and geranylgeranyl pyrophosphate (GGPP). Furthermore, Arabidopsis-derived AtVTE1 was introduced to catalyze the formation of δ-tocotrienols from MGGBQ. All heterologous genes were synthesized by a company to obtain plasmid templates containing the target genes. First, primers for the heterologous genes were designed using Snapgene software. Primers YLHPD-F / YLHPD-R were used to extract the genome of Yarrowia lipophila PO1f-Δku70 (published in the paper "Combining 26s rDNA and the Cre-loxP System for Iterative Gene Integration and Efficient Marker Curation in Yarrowia"). The endogenous gene YlHPD was amplified using primers PAHPD-F / PAHPD-R from a commissioned plasmid template containing the target gene. Similarly, the gene TrHPT was amplified using primers TrHPT-F / TrHPT-R, the gene SyHPT was amplified using primers SyHPT-F / SyHPT-R, and the gene AtVTE1 was amplified using primers AtVTE1-F / AtVTE1-R. Finally, the homologous arms of the D17 and E4 sites (as described in the paper "Remodeling metabolism for high-level resveratrol production in Yarrowia lipolytica") were amplified using primers D17-armup-F / D17-armup-R and E4-armup-F / E4-armdown-R. The upstream homologous arm sequence of the D17 site is shown in SEQ ID NO. As shown in NO.19, the downstream homologous arm sequence is shown in SEQ ID NO.20; the upstream homologous arm sequence at 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 from the target gene are amplified together by PCR. During yeast transformation, the PCR-amplified fragment and the corresponding Cas9 plasmid are added. The fragment containing the promoter P is then added. TEF -YlHPD-T XPR2 or P TEF -PaHPD-T XPR2 Integration into the D17 site of the strain will bring the promoter-containing fragment P TEF-TrHPT-T XPR2 / P TEF -SyHPT-T XPR2 or P TEF -AtVTE1-T XPR2 By integrating genes into the E4 site of the strains and combining them with those of the δ-tocotrienol biosynthetic pathway, 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 for the optimal gene combination.

[0086] The constructed Δku70-1-Δku70-4 engineered strains were fermented in YPD medium at 30℃ and 220 rpm for 96 h. After 24 h of fermentation, 10% olive oil was added to the medium for extraction of δ-tocotrienols. After fermentation, the fermentation broth and organic phase were poured into 50 mL centrifuge tubes and centrifuged at 12000 rpm for 5 min. The upper organic phase was then poured into another 5 mL centrifuge tube and centrifuged at 12000 rpm for 5 min. Subsequently, 100 μL of the organic phase was dissolved in 900 μL of dimethyl sulfoxide and diluted 10-fold, then filtered through a membrane into a liquid chromatography bottle for analysis. LC-MS analysis showed that all four strains synthesized δ-tocotrienols, as shown in Figures 2-3. The gene combination YlHPD-SyHPT-AtVTE1 exhibited the highest δ-tocotrienol yield, reaching 35.1 mg / L (Figure 4).

[0087] All primers are listed in Table 2.

[0088] Table 2 Primer sequences

[0089] Example 2: Enhancing HGA supply through shikimic acid pathway enhancement

[0090] 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 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 220 rpm for 96 h, and 10% olive oil was added for extraction of δ-tocotrienols, followed by HPLC analysis. The results (Figure 5) showed that the recombinant engineered strain VE-7 had the highest yield of δ-tocotrienols, at 58.2 mg / L.

[0091] Table 3 Primer sequences

[0092] Example 3: Enhancing MVA pathway flux to improve GGPP supply

[0093] To promote the supply of the precursor GGPP, the following genes were overexpressed: YlGGPS (nucleotide sequence shown in SEQ ID NO. 10), ERG12 (nucleotide sequence shown in SEQ ID NO. 11), tHMG1 (nucleotide sequence shown in SEQ ID NO. 12), IDI 1 (nucleotide sequence shown in SEQ ID NO. 13), ERG20 (nucleotide sequence shown in SEQ ID NO. 14), the GGPP synthase gene SaGGPS derived from Sulfolobus acidocaldarius, and the GGPP synthase gene XdGGPS derived from Xanthophyllomyces dendrorhous. The YlGGPS gene was amplified from the Yarrowia lipolitica PO1f-Δku70 genome using primers YlGGPS-F / YlGGPS-R (published in the paper "Combining 26s rDNA and the Cre-loxP System for Iterative Gene Integration and Efficient Marker Curation in Yarrowia lipolitica"). 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. The IDI gene was amplified from the Yarrowia lipolitica genome using primers IDI 1-F / IDI 1-R. 1. The gene ERG20 was amplified from the genome of *Yamylostella lipolytica* using primers ERG20-F / ERG20-R. The gene SaGGPS was amplified from a plasmid template containing the target gene synthesized by a commissioned supplier using primers SaGGPS-F / SaGGPS-R. The gene XdGGPS was amplified from a plasmid template containing the target gene synthesized by a commissioned supplier using primers XdGGPS-F / XdGGPS-R. The upstream and downstream homologous structures at the C7 site were amplified using primers C7-armup-F / C7-armdown-R, respectively. The upstream homologous arm (1000 bp in length, downstream homologous arm (999 bp in length) was amplified using primers E5-armup-F / E5-armdown-R, and the upstream and downstream homologous arms of the E5 site (1183 bp in length, downstream homologous arm (838 bp in length)) were amplified using primers C1-armup-F / C1-armdown-R, and the upstream and downstream homologous arms of the C1 site (1000 bp in length, downstream homologous arm (1000 bp in length)) were amplified using primers C1-armup-F / C1-armdown-R, respectively.Approximately 1 μg of the integrated fragment and approximately 600 ng of sgRNA were transformed into the *Yeast Transformation* strain VE-7 constructed in Example 2 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. Engineered strains VE-9 to VE-12 were then constructed. These 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 (Figure 6) showed that the recombinant engineered strain VE-12 produced 102.8 mg / L of δ-tocotrienols.

