Recombinant Yarrowia lipolytica engineering strain for producing ergosterol and application of recombinant Yarrowia lipolytica engineering strain

Abstract

The invention discloses a recombinant Yarrowia lipolytica engineering strain for producing ergosterol and application of the recombinant Yarrowia lipolytica engineering strain, and belongs to the technical field of genetic engineering and bioengineering. According to the method, sterol synthesis route genes are screened, the speed limiting step is determined, high-yield ergosterol is preliminarily achieved through combined expression, the flow direction of carbon metabolic flow is further pulled through lipid droplet engineering and cell area chamber engineering, and the yield of ergosterol is increased. On the basis of enzyme modification engineering, the catalytic efficiency of the ERG11 on the lanosterol is improved through substrate channel engineering and proton-dependent catalytic path construction. Through multi-copy integration of the genes in the speed limiting step, the yield of ergosterol in a shake flask of the constructed engineering strain reaches 433.1 mg / L, and the yield of ergosterol in a 5L fermentation tank system reaches 4.58 g / L.

Description

TECHNICAL FIELD

[0001] The present application relates to a recombinant L lipomyces starkeyi engineering strain for producing ergosterol and application thereof, and belongs to the technical field of genetic engineering and bioengineering. BACKGROUND

[0002] Ergosterol is also called ergosterol, and its chemical formula is C 28 H 44 O, and its relative molecular mass is 396.65. Its chemical name is 24β-methylcholesterol-5,7 en-3β-hydroxy. Ergosterol is an important component of fungal cell membrane, and has stable structure and strong specificity. It is generally colorless needle-like or flaky crystal. It is soluble in ethanol, diethyl ether, benzene and chloroform, and insoluble in water.

[0003] Ergosterol is a kind of tetracyclic triterpenoid compound, mainly existing in ganoderma lucidum, corn germ, sesame oil and the like. In addition, ergosterol and some biosynthetic intermediates thereof are important metabolites with great economic value. In the pharmaceutical industry, ergosterol is a precursor of vitamin D2 and steroid hormone drugs. In recent years, new functions of ergosterol have been found, such as 11-dehydroergosterol peroxide having significant anti-tumor activity, and several compounds having anti-HIV activity being structural analogues of ergosterol. Therefore, ergosterol has wide application prospects as an important precursor for developing new anti-cancer and anti-HIV drugs, and promotes in-depth research on fungal sterol biosynthesis, metabolism and regulation. Good efficacy. At present, there have been reports on the synthesis of ergosterol by using saccharomyces cerevisiae, but the yield of synthesized ergosterol is low. Therefore, how to synthesize ergosterol by using a safer biological method and obtain a higher yield and improve the efficiency of fermentation production is a problem to be solved at present. SUMMARY

[0004] The present application also provides a lanosterol 14α-demethylase mutant, which has a mutation of the 305th and / or 372nd amino acid relative to a lanosterol 14α-demethylase parent. ERG11 The present application also provides a lanosterol 14α-demethylase mutant, which has a mutation of the 305th and / or 372nd amino acid relative to a lanosterol 14α-demethylase parent.

[0005] In one embodiment, the amino acid sequence of the parent is shown in SEQ ID NO. 18.

[0006] In one embodiment, the mutation is that the 372nd serine (S) is mutated to valine (V), and / or the 305th threonine (T) is mutated to histidine (H).

[0007] The present application also provides a fusion enzyme, which is connected by a connecting peptide and at least two enzymes: lactate dehydrogenase LDH1, sterol acyltransferase related enzyme ARE1, C-24(28) sterol reductase ERG4.

[0008] In one embodiment, the connecting peptide includes, but is not limited to, a flexible connecting peptide (GGGGS)3, an alpha-helix connecting peptide (EAAAK)3, or an alpha-helix connecting peptide (PT)5P.

[0009] The present application also provides a gene encoding the mutant.

[0010] The present application also provides a recombinant microorganism expressing the mutant or expressing the fusion enzyme.

[0011] The present application provides a Yarrowia lipolytica engineering strain for efficiently synthesizing ergosterol. On the basis of the starting strain, the Yarrowia lipolytica engineering strain expresses methylglutaconyl-CoA reductase tHMG1 and isopentenyl diphosphate isomerase IDI1 , 3-ketosteroid reductase ERG27 , and lanosterol 14α-demethylase ERG11 or a mutant thereof.

[0012] In one embodiment, the methylglutaconyl-CoA reductase gene tHMG1 and the isopentenyl diphosphate isomerase gene IDI1 are integrated into the A3 site.

[0013] In one embodiment, the 3-ketosteroid reductase gene ERG27 and the lanosterol 14α-demethylase gene ERG11 or a mutant gene thereof are integrated into the D17 site.

[0014] In one embodiment, the Yarrowia lipolytica engineering strain further expresses C-24(28) sterol reductase ERG4 and C-22 sterol desaturase ERG5 .

[0015] In one embodiment, the Yarrowia lipolytica engineering strain further expresses lanosterol 14α-demethylase ERG11 and squalene epoxidase ERG1 .

[0016] In one embodiment, the Yarrowia lipolytica engineering strain further knocks out the formaldehyde dehydrogenase gene FLD1 .

[0017] In one embodiment, the Yarrowia lipolytica engineering strain expresses, on the basis of the starting strain, a fusion enzyme of lactate dehydrogenase LDH1 and sterol acyltransferase-related enzyme ARE1 connected by a flexible connecting peptide.

