A recombinant strain for producing squalene, a construction method thereof and applications thereof

Abstract

The present invention discloses a recombinant strain for producing squalene, a construction method thereof and an application thereof. The construction method includes: heterologously expressing the cDNA of each gene of the squalene synthesis pathway derived from filamentous fungi in yeast. The genes of the squalene synthesis pathway include: mevalonate-5-phosphate kinase gene, squalene synthase gene, acetoacetyl-CoA thiolase gene, mevalonate kinase gene, hydroxymethylglutaryl-CoA synthase gene, mevalonate pyrophosphate decarboxylase gene, farnesyl pyrophosphate synthase gene, isopentenyl pyrophosphate:dimethylallyl pyrophosphate isomerase gene, and truncated hydroxymethylglutaryl-CoA reductase gene; the filamentous fungus is Sarocladium oryzae or Stibella sp.1056; the squalene synthesis pathway excavated by the present invention can complete the efficient de novo synthesis of squalene and improve the squalene yield.

Description

Technical Field

[0001] The present invention relates to the technical field of biosynthesis applications, and particularly relates to a recombinant strain for producing squalene, a construction method thereof, and an application thereof. Background Art

[0002] Squalene (2,6,10,15,19,23 - hexamethyl - 2,6,10,14,18,22 - tetracosahexaene, C 30 H 50 ) is a kind of all - trans triterpenoid compound. It is not only a precursor of many active sterols or triterpenoid compounds such as lanosterol, ergosterol, and aspergillic acid, but also widely used in the fields of food, medicine, cosmetics, etc. due to its functional characteristics in regulating immunity, promoting cardiovascular health, anti - cancer, antioxidant, antibacterial, etc., such as health products, vaccine adjuvants, emollient creams, etc.

[0003] Since squalene was first discovered in shark liver in 1916, shark liver has become the main source of squalene, but this production method has been strictly prohibited by the Convention on International Trade in Endangered Species of Wild Fauna and Flora. With the analysis of the squalene synthesis pathway, people have tried to use microbial cell factories to efficiently produce squalene in order to reduce the pressure on the environment and be conducive to sustainable development.

[0004] In related technologies, as disclosed in document [1], the following content is available: An exogenous gene highly controllable expression system carried endogenously was used to reconstruct the squalene biosynthesis pathway in the peroxisome of Saccharomyces cerevisiae, heterologously expressing the Enterococcus faecalis from EfmvaE and EfmvaS , overexpressing the endogenous genes ERG8 , ERG9 , ERG12 , ERG19 , ERG20 and IDI1 of Saccharomyces cerevisiae, and the squalene yield was increased by 900 times, reaching 1090.41 ± 80.92 mg / L.

[0005] Therefore, further developing recombinant strains with high squalene production has great application value. Summary of the Invention

[0006] The purpose of the present invention is to provide a recombinant strain for producing squalene, a construction method thereof, and an application thereof to improve the squalene yield.

[0007] The technical solution adopted by the present invention is as follows:

[0008] In the first aspect,

[0009] The present invention provides a construction method of a recombinant strain for producing squalene, and the construction method includes:

[0010] Heterologously express the cDNA of each gene of the squalene synthesis pathway derived from filamentous fungi in yeast. The genes of the squalene synthesis pathway include: mevalonate-5-phosphate kinase gene, squalene synthase gene, acetoacetyl-CoA thiolase gene, mevalonate kinase gene, hydroxymethylglutaryl-CoA synthase gene, mevalonate pyrophosphate decarboxylase gene, farnesyl pyrophosphate synthase gene, isopentenyl pyrophosphate: dimethylallyl pyrophosphate isomerase gene, and truncated hydroxymethylglutaryl-CoA reductase gene;

[0011] The filamentous fungus is Sarocladium oryzae or Stibella sp. 1056;

[0012] The Sarocladium oryzae Genbank accession number of the genome is PRJNA305454; The mevalonate-5-phosphate kinase gene, squalene synthase gene, acetoacetyl-CoA thiolase gene, mevalonate kinase gene, hydroxymethylglutaryl-CoA synthase gene, mevalonate pyrophosphate decarboxylase gene, farnesyl pyrophosphate synthase gene, isopentenyl pyrophosphate: dimethylallyl pyrophosphate isomerase gene, and truncated hydroxymethylglutaryl-CoA reductase gene derived from Sarocladium oryzae are sequentially denoted as SOERG8 gene 、SOERG9 gene 、SOERG10 gene 、SOERG12 gene 、 SOERG13 gene 、SOERG19 gene 、SOERG20 gene 、SOIDI1 gene 、SOtHMGR1 gene, and the DNA sequences are sequentially as shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.10;

[0013] The Stibella Deposit number of sp. 1056 is CGMCC No.40422; The mevalonate-5-phosphate kinase gene, squalene synthase gene, acetoacetyl-CoA thiolase gene, mevalonate kinase gene, hydroxymethylglutaryl-CoA synthase gene, mevalonate pyrophosphate decarboxylase gene, farnesyl pyrophosphate synthase gene, isopentenyl pyrophosphate: dimethylallyl pyrophosphate isomerase gene, and truncated hydroxymethylglutaryl-CoA reductase gene derived from Stibella sp.1056 are sequentially denoted as 1056ERG8 gene 、1056ERG9 gene 、1056ERG10Gene 、1056ERG12 Gene 、1056ERG13 Gene 、1056ERG19 Gene 、1056ERG20 Gene 、1056IDI1 Gene 、1056tHMGR1 Genes, the DNA sequences are successively as shown in SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO.16, SEQ ID NO.17, SEQ ID NO.18, SEQ ID NO.20.

[0014] In some embodiments, the yeast is Saccharomyces cerevisiae. Further, in some embodiments, the Saccharomyces cerevisiae is auxotrophic Saccharomyces cerevisiae CEN.PK2-1D.