[0094] Table 4 Primer sequences

[0095] Example 4: Fusion and Expression of SyHPT and AtVTE1

[0096] To increase the yield of δ-tocotrienol, the key enzymes SyHPT and AtVTE1 of δ-tocotrienol were fused in *Yamylostella lipolytica* using rigid linker peptides TPTP, (TPTP)2, EAAAK, (EAAAK)2, or flexible linker peptides 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 sequences of the linker peptides were added as homologous arms to the primer sequences used to amplify the SyHPT and AtVTE1 genes. Approximately 1 μg of the integrated fragment and approximately 600 ng of sgRNA were transformed into the *Yeast Transformation* strain VE-12 constructed in Example 3 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 resulting VE-13-VE-20 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 (Figure 7) showed that the recombinant engineered strain VE-20 had the highest yield of δ-tocotrienols, at 137.9 mg / L.

[0097] Table 5 Primer sequences

[0098] Table 6. Nucleotide sequences of linking peptides

[0099] Example 5: Semi-rational Design of SyHPT

[0100] 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… K77Y The number of hydrogen bonds in SyHPT is greater than that in WT, with a binding free energy of -73.4 kcal / mol, compared to -71.7 kcal / mol for WT. This means that the mutant SyHPT... K77YIt exhibits higher stability and stronger binding to substrates. Subsequently, this mutant and the δ-tocotrienol synthesis pathway gene were integrated into the multicopy site ZETA of *Yarrowia lipolytica* (published in the paper "YaliCMulti and YaliHMulti: Stable, efficient multi-copy integration tools for engineering *Yarrowia lipolytica*"), while the LEU2 and URA3 tags were reintroduced, further enhancing the yield of δ-tocotrienol. Approximately 1 μg of the integrated fragment and approximately 600 ng of sgRNA were transformed into the *Yarrowia lipolytica* engineered strain VE-20 constructed in Example 4 using the Frozen-EZ Yeast Transformation II kit, plated on selection solid medium, and incubated at 30°C for 3 days until colonies appeared. The 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-tocotrienols. HPLC analysis showed that the yield of δ-tocotrienols in strain VE-23 reached 189.9 mg / L (Figure 8).

[0101] Table 7 Primer sequences

[0102] Example 6: Scale-up cultivation of recombinant bacteria in a 5L fermenter

[0103] To further increase the yield of δ-tocotrienol, strain VE-23 was used for fed-batch fermentation in a 5L fermenter.

[0104] 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.

[0105] Metal ion solutions: 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;

[0106] Vitamin solution: 50 mg / L biotin, 200 mg / L para-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.

[0107] Feed-supported culture medium: 200 g / L peptone, 100 g / L yeast extract.

[0108] Preparation of primary seed culture: Colonies were picked from YPD plates and inoculated into shake flasks containing 10 mL of YPD. The culture was carried out at 28 °C and 220 rpm for 24 hours to obtain primary seed culture.

[0109] Preparation of secondary seed culture: Transfer all of the primary seed culture to a shake flask containing 200 mL YPD and incubate at 28 °C and 220 rpm for 24 hours.

[0110] The secondary seed culture was transferred to a fermenter containing 2.2 L of fermentation medium. The pH was set to 5.0, dissolved oxygen to 20%, and fermentation was carried out at 28 °C. To capture δ-tocotrienols, 10% (v / v) olive oil was added to the medium after 24 h of fermentation for extraction. When the initial glucose was depleted, 800 g / L glucose and fed medium were added, controlling the glucose flow rate to maintain a glucose concentration between 0.3 and 0.8 g / L, and the fed medium flow rate to 12–15 mL / h. 50% ammonia was added to maintain the pH at 5.0; the stirring rate was adjusted according to dissolved oxygen (DO) to maintain DO at 20%.