[0018] In one embodiment, the Yarrowia lipolytica engineering strain expresses, on the basis of the starting strain, a fusion enzyme of sterol acyltransferase-related enzyme ARE1 and C-24(28) sterol reductaseERG4 Fusion enzyme.

[0019] In one embodiment, the engineered *Yamylostella lipolytica* strain is based on a starting strain and expresses lactate dehydrogenase LDH1 and C-24(28) sterol reductase linked by flexible linker peptides. ERG4 A fusion enzyme of ARE1, a sterol acyltransferase-associated enzyme.

[0020] In one embodiment, the nucleotide sequences of genes tHMG1 and IDI1 are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively; the nucleotide sequences of genes ERG4, ERG27, ERG11, ERG5, and ERG1 are shown in SEQ ID NO.4, SEQ ID NO.7, SEQ ID NO.9, SEQ ID NO.12, and SEQ ID NO.14, respectively; the formaldehyde dehydrogenase gene FLD1 The nucleotide sequence is shown in SEQ ID NO.17.

[0021] In one implementation, promoter P is used. TDH P TEF P MNDH2 or P GPD1 Initiate the expression of one or more genes from tHMG1, IDI1, ERG1, ERG11, ERG27, ERG4, and ERG5.

[0022] In one embodiment, the engineered *Yarrowia lipophila* strain is based on a starting strain and expresses a fusion enzyme in which lactate dehydrogenase 1 and sterol acyltransferase-associated enzyme 1 are linked by an α-helix linker peptide (PT)3P.

[0023] In one embodiment, the engineered *Yamylostella lipolytica* strain is based on the starting strain and expresses a fusion enzyme in which lactate dehydrogenase 1;C-24(28) sterol reductase is linked by an α-helix linker peptide (PT)3P.

[0024] In one implementation, tHMG1, IDI1, ERG9, ERG12 and ERG20 are integrated and expressed at multiple copy sites.

[0025] In one implementation, expression is integrated at multiple copy sites. ERG1 and ERG11 mutant S372V-T305H .

[0026] In one embodiment, the starting strain includes, but is not limited to, Yeastra lipolytica Po1f.

[0027] In one implementation, genes tHMG1, IDI1 Integrated at the D17 site, geneERG1, ERG11 It is integrated at the A3 site.

[0028] In one implementation, genes ERG11, ERG4 Integrating at the AXP site, gene ERG5, ERG27 It is integrated at the E3 site.

[0029] In one embodiment, the multiple copy site includes, but is not limited to, ZETA or 26S rDNA.

[0030] In one embodiment, the starting strain is *Yamylostella lipolytica* Po1f.

[0031] The present invention also provides the application of the engineered *Yamylostella lipolytica* strain in the production of ergosterol.

[0032] In one embodiment, the engineered *Yamylostella lipolytica* strain is fermented in a culture medium at 28-30°C for at least 72 hours.

[0033] In one embodiment, the engineered *Yamylostella lipolytica* strain is fermented in a culture medium at 30°C for 72-96 hours, or 72-120 hours.

[0034] In one embodiment, the culture medium includes, but is not limited to, YPD culture medium and inorganic salt culture medium.

[0035] In one embodiment, the inorganic salt culture medium contains glucose, ammonium sulfate, potassium salt, and magnesium salt; the potassium salt includes, but is not limited to, potassium dihydrogen phosphate; and the magnesium salt includes, but is not limited to, magnesium sulfate.

[0036] In one embodiment, glucose is fed to control the carbon source in the reaction system to 0.1~1 g / L.

[0037] This invention also claims protection for the use of the engineered *Yamylostella lipolytica* strain in the production of ergosterol-containing products in the food, pharmaceutical, and chemical industries.

[0038] In one embodiment, the application is for the preparation of vaccines or drugs containing ergosterol, or for the preparation of cosmetics containing ergosterol.

[0039] Beneficial effects: (1) In order to adjust the carbon distribution flow and increase ergosterol production, this invention systematically evaluated the effects of various genes in the sterol modification pathway on ergosterol and screened out the key rate-limiting steps. The carbon flow was readjusted by combining expression. Mutagenesis of key substrate channel residues guided by molecular dynamics (especially S372V ERG11) enhanced local flexibility and significantly increased ergosterol production.

[0040] (2) This invention achieves efficient synthesis of ergosterol by readjusting the carbon distribution flow in Yeast Extract. An artificial proton-dependent pathway was established by introducing the proton donor mutant S372V-T305H-Erg11, which, together with channel engineering, further increased the titer to 124 mg / L.

[0041] (3) The present invention further increases the ergosterol titer of the engineered bacteria to 148.3 mg / L by lipid droplet engineering and cell compartmentalization strategy, and increases the titer at the shake flask level to 433.1 mg / L by integrating multiple copies of the ergosterol synthesis pathway gene.

[0042] (4) The present invention also provides a low-cost ergosterol fermentation method, in which engineered bacteria are fed in batches in a 5-L bioreactor using an inorganic salt culture medium, so that the final titer after 168 h of fermentation is 4.58 g / L. Attached Figure Description

[0043] Figure 1 A schematic diagram of the metabolism of endogenous ergosterol synthesis in Yeast Extract.