[0015] In some embodiments, SOERG8 Gene 、SOERG9 Gene 、SOERG10 Gene 、SOERG12 Gene 、 SOERG13 Gene 、SOERG19 Gene 、SOERG20 Gene 、SOIDI1 Genes, SOtHMGR1 The endogenous target genes of the genes are successively CYS3 Genes, RPL38 Genes, TDH2 Genes, RPS10A Genes, TIF1 Genes, RPS25A Genes, RPS0A Genes, PFY1 Genes, TDH3 Genes; and are linked with IGG the element 5'-CAATCAAAC-3'. IGG The element is denoted as IGG6。

[0016] In some embodiments, 1056ERG8 Gene 、1056ERG9 Gene 、1056ERG10 Gene 、1056ERG12 Gene 、1056ERG13 Gene 、1056ERG19 Gene 、1056ERG20 Gene 、1056IDI1 Genes, 1056tHMGR1 The endogenous target genes of the genes are successively CYS3 Genes, RPL38 Genes, TDH2 Genes, RPS10A Genes, TIF1 Genes, RPS25A Genes, RPS0A Genes,PFY1 gene TDH3 gene; and ligated with the IGG element 5'-CAATCAAAC-3'. IGG The element is denoted as IGG6。

[0017] In some embodiments, each of the mevalonate-5-phosphate kinase gene, squalene synthase gene, acetoacetyl-CoA thiolase gene, mevalonate kinase gene, hydroxymethylglutaryl-CoA synthase gene, mevalonate pyrophosphate decarboxylase gene, farnesyl pyrophosphate synthase gene, isopentenyl pyrophosphate:dimethylallyl pyrophosphate isomerase gene, and truncated hydroxymethylglutaryl-CoA reductase gene is fused with the DNA sequence encoding the peroxisomal targeting signal MDH3. (MDH3 is linked to the carboxyl terminus of the protein such that the 9 enzymes are expressed in the peroxisome.)

[0018] In a more specific embodiment, the peroxisomal targeting signal is SKL.

[0019] In a second aspect,

[0020] the present invention also provides a recombinant strain for producing squalene, which is constructed by the above construction method.

[0021] In a third aspect,

[0022] the present invention also provides the application of the above recombinant strain in the production of squalene.

[0023] In a fourth aspect,

[0024] the present invention also provides a method for producing squalene, comprising fermenting the above recombinant strain and obtaining squalene from the fermentation broth.

[0025] In some embodiments, the fermentation medium is YPD medium, and the initial cell OD 600 value is 0.08 - 0.12; the fermentation temperature is 28 - 32 °C, and the cells are cultured at 150 - 250 rpm for 7 - 10 days. Preferably, the initial cell OD 600 value is 0.1, the fermentation temperature is 30 °C, and the cells are cultured at 200 rpm for 7 days.

[0026] In some embodiments, the components of the YPD medium include: 2% yeast extract, 2% peptone, 2% glucose.

[0027] In some embodiments, the seed medium of the recombinant strain is SC-Ura defective medium.

[0028] In some embodiments, the components of the SC-Ura defective medium include: 2% glucose, 6.7% yeast nitrogen base without amino acids, and 0.78 g / L uracil-deficient amino acid mixture.

[0029] The present invention has the following advantages and beneficial effects:

[0030] The squalene synthesis pathway discovered by the present invention can complete the efficient de novo synthesis of squalene, improve the squalene yield, and has great application value for developing recombinant strains with high squalene production.

[0031] The present invention uses an endogenous exogenous gene highly controllable expression system to Stibella sp. 1056 or Sarocladium oryzae The squalene synthesis pathways in (a total of 9 enzymes respectively: mevalonate-5-phosphate kinase 1056ERG8 / SOERG8; squalene synthase 1056ERG9 / SOERG9; acetoacetyl-CoA thiolase 1056ERG10 / SOERG10; mevalonate kinase 1056ERG12 / SOERG12; hydroxymethylglutaryl-CoA synthase 1056ERG13 / SOERG13; mevalonate pyrophosphate decarboxylase 1056ERG19 / SOERG19; farnesyl pyrophosphate synthase 1056ERG20 / SOERG20; isopentenyl pyrophosphate: dimethylallyl pyrophosphate isomerase 1056IDI1 / SOIDI1; truncated hydroxymethylglutaryl-CoA reductase 1056tHMGR1 / SOtHMGR1) were heterologously expressed respectively. Sarocladium oryzae After transferring the squalene synthesis pathway in into Saccharomyces cerevisiae, the total squalene production was 1241.37 ± 88.02 mg / L, and the squalene yield per unit dry weight of cells reached 301.02 ± 43.49 mg / g cdw; Stibella After transferring the squalene synthesis pathway in sp. 1056 into Saccharomyces cerevisiae, the total squalene production was 986.31 ± 35.77 mg / L, and the squalene yield per unit dry weight of cells reached 318.31 ± 66.14 mg / g cdw. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 It is the HPLC diagram of squalene production by different squalene engineering strains;

[0033] Among them, HCS1 is recorded in the literature [1], that is, a squalene biosynthesis pathway was reconstructed in the peroxisome of Saccharomyces cerevisiae by applying an endogenous exogenous gene highly controllable expression system, and the heterologous expression was derived from Enterococcus faecalis of EfmvaE and EfmvaS, overexpress the endogenous genes of Saccharomyces cerevisiae ERG8 , ERG9 , ERG12 , ERG19 , ERG20 and IDI1 . Detailed implementation manners

[0034] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be described clearly and completely below. Obviously, the described embodiments are some but not all of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the described embodiments of the present invention without creative efforts fall within the protection scope of the present invention.

[0035] Unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Moreover, the laboratory operation steps of molecular genetics, nucleic acid chemistry, and immunology used herein are all conventional steps widely used in the corresponding fields or are carried out according to the conditions recommended by the manufacturers.

[0036] The following are the term descriptions in this article:

[0037] In this article, regarding "the first aspect", "the second aspect", "the third aspect", etc., the terms "first", "second", "third", etc. are only used for descriptive purposes, and cannot be understood as indicating or implying relative importance or quantity, nor can they be understood as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first", "second", "third", "fourth", etc. only serve the purpose of non-exhaustive list description, and it should be understood that they do not constitute a closed limitation on quantity.

[0038] In this article, regarding "preferred", it is only for describing the implementation manners or embodiments with better effects. It should be understood that it does not constitute a limitation on the protection scope of the present invention.