[0111] After 120 hours of cultivation, the accumulation of δ-tocotrienol reached 466.8 mg / L (Figure 9), which is the highest yield of δ-tocotrienol reported in microorganisms to date.

[0112] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

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

6.

2. The urea-sandy phytotransferase mutant according to claim 1, characterized in that, The mutations include (a) and / or (b): (a) Mutate isoleucine at position 61, lysine at position 77, and / or isoleucine at position 146 to alanine; (b) Mutate the 77th lysine residue to aspartic acid, glutamic acid, tryptophan, tyrosine, or phenylalanine.

3. A gene encoding the homogentisic acid phytotransferase mutant of claim 1 or 2.

4. A recombinant microorganism expressing the homogentisic acid phytotransferase mutant of claim 1 or 2.

5. A modified *Yamylostella lipolytica* strain, characterized in that... It expresses 4-hydroxyphenylpyruvate dioxygenase, tocopherol cyclase, and homogentisic acid phytotransferase or a mutant of homogentisic acid phytotransferase according to any one of claims 1 to 2; The 4-hydroxyphenylpyruvate dioxygenase is either YlHPD derived from Yloenzyme lipolyticum or PaHPD derived from Pseudomonas putida. The homogentisic acid phytotransferase is either TrHPT, derived from wheat, or SyHPT, derived from Synesthesia spp.; the tocopherol cyclase is AtVTE1, derived from Arabidopsis thaliana.

6. The engineered *Yamylostella lipolytica* strain according to claim 5, characterized in that, It also has one or more of the following improvements: (a) Overexpression of shikimic acid pathway genes ARO1, ARO4, ARO7, and the tyrosine feedback-inhibiting mutant ARO4 K221L and ARO7 G139S One or more genes in; (b) Overexpression of key genes in the MVA pathway, including: GGPP synthase gene, mevalonate kinase gene ERG12, truncated mevalonate-coA reductase gene tHMG1, isoprene diphosphate isomerase gene IDI1, farnesyl diphosphate synthase gene YlERG20, GGPP synthase gene SaGGPS derived from *Sulfolobus acidocaldarius*, or GGPP synthase gene XdGGPS derived from *Xanthophyllomyces dendrorhous*. (c) Fusing SyHPT and AtVTE1 linked by a linker peptide, wherein the linker peptide includes TPTP, (TPTP)2, EAAAK, (EAAAK)2, GSG, (GSG)2, GGGGS or (GGGGS)2.

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

8. The engineered *Yamylostella lipolytica* strain according to claim 7, characterized in that, It expresses 4-hydroxyphenylpyruvate dioxygenase YlHPD, tocopherol cyclase AtVTE1, and the homogentisic acid phytotransferase mutant of claim 1, and overexpresses the shikimic acid pathway gene; the shikimic acid pathway gene is (a) or (b): (a) Genes ARO1, ARO4, and ARO7; (b) Genes ARO1 and ARO4 K221L and ARO7 G139S ; The nucleotide sequence encoding the 4-hydroxyphenylpyruvate dioxygenase YlHPD is shown in SEQ ID NO.1; the nucleotide sequence encoding the tocopherol cyclase AtVTE1 is shown in SEQ ID NO.5; the nucleotide sequences of genes ARO1, ARO4, and ARO7 are shown in SEQ ID NO.7 to 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.

9. The engineered *Agropyron cristatum* strain according to any one of claims 5 to 7, characterized in that, Overexpression of the mevalonate kinase gene ERG12 and the GGPP synthase gene YlGGPS; or Overexpression of the mevalonate kinase gene ERG12, the GGPP synthase gene YlGGPS, the truncated mevalonate coenzyme A reductase gene tHMG1, and the isoprene diphosphate isomerase gene IDI1.

10. The engineered *Yamylostella lipolytica* strain according to any one of claims 5 to 9, characterized in that, It also overexpressed the farnesyl diphosphate synthase gene YlERG20, as well as the GGPP synthase gene SaGGPS derived from *Sulfolobus acidocaldarius* or the GGPP synthase gene XdGGPS derived from *Xanthophyllomyces dendrorhous*.

11. The engineered *Yamylostella lipolytica* strain according to claim 5, 8, or 10, characterized in that, The fusion expression of the homogentisic acid phytotransferase mutant K77Y and the Arabidopsis-derived tocopherol cyclase AtVTE was achieved.

12. A method for preparing δ-tocotrienol, characterized in that, The engineered *Yamylostella lipolytica* strain according to any one of claims 5 to 11 is fermented in a fermentation medium at 28–30°C for at least 72 h.

13. The method according to claim 12, characterized in that, Glucose is also added during the fermentation process.

14. The use of the homogentisic acid phytotransferase mutant of claim 1 or 2, or the gene of claim 3, or the recombinant microorganism of claim 4, or the engineered *Yamylostella lipolytica* of any one of claims 5 to 11, or the method of any one of claims 11 to 12 in the production of products containing δ-tocotrienols in the food, pharmaceutical, and chemical industries.

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