[0044] Figure 2 This is a schematic diagram showing the docking results of ERG11 enzyme with lanosterol molecules.

[0045] Figure 3 Figure 1 shows the ergosterol production of different recombinant *Yacinthia lipolytica* strains under YPD culture; among them, the ergosterol production of strains RB01, RB02, RB03, and RB06 were 43.7 mg / L, 49.8 mg / L, 71.7 mg / L, and 94.2 mg / L, respectively.

[0046] Figure 4 Synthesis of ergosterol from strain RB19 optimized for use in a 5L fermenter.

[0047] Figure 5 The effects of different linker peptides or combinations on the synthesis of ergosterol in recombinant bacteria. Detailed Implementation

[0048] (a) Culture medium 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.

[0049] YNB medium: Yeast Nutrition Base 67.4 g / L, glucose 20 g / L. Depending on the auxotrophic type of the strain to be cultured, appropriate amino acids (5 g / L uracil, 5 g / L leucine) are added to the medium.

[0050] YPD medium: peptone 20 g / L, yeast extract 10 g / L, glucose 20 g / L.

[0051] Inorganic salt culture medium: ammonium sulfate 5 g / L, potassium dihydrogen phosphate 3.5 g / L, magnesium sulfate 5 g / L, glucose 40 g / L.

[0052] Trace metal solutions: 4.5 g / L CaCl₂·2H₂O, 4.5 g / L ZnSO₄·7H₂O, 3 g / L FeSO₄·7H₂O, 1 g / L H₃BO₃, 1 g / L MnCl₂·4H₂O, 0.4 g / L Na₂MoO₄·2H₂O, 0.3 g / L CoCl₂·6H₂O, 0.1 g / L CuSO₄·5H₂O, 0.1 g / L KI, 15 g / L LEDTA. Adjust pH to 4, filter for sterilization, and store at 4°C protected from light.

[0053] 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 hydrochloride, 1 g / L thiamine hydrochloride, 25 g / L inositol. Adjust pH to 6.5, filter for sterilization, and store at 4°C protected from light.

[0054] (II) Transformation steps of *Yacinthia lipolytica*: Pick a single yeast colony from an agar plate and inoculate it into 3-4 mL of YPD liquid medium. Incubate at 30°C for 16-22 h to obtain the primary seed culture. Transfer the primary seed culture at a 2% inoculation rate to 10 mL of YPD liquid medium and incubate at 30°C for 4-6 h to obtain the secondary seed culture. Centrifuge 500 μL of the secondary seed culture at 5000 rpm for 4 min and discard the supernatant. Then prepare the transformation buffer in a sterile EP tube. Transformation buffer: 90 μL of 50% sterile PEG4000, 5 μL of 2 M lithium acetate, and 5 μL of boiled linear single-stranded DNA, vortex to mix. Add the transformation buffer, 0.4-0.6 μg of circular plasmid or 3-10 μg of linear DNA fragment, vortex to mix. Incubate in a 30°C water bath for 30-45 min, vortexing for 15 s every 10 min. Incubate in a 39°C water bath for 10 minutes. Spread the mixture onto the appropriate selective plates and incubate at 30°C for approximately 2-3 days.

[0055] (III) Ergosterol HPLC determination: The determination was performed using Shimadzu high-performance liquid chromatography. HPLC conditions: Column: InertSustain C18 250 mm × 4.6 mm column (particle size 5 μm); mobile phase B, methanol containing 1‰ trifluoroacetic acid; flow rate: 1 mL / min; column temperature: 30℃; injection volume: 10 μL; detector wavelength: 281 nm.

[0056] (v) The strain information is shown in Table 1.

[0057] Table 1. Strains involved in this invention

[0058] Table 2 Primer sequences

[0059] Table 3 Promoters and Sequences

[0060] Example 1 Construction of recombinant engineered strain RB01 Using *Yarrowia lipolytica* Po1f (published in the paper "Remodeling metabolism for high-level resveratrol production in Yarrowia lipolytica") as the starting strain, expression cassette P... TEF - tHMG1 -T XPR2- P TEF - IDI1 -T XPR2 The chassis strain, integrated at the A3 site, was named RB01.

[0061] The specific construction method is as follows: using the original genome of the Polf1 strain, the HMG1 gene fragment (nucleotide sequence as shown in SEQ ID NO.1) was amplified from the genome using primers HMG1-F / HMG1-R, and the IDI1 gene fragment (nucleotide sequence as shown in SEQ ID NO.2) was amplified using primers IDI1-F / IDI1-R. The promoter was then amplified using primers PTEF-F / PTEF-R. P TEFThe upstream and downstream homologous arms of the A3 site were amplified using primers D17-armup-F / D17-armup-R and A3-armdown-F / A3-armdown-R, respectively. The PCR products were recovered by ethanol precipitation. The above fragments were assembled using Gibson chromatography. Approximately 1 μg of the integrated fragment, consisting of gene fragments IDI1 and tHMG1 linked to the A3 homologous arm, and approximately 300 ng of sgRNA were transformed into the *Yamylostella lipolytica* engineered strain Polf1 using lithium acetate. The transformed fragment was plated on selection solid medium and incubated at 30°C for 3 days until colonies appeared. The correctly cloned strain was named RB01. Single colonies were picked and transferred to 5 mL of the corresponding YNB medium. After 24 h, the inoculum was transferred to 25 mL of YPD medium at a 1% inoculation rate. After culturing for 120 h, 500 μL of fermentation broth was collected and centrifuged at 12000 × g for 2 min. The precipitate was then collected and 1 mL of potassium hydroxide saponification solution was added. Saponification was carried out at 89 °C for 4 h, with resuspension by shaking every half hour. Then, 2.5 mL of anhydrous diethyl ether was added, and the mixture was vortexed at 2000 rpm for 10 min. After the solution was allowed to stand and separate into layers, the supernatant was collected, filtered through a 0.22 μm organic filter membrane, and analyzed by HPLC. The results showed that after integrating tHMG1 and IDI1, the squalene yield increased from 16.7 mg / L of the starting strain Po1f to 260.1 mg / L, while the ergosterol yield increased to 41.9 mg / L.