[0039] The documents cited in the present invention are:

[0040] Reference [1]: Yue, Q., J. Meng, Y. Qiu, M. Yin, L. Zhang, W. Zhou, Z. An, Z. Liu, Q. Yuan, W. Sun, C. Li, H. Zhao, I. Molnar, Y. Xu and S. Shi (2023). A polycistronic system for multiplexed and precalibrated expression of multigene pathways in fungi. Nat Commun 14(1): 4267

[0041] Reference [2]: Xu Yuquan, Yue Qun, Zhang Liwen, Song Kainan, Ai Yutong, A filamentous fungus producing high yields of terpene and polypeptide metabolites, ZL202310601793.1, Authorization announcement date: August 15, 2023

[0042] Reference [3]: Hittalmani, S., Mahesh, H.B., Mahadevaiah, C. and Prasannakumar, M.K. (2016). De novo genome assembly and annotation of rice sheath rot fungus Sarocladium oryzae reveals genes involved in helvolic acid and cerulenin biosynthesis pathways. BMC Genomics 17: 271

[0043] Examples of the present invention Stibella sp. 1056, this strain was preserved by the China General Microbiological Culture Collection Center (CGMCC) on November 11, 2022 (the preservation address is: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing), and the preservation number is CGMCC No. 40422. After detection, it survived. And it is recorded in Reference [2]. It is derived from the Institute of Biotechnology, Chinese Academy of Agricultural Sciences.

[0044] Examples of the present invention Sarocladium oryzae , this strain is the fungus reported in Reference [3], and the Genbank accession number of its genome is PRJNA305454.

[0045] Squalene is an important precursor of triterpenoids, and filamentous fungi are an important source of triterpenoids. The filamentous fungus isolated by the inventor from the soilStibella The yield of fumigaclavine C by Aspergillus fumigatus sp. 1056 under rice medium conditions can reach 4.52 g / kg, and its yield of fumigaclavine C under YES medium conditions can reach 610 mg / L (Reference [2]); Sarocladium oryzae The produced fumigaclavine C is the main virulence factor for its causing rice sheath blight (Reference [3]). Based on this, the inventors speculated that squalene, as a precursor for the biosynthesis of fumigaclavine C, has an efficient Stibella sp.1056 、Sarocladium oryzae biosynthesis pathway. In the examples of the present invention, the pathway was introduced into Saccharomyces cerevisiae to increase the squalene yield and develop a recombinant strain with a higher squalene yield.

[0046] The present invention provides two recombinant strains for producing squalene. Among them,

[0047] the genotype of one recombinant strain is CEN.PK2-1D RPS10A::IGG6 - SOERG12 ; CYS3::IGG6 - SOERG8 ; RPS25A::IGG6 - SOERG19 ; PFY1::IGG6 - SOIDI1 ; RPS0A::IGG6 - SOERG20 ; RPL38:: IGG6 - SOERG9 ; TDH2::IGG6 - SOERG10 ; TIF1::IGG6 - SOERG13 ; TDH3::IGG6 - SOtHMGR1 .

[0048] the genotype of the other recombinant strain is CEN.PK2-1D RPS10A::IGG6 - 1056ERG12 ; CYS3::IGG6 - 1056ERG8 ; RPS25A::IGG6 - 1056ERG19 ; PFY1::IGG6-1056IDI1 ; RPS0A::IGG6-1056ERG20 ; RPL38::IGG6-1056ERG9 ; TDH2::IGG6-1056ERG10 ; TIF1::IGG6-1056ERG13 ; TDH3::IGG6- 1056tHMGR1 .

[0049] In the following examples, the experimental methods are all conventional methods unless otherwise specified. The test materials used in the following examples are all obtained from regular biochemical reagent stores unless otherwise specified. In the following examples, quantitative tests are all set with three repeated experiments, and the results are averaged.

[0050] Vector pJET1.2 / blunt Cloning Vector: purchased from Thermo Fisher Scientific;

[0051] Plasmids pCAS, pScURA3 and psgtRNA are shown in Reference [1].

[0052] Saccharomyces cerevisiae CEN.PK2-1D: MATα; ura3-52; trp1-289; leu2-3,112; his3Δ1; MAL2-8C; SUC2。

[0053] Enzymes and kits:

[0054] The high-fidelity DNA amplification MIX and seamless cloning kit were purchased from Novoprotein;

[0055] DNA loading buffer and DNA marker were purchased from CW Biotech;

[0056] The DNA purification gel extraction kit was purchased from Thermo Fisher;

[0057] The RNA extraction reagent Trizol and the PureLink TM RNA Mini Kit were purchased from Thermo Fisher;

[0058] The reverse transcription kit PrimeScript reagent kit was purchased from TaKaRa;

[0059] NEBridge ® The Golden Gate assembly kit (BsaI-HF ® v2) was purchased from NEB.

[0060] The competent cells of Escherichia coli DH5α were purchased from CW Biotech.

[0061] The solvent n-hexane used in the experiment was produced by Sinopharm Chemical Reagent Co., Ltd. Beijing. The chromatographically pure acetonitrile and methanol were produced by Fisher Chemical.

[0062] Media:

[0063] SC-Ura dropout medium (2% glucose, 6.7% yeast nitrogen base without amino acids, 0.78 g / L uracil dropout amino acid mixture);

[0064] YPD medium (2% yeast extract, 2% Peptone, 2% glucose);

[0065] YES medium (2% yeast extract, 0.05% magnesium sulfate heptahydrate, 15% sucrose, 0.001% zinc sulfate heptahydrate, 0.0005% copper sulfate pentahydrate).

[0066] If preparing solid medium, add 2% agar powder.

[0067] In the examples of the present invention, SOHMGR1The DNA sequence of the gene is shown in SEQ ID NO.9; 1056HMGR1 The DNA sequence of the gene is shown in SEQ ID NO.19.

[0068] Example 1

[0069] Cloning of the cDNA Sequences of Genes in the Squalene Biosynthesis Pathway

[0070] 1.1. Experimental Purpose

[0071] Under the condition that Stibella sp. 1056 produces fumigaclavine, its total RNA was extracted. After reverse transcription, nine enzymes in its squalene biosynthesis pathway were amplified and cloned respectively using gene-specific primers to obtain 1056ERG12 , 1056ERG8 , 1056ERG19 , 1056IDI1 , 1056ERG20 , 1056ERG9 , 1056ERG10 , 1056ERG13 and 1056HMGR1 transcripts.