[0062] Example 2 Screening of the key rate-limiting enzyme in the synthesis of ergosterol Based on the strain RB01 constructed in Example 1, integration was performed at the D17 site. ERG3 , ERG4 , ERG6 , ERG26 , ERG27 , ERG24 , ERG11 , ERG4 , ERG7 , ERG5 , ERG25 , ERG1 Genes, constructing engineered strain RB01- ERG3 RB01- ERG4 RB01- ERG6 RB01- ERG26 RB01- ERG27 RB01- ERG24 RB01- ERG11 RB01- ERG4 RB01- ERG7 RB01- ERG5 RB01- ERG25 RB01- ERG1.

[0063] The specific construction method is as follows: using the original genome of the Polf1 strain, the genome was amplified by PCR using primers ERG3-F / ERG3-R to obtain the amplified genome. ERG3 Gene fragment (nucleotide sequence as shown in SEQ ID NO.3), amplified using primers ERG4-F / ERG4-R. ERG4 Gene fragment (nucleotide sequence as shown in SEQ ID NO.4), amplified using primers ERG6-F / ERG6-R. ERG6 Gene fragment (nucleotide sequence as shown in SEQ ID NO.5), amplified using primers ERG26-F / ERG26-R. ERG26 Gene fragment (nucleotide sequence as shown in SEQ ID NO. 6), amplified using primers ERG27-F / ERG27-R. ERG27 Gene fragment (nucleotide sequence as shown in SEQ ID NO.7), amplified using primers ERG24-F / ERG24-R. ERG24 Gene fragment (nucleotide sequence as shown in SEQ ID NO. 8), amplified using primers ERG11-F / ERG11-R. ERG11 Gene fragment (nucleotide sequence as shown in SEQ ID NO. 9), amplified using primers ERG2-F / ERG2-R. ERG2 Gene fragment (nucleotide sequence as shown in SEQ ID NO.10), amplified using primers ERG7-F / ERG7-R. ERG7 Gene fragment (nucleotide sequence as shown in SEQ ID NO.11), amplified using primers ERG5-F / ERG5-R. ERG5 Gene fragment (nucleotide sequence as shown in SEQ ID NO.12), amplified using primers ERG25-F / ERG25-R. ERG25 Gene fragment (nucleotide sequence as shown in SEQ ID NO.13), amplified using primers ERG1-F / ERG1-R. ERG1 The gene fragment (nucleotide sequence shown in SEQ ID NO.14) was amplified using primers PTEF-F / PTEF-R to amplify the promoter. P TEFThe upstream and downstream homologous arms of the D17 site were amplified using primers D17-armup-F / D17-armup-R and D17-armdown-F / D17-armdown-R, respectively. The PCR products were recovered by ethanol precipitation. Using Gibson assembly, approximately 1 μg of the integrated fragments (ERG4, ERG6, ERG26, ERG27, ERG24, ERG11, ERG4, ERG7, ERG5, ERG25, and ERG1 genes linked to the D17 homologous arms) and approximately 300 ng of sgRNA were transformed into the *Yarrowia lipolytica* strain RB01 constructed in Example 1 using lithium acetate. The transformed strain was plated on screening solid medium and incubated at 30°C for 3 days until colonies appeared. The correctly cloned strain was named RB01-. ERG3 RB01- ERG4 RB01- ERG6 RB01- ERG26 RB01- ERG27 RB01- ERG24 RB01- ERG11 RB01- ERG4 RB01- ERG7 RB01- ERG5 RB01- ERG25 RB01- ERG1 Single colonies were picked and transferred to 5 mL of the corresponding YNB medium. After 24 h, they were transferred to 25 mL of YPD medium at a 1% inoculation rate. After culturing for 120 h, 500 μL of fermentation broth was collected and centrifuged at 12000 × g for 2 min. The precipitate was then collected and 1 mL of potassium hydroxide saponification solution was added. Saponification was carried out at 89 °C for 4 h, with resuspending by shaking every half hour. Then, 2.5 mL of anhydrous diethyl ether was added, and the mixture was vortexed at 2000 rpm for 10 min. After the solution was allowed to stand and separate into layers, the supernatant was collected, filtered through a 0.22 μm organic filter membrane, and analyzed by HPLC. The results are shown in Table 4. ERG1 The strain of the gene produced ergosterol at a yield of 62.2 ± 1.6 mg / L.

[0064] Table 4. Effects of genes with different sterol modification pathways on ergosterol production.

[0065] Example 3 Construction of a recombinant *Aylocereus lipolyticus* strain for ergosterol synthesis To further increase ergosterol production and readjust intracellular carbon metabolic flux, the D17, C2, and AXP sites of strain RB01 were used as lanosterol 14α demethylases. ERG11 and 3-ketosterol reductaseERG27, δ(24(24(1)))-sterol reductase ERG4 and C-22 sterol desaturase ERG5, squalene monooxygenase ERG1 and lanosterol 14α demethylase ERG11 The integration site was identified, and engineered strains RB02, RB03, and RB06 were constructed. Specifically, the PCR method involved using the Polf1 strain genome as a template and amplifying the genome with primers ERG4-F / ERG4-R. ERG4 Gene fragment (nucleotide sequence as shown in SEQ ID NO.4), amplified using primers ERG27-F / ERG27-R. ERG27 Gene fragment (nucleotide sequence as shown in SEQ ID NO.7), amplified using primers ERG11-F / ERG11-R. ERG11 Gene fragment (nucleotide sequence as shown in SEQ ID NO.9), primers ERG5-F / ERG5-R amplification ERG5 Gene fragment (nucleotide sequence as shown in SEQ ID NO.12), primers ERG1-F / ERG1-R amplification ERG1 The gene fragment (nucleotide sequence shown in SEQ ID NO.14) was amplified using primers PTEF-F / PTEF-R to amplify the promoter. P TEFThe upstream and downstream homologous arms of the D17 site were amplified using primers D17-armup-F / D17-armup-R and D17-armdown-F / D17-armdown-R, respectively. Similarly, the upstream and downstream homologous arms of the C2 site were amplified using primers C2-armup-F / C2-armup-R and C2-armdown-F / C2-armdown-R, and the upstream and downstream homologous arms of the AXP site were amplified using primers AXP-armup-F / AXP-armup-R and AXP-armdown-F / AXP-armdown-R. The PCR products were recovered by ethanol precipitation. The above fragments were assembled using Gibson. Approximately 1 μg of integrated fragments, consisting of ERG4, ERG27, ERG11, ERG5, and ERG1 genes linked to the homologous arms of C2, AXP, and D17 respectively, along with approximately 300 ng of sgRNA, were transformed into the Yeast Extract RB01 using lithium acetate. The fragments were plated on a selection solid medium and cultured at 30°C for 3 days until colonies appeared. The correctly cloned strains were named RB02, RB03, and RB06. Single colonies were picked and transferred to 5 mL of the corresponding YNB medium. After 24 h, the inoculum was transferred to 25 mL of YPD medium at a 1% inoculation rate. After culturing for 120 h, 500 μL of fermentation broth was collected and centrifuged at 12000 × g for 2 min. The precipitate was then collected and 1 mL of potassium hydroxide saponification solution was added. Saponification was carried out at 89 °C for 4 h, with resuspending by shaking every half hour. Then, 2.5 mL of anhydrous diethyl ether was added, and the mixture was vortexed at 2000 rpm for 10 min. After allowing the solution to stand and separate into layers, the supernatant was collected, filtered through a 0.22 μm organic filter membrane, and analyzed by HPLC. The results showed that the yield of strain RB06 increased to 94.2 mg / L.

[0066] Example 4: Substrate channel modification to improve the catalytic efficiency of enzyme ERG11 To improve the catalytic efficiency of the enzyme ERG11, the protein structure of ERG11 was predicted and molecularly docked with lanosterol. Electron cloud analysis of the docking results identified the substrate channel of ERG11 and its surrounding residues. These residues included P225, Y64, F228, F374, and S372, located at different positions within the substrate channel. ERG11 was amplified using wild-type ERG11 (SEQ ID NO. 9) as a template, employing primers S372A-F / S372A-R. S372A The mutant, and referring to the method for constructing strain RB06 in Example 2, was used to construct ERG11. S372AThe gene fragment was linked to the upstream and downstream homologous arms of AXP. The PCR product was recovered by ethanol precipitation. Approximately 1 μg of the integrated fragment and approximately 300 ng of sgRNA were transformed into the engineered *Yamylostella lipolytica* strain RB01 using lithium acetate. The transformed strain was plated on a selection solid medium and incubated at 30°C for 3 days until colonies appeared. The correct clone was named RB01-S372V.

[0067] Recombinant bacteria expressing the ERG11 mutation shown in Table 4 were constructed using the same strategy described above. Single colonies were transferred to 5 mL of the corresponding YNB medium. After 24 h, a 1% inoculum was transferred to 25 mL of YPD medium. After culturing for 120 h, 500 μL of fermentation broth was collected and centrifuged at 12000 × g for 2 min. The precipitate was then collected and 1 mL of potassium hydroxide saponification solution was added. Saponification was carried out at 89℃ for 4 h, with resuspension by shaking every half hour. Then, 2.5 mL of anhydrous diethyl ether was added, and the mixture was vortexed at 2000 rpm for 10 min. After allowing the solution to stand and separate into layers, the supernatant was collected, filtered through a 0.22 μm organic filter, and analyzed by HPLC. The results showed that the mutation at the S327V site had the most significant effect on yield improvement, with ergosterol yield reaching 50.5 mg / L, an increase of 15.6% compared to the control strain (RB01).