[0072] Through bioinformatics analysis, the Sarocladium oryzae transcripts of SOERG12 , SOERG8 , SOERG19 , SOIDI1 , SOERG20 , SOERG9 , SOERG10 , SOERG13 , SOHMGR1 in the

[0073] genome (Genbank accession number: PRJNA305454) were predicted and gene synthesis was carried out.

[0074] 1.2. Experimental Method Stibella Squalene Biosynthesis Pathway of

[0075] (1) Stibella Obtaining Total cDNA of

[0076] The mycelia of Stibella sp. 1056 cultured in YES medium for 5 days were collected by filtration. After extracting total RNA according to the instructions of the RNA extraction kit, reverse transcription was carried out according to the instructions of the reverse transcription kit to obtain total cDNA.

[0077] (2) Stibella Cloning of the cDNA of Genes in the Squalene Biosynthesis Pathway of

[0078] Using the total StibellaUsing sp. 1056 cDNA as a template, according to 1056ERG12 , 1056ERG8 , 1056ERG19 , 1056IDI1 , 1056ERG20 , 1056ERG9 , 1056ERG10 , 1056ERG13 and 1056HMGR1 gene sequences, primers were designed, and the total cDNA obtained was used as a template for PCR amplification to obtain cDNA from the start codon of the gene to the base before the stop codon, and then they were respectively ligated to the pJET1.2 vector. After transformation, positive clones were picked for sequencing.

[0079] In the above method, the gene 1056ERG8 was amplified using primer 1056ERG8-F (shown in SEQ ID NO.21) and primer 1056ERG8-R (shown in SEQ ID NO.22), the gene 1056IDI1 was amplified using primer 1056IDI1-F (shown in SEQ ID NO.23) and primer 1056IDI1-R (shown in SEQ ID NO.24), the gene 1056ERG9 was amplified using primer 1056ERG9-F (shown in SEQ ID NO.25) and primer 1056ERG9-R (shown in SEQ ID NO.26), the gene 1056ERG20 was amplified using primer 1056ERG20-F (shown in SEQ ID NO.28) and primer 1056ERG20-R (shown in SEQ ID NO.27), the gene 1056ERG12 was amplified using primer 1056ERG12-F (shown in SEQ ID NO.29) and primer 1056ERG12-R (shown in SEQ ID NO.30), the gene 1056ERG19 was amplified using primer 1056ERG10-F (shown in SEQ ID NO.33) and primer 1056 ERG10-R (shown in SEQ ID NO.34), the gene 1056ERG10 was amplified using primer 1056ERG13-F (shown in SEQ ID NO.35) and primer 1056ERG13-R (shown in SEQ ID NO.36), the gene 1056ERG13 was amplified using primer 1056HMGR1-F (shown in SEQ ID NO.37) and primer 1056HMGR1-R (shown in SEQ ID NO.38), and the gene 1056HMGR1 .

[0080] 1.2.2 Sarocladium oryzae Squalene synthesis pathway

[0081] Sarocladium oryzae Prediction and synthesis of cDNA of squalene synthesis pathway genes

[0082] Using the auxotrophic Saccharomyces cerevisiae CEN.PK2-1D ERG12 、 ERG8 、 ERG19 、 IDI1 、 ERG20 、 ERG9 、 ERG10 、 ERG13 、 HMGR1 gene pairs Sarocladium oryzae to perform a tblastn alignment on the genome (Genbank accession number PRJNA305454) to predict SOERG12 、 SOERG8 、 SOERG19 、 SOIDI1 、 SOERG20 、 SOERG9 、 SOERG10 、 SOERG13 and SOHMGR1 sequences. The open reading frames were predicted and gene synthesized by FGENESH to obtain positive clones containing cDNA fragments of these genes.

[0083] 1.3. Experimental results and analysis

[0084] After culturing sp. 1056 in YES medium Stibella it was found that 9 enzymes in the squalene synthesis pathway were expressed. The correct cDNA of these 9 genes was obtained by sequencing.

[0085] The cDNA of squalene synthesis pathway genes was obtained through bioinformatics analysis prediction and gene synthesis Sarocladium oryzae

[0086] Example 2

[0087] Reconstruction of squalene synthesis pathway

[0088] 2.1. Experimental purpose

[0089] Using the auxotrophic Saccharomyces cerevisiae CEN.PK2-1D as the chassis cell, selecting RPS10A 、 CYS3 、 RPS25A 、 PFY1 、 RPS0A 、 RPL38 、 TDH2 、 TIF1 、 TDH3 respectively as Sarocladium oryzae ​Squalene synthesis pathway: SOERG12 , SOERG8 , SOERG19 , SOIDI1 , SOERG20 , SOERG9 , SOERG10 , SOERG13 , SOHMGR1 (or SOtHMGR1 ); or Stibella Squalene synthesis pathway of sp. 1056: 1056ERG12 , 1056ERG8 , 1056ERG19 , 1056IDI1 , 1056ERG20 , 1056ERG9 , 1056ERG10 , 1056ERG13 , 1056HMGR1 (or 1056tHMGR1 ), the endogenous target genes, and using the corresponding endogenous genes of Saccharomyces cerevisiae as controls, connecting with the IGG6 element 5’-CAATCAAAC-3’, and adding a peroxisome targeting signal MDH3 to each target gene, transferring the target genes into yeast by CRISPR gene editing technology, and detecting the squalene produced in the positive transformants by HPLC.

[0090] 2.2. Experimental methods:

[0091] (1) Obtaining of plasmids and donor DNA for CRISPR gene editing

[0092] According to the sequences of endogenous target genes, target genes, IGG elements ( IGG6 , 5’-CAATCAAAC-3’), MDH3 , primers were designed. A homologous fragment of the target gene and the IGG6 sequence were added to the 5’ end of the forward primer, and a homologous fragment of the target gene and the MDH3 sequence were added to the 5’ end of the reverse primer. Using the cDNA positive clone of the squalene synthesis pathway gene as a template, PCR amplification of the donor DNA was carried out.