[0068] Table 5 Effects of different mutants on ergosterol production

[0069] Example 5 Construction of a proton-dependent catalytic pathway Functional amino acid residues in the ERG11 catalytic pocket were identified using alanine scanning, including Y118, Y132, G301, I370, and T305. Subsequently, based on fermentation conditions, the physical properties of the functional amino acid residues, and their positions within the catalytic pocket, it was determined that lanosterol catalysis in *Yarrowia lipolytica* primarily occurs via a proton-independent catalytic pathway. To improve the catalytic efficiency of ERG11, a proton-independent catalytic pathway was constructed. The aforementioned constructed ERG11... S372A Based on the mutant, the combined mutant ERG11 was amplified using T305H-F / T305H-R primers. S372A-T305H Following the method used in Example 2 to construct strain RB06, the upstream and downstream homologous arms of AXP were connected with ERG11. S372A-T305H Gene fragments were linked. PCR products were recovered by ethanol precipitation. Approximately 1 μg of the integrated fragment and approximately 300 ng of sgRNA were transformed into the engineered *Yamylostella lipolytica* strain RB01 using lithium acetate. The transformed fragments were plated on selection solid medium and incubated at 30°C for 3 days until colonies appeared. The correct clone was named RB01-S372V-T305H.

[0070] Recombinant bacteria expressing the ERG11 mutation shown in Table 5 were constructed using the same strategy described above. Single colonies were transferred to 5 mL of the corresponding YNB medium. After 24 h, a 1% inoculum was transferred to 25 mL of YPD medium. After culturing for 120 h, 500 μL of fermentation broth was collected and centrifuged at 12000 × g for 2 min. The precipitate was then collected and 1 mL of potassium hydroxide saponification solution was added. Saponification was carried out at 89 °C for 4 h, with resuspension by shaking every half hour. Then, 2.5 mL of anhydrous diethyl ether was added, and the mixture was vortexed at 2000 rpm for 10 min. After allowing the solution to stand and separate into layers, the supernatant was collected, filtered through a 0.22 μm organic filter, and analyzed by HPLC. The results showed that the mutation at the T305H site had the most significant effect on yield improvement, increasing ergosterol yield by 17.4% to 59.3 mg / L.

[0071] Table 6. Effects of different mutant combinations on ergosterol production

[0072] Example 6: Comparison of yield between the superior mutant ERG11-S372V-T305H and the wild type.

[0073] To verify the difference in catalytic effect between the modified mutant and the wild type, a wild-type ERG11 and a dominant mutant ERG11-S372V-T305H were integrated into the FLD1 site (nucleotide sequence shown in SEQ ID NO.17) of the strain RB06 constructed in Example 3. The specific steps were as follows: the genes at both ends were amplified using ERG11-F / ERG11-R. The PCR products were recovered by ethanol precipitation. The above fragment was assembled using Gibson. Approximately 1 μg of the integrated fragment, linked by the upper and lower homologous arms of FLD1, along with approximately 300 ng of sgRNA, was transformed into the *Yamylostella lipolytica* engineered strain RB06 using lithium acetate. The transformed fragment was plated on selection solid medium and incubated at 30°C for 3 days until colonies appeared. The correctly cloned strains were named RB06-WT and RB06-VA, respectively. Single colonies were picked and transferred to 5 mL of the corresponding YNB medium. After 24 h, the inoculum was transferred to 25 mL of YPD medium at a 1% inoculation rate. After culturing for 120 h, 500 μL of fermentation broth was collected and centrifuged at 12000 × g for 2 min. The precipitate was then collected and 1 mL of potassium hydroxide saponification solution was added. Saponification was carried out at 89 °C for 4 h, with resuspension by shaking every half hour. Then, 2.5 mL of anhydrous diethyl ether was added, and the mixture was vortexed at 2000 rpm for 10 min. After the solution was allowed to stand and separate into layers, the supernatant was collected, filtered through a 0.22 μm organic filter membrane, and analyzed by HPLC. The results showed that the ergosterol production of the strain RB06-VA, which integrated the dominant mutant, was increased by 38.1% compared with that of RB06-WT, which integrated the wild-type ERG11.

[0074] Example 7 Construction of recombinant engineered bacteria RB08 To increase the throughput of ergosterol synthesis, the FLD1 gene (nucleotide sequence shown in SEQ ID NO.17) was knocked out. The upstream and downstream homologous arms of the FLD1 site were amplified using FLD1-armup-F / FLD1-armup-R and FLD1-armdown-F / FLD1-armdown-R, respectively. PCR products were recovered by ethanol precipitation. The fragments were assembled using Gibson. Approximately 1 μg of the integrated fragment, linked by the upstream and downstream homologous arms of FLD1, along with approximately 300 ng of sgRNA, was transformed into the *Yamylostella lipolytica* engineered strain RB06 using lithium acetate. The transformed strain was plated on selection solid medium and incubated at 30°C for 3 days until colonies appeared. The correctly cloned strain was named RB08. Single colonies were picked and transferred to 5 mL of the corresponding YNB medium. After 24 h, the inoculum was transferred to 25 mL of YPD medium at a 1% inoculation rate. After culturing for 120 h, 500 μL of fermentation broth was collected and centrifuged at 12000 × g for 2 min. The precipitate was then collected and 1 mL of potassium hydroxide saponification solution was added. Saponification was carried out at 89 °C for 4 h, with resuspension by shaking every half hour. Then, 2.5 mL of anhydrous diethyl ether was added, and the mixture was vortexed at 2000 rpm for 10 min. After standing to allow the solution to separate into layers, the supernatant was collected, filtered through a 0.22 μm organic filter membrane, and analyzed by HPLC. The results showed that after knocking out FLD1, the ergosterol yield of strain RB08 increased by 25.3% compared to strain RB06, reaching 113 mg / L.