[0093] In the above method:

[0094] Reconstruct Sarocladium oryzae Related primers for the squalene synthesis pathway: The donor DNA was amplified using the primer SO-TDH2-ERG10-F (shown in SEQ ID NO.39) and the primer SO-TDH2-ERG10-R (shown in SEQ ID NO.40). TDH2- IGG6-SOERG10, the donor DNA was amplified using primer SO-RPS10A-ERG12-F (shown in SEQ ID NO.41) and primer SO-RPS10A-ERG12-R (shown in SEQ ID NO.42) RPS10A-IGG6-SOERG12 , the donor DNA was amplified using primer SO-TIF1-ERG13-F (shown in SEQ ID NO.43) and primer SO-TIF1-ERG13-R (shown in SEQ ID NO.44) TIF1-IGG6-SOERG13 , the donor DNA was amplified using primer SO-RPS0A-ERG20-F (shown in SEQ ID NO.45) and primer SO-RPS0A-ERG20-R (shown in SEQ ID NO.46) RPS0A-IGG6-SOERG20 , the donor DNA was amplified using primer SO-CYS3-ERG8-F (shown in SEQ ID NO.47) and primer SO-CYS3-ERG8-R (shown in SEQ ID NO.48) CYS3-IGG6-SOERG8 , the donor DNA was amplified using primer SO-RPS25A-ERG19-F (shown in SEQ ID NO.49) and primer SO-RPS25A-ERG19-R (shown in SEQ ID NO.50) RPS25A-IGG6-SOERG19 , the donor DNA was amplified using primer SO-PFY1-IDI1-F (shown in SEQ ID NO.51) and primer SO-PFY1-IDI1-R (shown in SEQ ID NO.52) PFY1-IGG6-SOIDI1 , the donor DNA was amplified using primer SO-RPL38-ERG9-F (shown in SEQ ID NO.53) and primer SO-RPL38-ERG9-R (shown in SEQ ID NO.54) RPL38-IGG6-SOERG9 , the donor DNA was amplified using primer SO-TDH3-tHMGR1-F (shown in SEQ ID NO.55) and primer SO-TDH3-tHMGR1-R (shown in SEQ ID NO.56) TDH3-IGG6-SOtHMGR1 , the donor DNA was amplified using primer SO-TDH3-HMGR1-F (shown in SEQ ID NO.57) and primer SO-TDH3-HMGR1-R (shown in SEQ ID NO.58) TDH3-IGG6-SOHMGR1 .

[0095] Reconstruction Stibella Related primers for the squalene synthesis pathway of sp. 1056: The donor DNA was amplified using primer 1056-CYS3-ERG8-F (shown in SEQ ID NO.59) and primer 1056-CYS3-ERG8-R (shown in SEQ ID NO.60) CYS3-IGG6-1056ERG8, the donor DNA was amplified using primer 1056 - PFY1 - IDI1 - F (shown in SEQ ID NO.61) and primer 1056 - PFY1 - IDI1 - R (shown in SEQ ID NO.62) PFY1-IGG6-1056IDI1 , the donor DNA was amplified using primer 1056 - RPL38 - ERG9 - F (shown in SEQ ID NO.63) and primer 1056 - RPL38 - ERG9 - R (shown in SEQ ID NO.64) RPL38-IGG6-1056ERG9 , the donor DNA was amplified using primer 1056 - RPS0A - ERG20 - F (shown in SEQ ID NO.66) and primer 1056 - RPS0A - ERG20 - R (shown in SEQ ID NO.65) RPS0A-IGG6-1056ERG20 , the donor DNA was amplified using primer 1056 - RPS10A - ERG12 - F (shown in SEQ ID NO.67) and primer 1056 - RPS10A - ERG12 - R (shown in SEQ ID NO.68) RPS10A-IGG6-1056ERG12 , the donor DNA was amplified using primer 1056 - RPS25A - ERG19 - F (shown in SEQ IDNO.69) and primer 1056 - RPS25A - ERG19 - R (shown in SEQ ID NO.70) RPS25A- IGG6-1056ERG19 , the donor DNA was amplified using primer 1056 - TDH2 - ERG10 - F (shown in SEQ ID NO.71) and primer 1056 - TDH2 - ERG10 - R (shown in SEQ ID NO.72) TDH2-IGG6-1056ERG10 , the donor DNA was amplified using primer 1056 - TIF1 - ERG13 - F (shown in SEQ ID NO.73) and primer 1056 - TIF1 - ERG13 - R (shown in SEQ ID NO.74) TIF1-IGG6-1056ERG13 , the donor DNA was amplified using primer 1056 - TDH3 - HMGR1 - F (shown in SEQ ID NO.75) and primer 1056 - TDH3 - HMGR1 - R (shown in SEQ ID NO.76) TDH3-IGG6-1056HMGR1 , the donor DNA was amplified using primer 1056 - TDH3 - tHMGR1 - F (shown in SEQ ID NO.77) and primer 1056 - TDH3 - HMGR1 - R (shown in SEQ ID NO.76) TDH3-IGG6-1056tHMGR1 。

[0096] Primers related to reconstructing the endogenous squalene synthesis pathway in Saccharomyces cerevisiae: The donor DNA was amplified using primer CEN-TDH2-ERG10-F (shown in SEQ ID NO.78) and primer CEN-TDH2-ERG10-R (shown in SEQ ID NO.79). TDH2-IGG6- CENERG10 , The donor DNA was amplified using primer CEN-RPS10A-ERG12-F (shown in SEQ ID NO.80) and primer CEN-RPS10A-ERG12-R (shown in SEQ ID NO.81). RPS10A-IGG6-CENERG12 , The donor DNA was amplified using primer CEN-TIF1-ERG13-F (shown in SEQ ID NO.82) and primer CEN-TIF1-ERG13-R (shown in SEQ ID NO.83). TIF1- IGG6-CENERG13 , The donor DNA was amplified using primer CEN-RPS0A-ERG20-F (shown in SEQ ID NO.84) and primer CEN-RPS0A-ERG20-R (shown in SEQ ID NO.85). RPS0A-IGG6-CENERG20 , The donor DNA was amplified using primer CEN-CYS3-ERG8-F (shown in SEQ ID NO.86) and primer CEN-CYS3-ERG8-R (shown in SEQ ID NO.87). CYS3-IGG6-CENERG8 , The donor DNA was amplified using primer CEN-RPS25A-ERG19-F (shown in SEQ ID NO.88) and primer CEN-RPS25A-ERG19-R (shown in SEQ ID NO.89). RPS25A-IGG6-CENERG19 , The donor DNA was amplified using primer CEN-PFY1-IDI1-F (shown in SEQ ID NO.90) and primer CEN-PFY1-IDI1-R (shown in SEQ ID NO.91). PFY1-IGG6-CENIDI1 , The donor DNA was amplified using primer CEN-RPL38-ERG9-F (shown in SEQ ID NO.92) and primer CEN-RPL38-ERG9-R (shown in SEQ ID NO.93). RPL38-IGG6-CENERG9 , The donor DNA was amplified using primer CEN-TDH3-tHMGR1-F (shown in SEQ ID NO.94) and primer CEN-TDH3-tHMGR1-R (shown in SEQ ID NO.95). TDH3-IGG6-CENtHMGR1 .

[0097] According to those used in reference [1] RPS10A , CYS3 , RPS25A , PFY1 ,RPS0A , RPL38 , TDH2 , TIF1 , TDH3 gRNA design primers for TIF1 , using pScURA3 and psgtRNA as templates, construct plasmid pCAS-sgRNA according to the method described in the text. TDH2+ TIF1 pCAS-sgRNA RPS10A + CYS3 + RPS25A pCAS-sgRNA PFY1 + RPS0A + RPL38 pCAS-sgRNA TDH3 .

[0098] In the above method, use primer GRNA-TDH2-BSAI-F (shown in SEQ ID NO.96) and primer GRNA-TIF1-BSAI-R4 (shown in SEQ ID NO.97) to amplify a DNA fragment using psgtRNA as a template. TDH2-TIF1 , use primer GRNA-TIF1-BSAI-F4 (shown in SEQ ID NO.98) and primer SGT4R.URA3R (shown in SEQ ID NO.99) to amplify a DNA fragment using pScURA3 as a template. TIF1-URA3 , use the Golden Gate assembly kit to ligate these two DNA fragments to plasmid pCAS to obtain pCAS-sgRNA. TDH2+TIF1 . Use primer GRNA-RPS0A-BSAI-F (shown in SEQ ID NO.100) and primer GRNA-RPL38-BSAI-R4 (shown in SEQ ID NO.101) to amplify a DNA fragment using psgtRNA as a template. RPS0A-RPL38 , use primer GRNA-RPL38-BSAI-F4 (shown in SEQ ID NO.102) and primer GRNA-PFY1-BSAI-R4 (shown in SEQ ID NO.103) to amplify a DNA fragment using psgtRNA as a template. RPL38-PFY1 , use primer GRNA-PFY1-BSAI-F4 (shown in SEQ ID NO.104) and primer SGT4R.URA3R (shown in SEQ ID NO.99) to amplify a DNA fragment using pScURA3 as a template. PFY1-URA3 , use the Golden Gate assembly kit to ligate these 3 DNA fragments to plasmid pCAS to obtain pCAS-sgRNA. PFY1 + RPS0A + RPL38Using the primer GRNA-RPS10A-BSAI-F1 (shown in SEQ ID NO.105) and the primer GRNA-RPS25A-BSAI-R (shown in SEQ ID NO.106), a DNA fragment was amplified using psgtRNA as a template RPS10A-RPS25A , using the primer GRNA-RPS25A-BSAI-F (shown in SEQ ID NO.107) and the primer GRNA-CYS3-BSAI-R (shown in SEQ ID NO.108), a DNA fragment was amplified using psgtRNA as a template RPS25A-CYS3 , using the primer GRNA-CYS3-BSAI-F (shown in SEQ ID NO.109) and the primer SGT4R.URA3R (shown in SEQ ID NO.99), a DNA fragment was amplified using pScURA3 as a template CYS3-URA3 , using the Golden Gate assembly kit, these 3 DNA fragments were ligated to the plasmid pCAS to obtain pCAS-sgRNA RPS10A + CYS3 + RPS25A . Using the primer GRNA-TDH3-BSAI-F1 (shown in SEQ ID NO.110) and the primer SGT4R.URA3R (shown in SEQ ID NO.99), a DNA fragment was amplified using pScURA3 as a template TDH3-URA3 , using the Golden Gate assembly kit, this DNA fragment was ligated to the plasmid pCAS to obtain pCAS-sgRNA TDH3 .

[0099] (2) Yeast transformation

[0100] Yeast electroporation and plasmid curing were carried out according to the method in reference [1]. Taking the transfer of Sarocladium oryzae the squalene synthesis pathway as an example, first, pCAS-sgRNA TDH2+TIF1 and its corresponding donor DNA TDH2-IGG6-SOERG10 and TIF1-IGG6-SOERG13 were transferred into CEN.PK2-1D, and positive transformants S1 were obtained by colony PCR screening respectively; after plasmid elimination, using it as a host, pCAS-sgRNA RPS10A + CYS3 + RPS25A and its corresponding donor DNA RPS10A-IGG6-SOERG12 , CYS3-IGG6-SOERG8 and RPS25A-IGG6-SOERG19 were transferred respectively, and positive transformants S2 were obtained by colony PCR screening respectively; after plasmid elimination, using it as a host, pCAS-sgRNA PFY1 + RPS0A + RPL38and its corresponding donor DNA PFY1-IGG6-SOIDI1 、 RPS0A-IGG6-SOERG20 and RPL38-IGG6-SOERG9 , positive transformant S3 was obtained by colony PCR screening respectively; after eliminating the plasmid, using S3 as the host, pCAS-sgRNA TDH3 and its corresponding donor DNA TDH3-IGG6-SOHMGR1 (or TDH3-IGG6-SOtHMGR1 ), positive transformants HPSS1 (containing SOHMGR1 ), HPSS2 (containing SOtHMGR1 ) were obtained by colony PCR screening.