[0075] Example 8: Construction of engineered strain RB11 through cell compartmentalization engineering To reduce the concentration of ergosterol in the cytoplasm and thus alleviate the negative feedback pressure on the transcription of sterol pathway synthesis genes, cell compartmentalization engineering was used to overexpress the following genes linked by linker peptides (GGGGS)3, (EAAAK)3, and (PT)5P: LDH1 (nucleotide sequence as shown in SEQ ID NO.15), ARE1 (nucleotide sequence as shown in SEQ ID NO.16), and ERG4 (nucleotide sequence as shown in SEQ ID NO.4).

[0076] Table 7. Nucleotide sequences of different linking peptides

[0077] Genes were amplified from the genome of *Yarrowia lipolyticis* using primers ERG4-F / ERG4-R. ERG4 Fragments were amplified from the genome of *Yamylostella lipolytica* using ARE1-F / ARE1-R. ARE1 Fragments were amplified from the genome of *Yamylostella lipolytica* using LDH1-F / LDH1-R. LDH1The fragment was amplified using F13-armup-F / F13-armup-R and F13-armdown-F / F13-armdown-R to obtain the upstream and downstream homologous arms of the E3 site. The PCR products were recovered by ethanol precipitation. Gibson assembly was performed. Approximately 1 μg of the integrated fragment and approximately 300 ng of sgRNA were transformed into the *Yarrowia lipolytica* strain RB08 constructed in Example 5 using lithium acetate. The mixture was plated on selection solid medium and incubated at 30°C for 3 days until colonies appeared. Correct clones were named RB08–RB17. Single colonies were picked and transferred to 5 mL of the corresponding YNB medium. After 24 h, the inoculum was transferred to 25 mL of YPD medium at a 1% inoculation rate. After culturing for 120 h, 500 μL of fermentation broth was collected and centrifuged at 12000 × g for 2 min. The precipitate was then collected and 1 mL of potassium hydroxide saponification solution was added. Saponification was carried out at 89 °C for 4 h, with resuspending by shaking every half hour. Then, 2.5 mL of anhydrous diethyl ether was added, and the mixture was vortexed at 2000 rpm for 10 min. After the solution was allowed to stand and separate into layers, the supernatant was collected, filtered through a 0.22 μm organic filter membrane, and analyzed by HPLC. The results are as follows: Figure 5 As shown, the ergosterol production of strain RB11, which expresses ARE1 and ERG4 through (GGGGS)3 fusion, was increased by 34.2% to 138.3 mg / L.

[0078] Example 9: Multi-copy integration of key genes enhances sterol modification pathways Liquid chromatography-mass spectrometry (LC-MS) analysis revealed that a significant amount of squalene in the fermentation broth of strain RB11 remained unconverted to ergosterol. A multi-copy integration strategy was employed to further increase the synthesis throughput of ergosterol. Since the *Yarrowia lipolytica* strain constructed in this application is leucine and uracil-deficient, rDNA-F / rDNA-R primers were used with the pYlST1206 plasmid (disclosed in the paper "A set of efficient and stable multi-copy integration toolkits"). Yarrowia lipolytica Using 》 as a template, a multicopy plasmid backbone was amplified, and the leucine tag, key genes ERG11-S372V-T305H, ERG4, ERG5, ERG1, and ERG27 were linked to the homologous arm of the multicopy site rDNA to construct the multicopy plasmid pYLXP'-rDNA-PSERG.

[0079] Using ZETA-F / ZETA-R primers with pYlST889 plasmid (published in the paper "Remodeling metabolism for high-level resveratrol production in Yarrowia lipolytica") as a template, a multicopy plasmid backbone was amplified. The uracil tag, key genes IDI1 and tHMG1 were linked to the ZETA homologous arm of the multicopy site to construct the multicopy plasmid pYLXP'-ZETA-PRERG. Multiple copies of the 26S rDNA at the site of the strain RB11 constructed in Example 7 were integrated. Twenty-four single colonies were randomly selected from the transformation plate and inoculated into 5 mL of the corresponding YNB medium. After 24 h, the colonies were transferred to 25 mL of YPD medium at a 1% inoculation rate. After culturing for 120 h, 500 μL of the fermentation broth was collected and centrifuged at 12000 × g for 2 min. The precipitate was then collected and 1 mL of potassium hydroxide saponification solution was added. Saponification was carried out at 89 °C for 4 h, with resuspension by shaking every half hour. Then, 2.5 mL of anhydrous diethyl ether was added, and the mixture was vortexed at 2000 rpm for 10 min. After allowing the solution to stand and separate into layers, the supernatant was collected, filtered through a 0.22 μm organic filter membrane, and analyzed by HPLC. The results showed that the yield of ergosterol in the obtained recombinant engineered strain RB18 was 218.9 mg / L. Multiple copies of the ERG1 and ERG11-S372V-T305H genes were integrated into the ZETA site of the recombinant engineered bacterium RB18 to construct the recombinant engineered bacterium RB19. Twenty-four single colonies were randomly selected from transformation plates and inoculated into 5 mL of the corresponding YNB medium. After 24 h, the colonies were transferred to 25 mL of YPD medium at a 1% inoculum size and cultured for 120 h. 500 μL of the fermentation broth was collected and centrifuged at 12000 × g for 2 min. The precipitate was then collected and 1 mL of potassium hydroxide saponification solution was added. Saponification was carried out at 89 °C for 4 h, with resuspension by shaking every half hour. Then, 2.5 mL of anhydrous diethyl ether was added, and the mixture was vortexed at 2000 rpm for 10 min. After standing to allow separation, the supernatant was collected, filtered through a 0.22 μm organic filter membrane, and analyzed by HPLC. The results showed that the ergosterol yield of the recombinant engineered bacterium RB19 was 433.1 mg / L.