[0101] Primers used to identify positive transformants: primer TDH3-F (shown in SEQ ID NO.111) and primer TDH3-R (shown in SEQ ID NO.112) were used to detect whether SOHMGR1 (or SOtHMGR1 ) was correctly integrated, primer TDH2-F (shown in SEQ IDNO.113) and primer TDH2-R (shown in SEQ ID NO.114) were used to detect whether SOERG10 was correctly integrated, primer TIF1-F (shown in SEQ ID NO.115) and primer TIF1-R (shown in SEQ ID NO.116) were used to detect whether SOERG13 was correctly integrated, primer RPS10A-F (shown in SEQ ID NO.117) and primer RPS10A-R (shown in SEQ ID NO.118) were used to detect whether SOERG12 was correctly integrated, primer CYS3-F (shown in SEQ ID NO.119) and primer CYS3-R (shown in SEQ ID NO.120) were used to detect whether SOERG8 was correctly integrated, primer RPS25A-F (shown in SEQ ID NO.121) and primer RPS25A-R (shown in SEQ ID NO.122) were used to detect whether SOERG19 was correctly integrated, primer RPS0A-F (shown in SEQ ID NO.123) and primer RPS0A-R (shown in SEQ ID NO.124) were used to detect whether SOERG20 was correctly integrated, primer RPL38-F (shown in SEQID NO.125) and primer RPL38-R (shown in SEQ ID NO.126) were used to detect whether SOERG9 was correctly integrated, primer PFY1-F (shown in SEQ ID NO.127) and primer PFY1-R (shown in SEQ ID NO.128) were used to detect whether SOIDI1 was correctly integrated.

[0102] It was transferred in the same method StibellaSqualene synthesis pathway. First, pCAS-sgRNA TDH2 + TIF1 and its corresponding donor DNA TDH2 - IGG6 - 1056ERG10 and TIF1 - IGG6 - 1056ERG13 were transferred into CEN.PK2-1D, and positive transformants E1 were obtained by colony PCR screening respectively; after eliminating the plasmid, using it as the host, pCAS-sgRNA RPS10A + CYS3 + RPS25A and its corresponding donor DNA RPS10A - IGG6 - 1056ERG12 、 CYS3 - IGG6 - 1056ERG8 and RPS25A - IGG6 - 1056ERG19 were transferred respectively, and positive transformants E2 were obtained by colony PCR screening; after eliminating the plasmid, using it as the host, pCAS-sgRNA PFY1 + RPS0A + RPL38 and its corresponding donor DNA PFY1 - IGG6 - 1056IDI1 、 RPS0A - IGG6 - 1056ERG20 and RPL38 - IGG6 - 1056ERG9 were transferred respectively, and positive transformants E3 were obtained by colony PCR screening; after eliminating the plasmid, using E3 as the host, pCAS-sgRNA TDH3 and its corresponding donor DNA TDH3 - IGG6 - 1056HMGR1 (or TDH3 - IGG6 - 1056tHMGR1 ) were transferred, and positive transformants HPSE1 (containing 1056HMGR1 ), HPSE2 (containing 1056tHMGR1 ) were obtained by colony PCR screening.

[0103] Primers used to identify positive transformants: Primer TDH3-F (shown in SEQ ID NO.111) and primer TDH3-R (shown in SEQ ID NO.112) were used to detect whether 1056HMGR1 (or 1056tHMGR1 ) was integrated correctly, primer TDH2-F (shown in SEQID NO.113) and primer TDH2-R (shown in SEQ ID NO.114) were used to detect whether 1056ERG10 was integrated correctly, primer TIF1-F (shown in SEQ ID NO.115) and primer TIF1-R (shown in SEQ ID NO.116) were used to detect whether 1056ERG13 was integrated correctly, primer RPS10A-F (shown in SEQ ID NO.117) and primer RPS10A-R (shown in SEQ ID NO.118) were used to detect whether 1056ERG12 was integrated correctly, primer CYS3-F (shown in SEQ ID NO.119) and primer CYS3-R (shown in SEQ IDNO.120) were used to detect1056ERG8 Whether the integration is correct. Primer RPS25A-F (shown in SEQ ID NO. 121) and primer RPS25A-R (shown in SEQ ID NO. 122) are used for detection. 1056ERG19 Whether the integration is correct. Primer RPS0A-F (shown in SEQ ID NO. 123) and primer RPS0A-R (shown in SEQ ID NO. 124) are used for detection. 1056ERG20 Whether the integration is correct. Primer RPL38-F (shown in SEQ ID NO. 125) and primer RPL38-R (shown in SEQ ID NO. 126) are used for detection. 1056ERG9 Whether the integration is correct. Primer PFY1-F (shown in SEQ ID NO. 127) and primer PFY1-R (shown in SEQ ID NO. 128) are used for detection. 1056IDI1 Whether the integration is correct.

[0104] Transfer into the endogenous squalene synthesis pathway of Saccharomyces cerevisiae in the same method to obtain HPSC2 (containing tHMGR1 ).

[0105] After eliminating the plasmids from the obtained positive HPSS1, HPSS2, HPSE1, HPSE2 and HPSC2 yeast transformants, streak-culture them on a new SC-Ura defective medium and culture in an incubator at 30 °C for about 2 days.

[0106] (3)Fermentation culture

[0107] First, inoculate an appropriate amount of HPSS1, HPSS2, HPSE1, HPSE2 and HPSC2 yeast transformants into 3 mL of SC-Ura defective medium and culture at 30 °C and 200 rpm for about 16 h as the seed culture solution. Take an appropriate amount of the seed culture solution and inoculate it into 50 mL of YPD medium to make the initial cell OD value about 0.1, and culture at 30 °C and 200 rpm for 7 days; extract the fermentation product with n-hexane, blow-dry with nitrogen to obtain the crude extract, and redissolve the crude extract with 1 mL of methanol.

[0108] (4)Liquid-phase detection of squalene

[0109] Take 1 mL of the above-obtained fermentation product, after high-speed centrifugation and filtration through a 0.22 µm filter membrane, detect it by high-performance liquid chromatography.

[0110] The liquid chromatography analysis instrument was the Agilent 1260 high performance liquid chromatography, and the chromatographic column was Krmasil 100-5 C18 column 5 µm (250×4.6 mm); the total flow rate of the mobile phase was 2 mL / min; the mobile phase was a mixture of 40% methanol and 60% acetonitrile for isocratic elution, and the elution time was 30 min; the detection wavelength was 210 nm, the column temperature was 30 °C, and the injection volume of the sample was 5 μL. The results are as Figure 1 shown.