[0080] Example 10: Scale-up and optimization of 5L fermenter The ergosterol production capacity of strain RB19 constructed in Example 9 was verified in a 5L fermenter. Single colonies were picked from plates and inoculated into 4 mL of YPD liquid medium, and cultured at 30°C for 18–24 h as the primary seed culture. The primary seed culture was transferred to a shake flask containing 200 mL of YNB liquid medium and cultured at 30°C and 220 rpm for 24 h as the secondary seed culture. The secondary seed culture was inoculated into a 5L fermenter with an initial inoculum of 8%–10%, and the initial OD... 600 The dissolved oxygen level, pH, and temperature were monitored using electrodes controlled at 0.6-0.8. The fermenter initially contained 2.2 L of LYPD medium or 2.2 L of inorganic salt medium, with an additional 3 mL of trace metal solution and 3 mL of vitamin solution added to promote cell growth. Once the initial glucose was depleted, glucose was added at a constant flow rate to maintain a concentration of 0.1 g / L–1 g / L. NH3·H2O was used to maintain the pH of the fermentation broth at 4.5–5.0, and dissolved oxygen was controlled at 10–20% by adjusting the stirring speed and aeration rate. Samples were taken every 6 hours, with three samples taken as parallels to measure glucose concentration and OD. 600 The results showed that the strain produced 3.88 g / L of ergosterol after 168 h of fermentation in YPD medium, while the ergosterol yield reached 4.58 g / L after 168 h of fermentation in inorganic salt medium. The OD... 600 Reaching 180 makes it more conducive to the low-cost industrial production of ergosterol.

[0081] Comparative Example 1: The specific implementation method is the same as in Example 3, except that, based on strain RB03, the NAD-specific glutamate dehydrogenase gene is integrated and expressed at the AXP site. GDH1 (Gene ID:2912559), NADP-specific glutamate dehydrogenase gene GDH2 (Gene ID: 2908646). The constructed strains RB04 and RB05 were fermented according to the method in Example 3, and the results showed that the yields were 65.5 mg / L and 60.2 mg / L, respectively.

[0082] Comparative Example 2: The specific implementation method is the same as in Example 3, except that, based on strain RB06, the diacylglycerol O-acyltransferase 1 gene is integrated and expressed at the E3 site. DGA1 (Gene ID: 2910950) and diacylglycerol O-acyltransferase gene 2 DGA2(Gene ID: 2911905). The constructed strain RB07 was fermented according to the method in Example 3, and the results showed that the yield was 90.6 mg / L.

[0083] 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. Mutant of the lanosterol 14α-demethylase characterized in that, The amino acid mutation at position 305 and / or 372 relative to the lanosterol 14a-demethylase YlCYP51 parent.

2. A fusion enzyme characterized in that, At least two enzymes of lactic acid dehydrogenase LDH1, sterol acyltransferase related enzyme ARE1, C-24(28) sterol reductase ERG4 connected by a connecting peptide, the connecting peptide includes but is not limited to flexible connecting peptide (GGGGS) 3, alpha helix connecting peptide (EAAAK) 3 or alpha helix connecting peptide (PT) 5P.

3. A gene encoding the mutant of claim 1 or the fusion enzyme of claim 2.

4. A recombinant microorganism expressing the mutant of claim 1 or the fusion enzyme of claim 2.

5. An engineered Yarrowia lipolytica strain with high efficiency of synthesizing ergosterol, characterized in that, On the basis of the starting strain, methylglutaryl coenzyme A reductase tHMG1 and isopentenyl diphosphate isomerase IDI1 , 3-ketosteroid reductase ERG27 , and lanosterol 14α-demethylase ERG11 or a mutant thereof.

6. The engineered Yarrowia lipolytica yeast strain of claim 5, wherein, C-24(28) sterol reductase ERG4 and C-22 sterol desaturase ERG5 .

7. The engineered Yarrowia lipolytica yeast strain of claim 5, wherein, The fusion enzyme of lactic acid dehydrogenase LDH1, sterol acyltransferase related enzyme ARE1 and C-24(28) sterol reductase ERG4 connected by a flexible connecting peptide is expressed.

8. The engineered Yarrowia lipolytica yeast strain of claim 6 or 7, wherein, The starting strain is Yarrowia lipolytica Po1f.

9. A method of preparing ergosterol, characterized by, The Yarrowia lipolytica engineering strain of claims 5-8 is fermented in a culture medium at 28-30°C for at least 72 h.

10. Use of the Yarrowia lipolytica engineering strain of any one of claims 5-8 or the method of claim 9 in the preparation of ergosterol or ergosterol-containing products.

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