[0111] 2.3. Experimental results and analysis

[0112] In the prior art, the total production of squalene by the starting strain CEN.PK2-1D was reported to be 0.34 ± 0.10 mg / L (Literature [1]); through Figure 1 , Table 1 shows that squalene was successfully detected in HPSS1, HPSS2, HPSE1, HPSE2 and HPSC2 by HPLC. The total production of HPSS1 was 9.79 ± 0.93 mg / L, the total production of HPSS2 was 1241.37 ± 88.02 mg / L, the total production of HPSE1 was 12.23 ± 3.18 mg / L, the total production of HPSE2 was 986.31 ± 35.77 mg / L, and the total production of HPSC2 was 1061.96 ± 113.77 mg / L. The total production of squalene in HPSS2 increased by 13.84% compared with that of HCS1 and by 16.89% compared with that of HPSC2. It shows that compared with the yeast endogenous squalene synthesis pathway, Sarocladium oryzae the introduction of the squalene synthesis pathway can significantly increase the total production of squalene, providing a new idea for the industrial production of squalene cell factories.

[0113] Calculation of squalene production per unit dry weight of cells: total production of squalene in 1 ml of culture / dry weight of 1 ml of cells. The results are shown in Table 1.

[0114] Table 1 Total production of squalene and squalene production per unit dry weight of cells in HCS1, HPSS2, HPSE2 and HPSC2

[0115]

[0116] As can be seen from Table 1, compared with HPSS2, HCS1 and HPSC2, the total production of squalene in HPSE2 was lower than the three, but the squalene production per unit dry weight of cells in HPSE2 was higher than them, which was 318.31±66.14 mg / g cdw. When those skilled in the art increase the cell concentration of HPSE2 by genetic engineering means, then StibellaThe introduction of the squalene synthesis pathway in sp. 1056 will also become an important means to increase the squalene production in yeast.

[0117] Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present invention.

Claims

1. A method for constructing a recombinant strain for producing squalene, characterized in that: The construction method comprises: heterologously expressing cDNAs of genes of the squalene synthesis pathway from filamentous fungi in auxotrophic Saccharomyces cerevisiae CEN.PK2-1D, wherein the genes of the squalene synthesis pathway include: mevalonate-5-phosphate kinase gene, squalene synthase gene, acetoacetyl-CoA thiolase gene, mevalonate kinase gene, hydroxymethylglutaryl-CoA synthetase gene, mevalonate pyrophosphate decarboxylase gene, farnesyl pyrophosphate synthetase gene, isopentenyl pyrophosphate: dimethylpropylene pyrophosphate isomerase gene and truncated hydroxymethylglutaryl-CoA reductase gene; The filamentous fungus is Sarocladium oryzae or Stibella sp. 1056; Sarocladium oryzae The Genbank accession number of the genome is PRJNA305454; Stibella The deposit number of sp. 1056 is CGMCC No.40422; From Sarocladium oryzae The mevalonate-5-phosphate kinase gene, squalene synthase gene, acetoacetyl-CoA thiolase gene, mevalonate kinase gene, hydroxymethylglutaryl-CoA synthetase gene, mevalonate pyrophosphate decarboxylase gene, farnesyl pyrophosphate synthetase gene, isopentenyl pyrophosphate: dimethylpropylene pyrophosphate isomerase gene and truncated hydroxymethylglutaryl-CoA reductase gene are denoted as SOERG8 Gene 、SOERG9 Gene 、SOERG10 Gene 、SOERG12 Gene 、SOERG13 Gene 、SOERG19 Gene 、SOERG20 Gene 、SOIDI1 Gene 、SOtHMGR1 Gene, DNA sequence is shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.10; From Stibella sp. 1056, the mevalonate-5-phosphate kinase gene, squalene synthase gene, acetoacetyl-CoA thiolase gene, mevalonate kinase gene, hydroxymethylglutaryl-CoA synthetase gene, mevalonate pyrophosphate decarboxylase gene, farnesyl pyrophosphate synthetase gene, isopentenyl pyrophosphate: dimethylpropylene pyrophosphate isomerase gene, and truncated hydroxymethylglutaryl-CoA reductase gene are denoted as 1056ERG8 Gene 、1056ERG9 Gene 、1056ERG10 Gene 、 1056ERG12 Gene 、1056ERG13 Gene 、1056ERG19 Gene 、1056ERG20 Gene 、1056IDI1 Gene 、 1056tHMGR1 The genes, the DNA sequences are shown in SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO.16, SEQ ID NO.17, SEQ ID NO.18, and SEQ ID NO.20 respectively.

2. The method for constructing a recombinant strain for producing squalene according to claim 1, characterized in that: SOERG8 Gene 、SOERG9 Gene 、SOERG10 Gene 、SOERG12 Gene 、SOERG13 Gene 、SOERG19 Gene 、 SOERG20 Gene 、SOIDI1 Gene, SOT HMGR1 The endogenous target genes of the gene are CYS3 Gene, RPL38 Gene, TDH2 Gene, RPS10A Gene, TIF1 Gene, RPS25A Gene, RPS0A Gene, PFY1 Gene, TDH3 Gene; by IGG Element 5′-CAATCAAAC-3′ was used for ligation; 1056ERG8 Gene 、1056ERG9 Gene 、1056ERG10 Gene 、1056ERG12 Gene 、1056ERG13 Gene 、 1056ERG19 Gene 、1056ERG20 Gene 、1056IDI1 Gene, 1056tHMGR1 The endogenous target genes of the gene are CYS3 Gene, RPL38 Gene, TDH2 Gene, RPS10A Gene, TIF1 Gene, RPS25A Gene, RPS0A Gene, PFY1 Gene, TDH3 Gene; by IGG The element 5'-CAATCAAAC-3' was ligated.

3. The method for constructing a recombinant strain for producing squalene according to claim 1, characterized in that: Each of the mevalonate-5-phosphate kinase gene, squalene synthase gene, acetoacetyl-CoA thiolase gene, mevalonate kinase gene, hydroxymethylglutaryl-CoA synthetase gene, mevalonate pyrophosphate decarboxylase gene, farnesyl pyrophosphate synthetase gene, isopentenyl pyrophosphate: dimethylpropylene pyrophosphate isomerase gene and truncated hydroxymethylglutaryl-CoA reductase gene is fused to a DNA sequence encoding the peroxisomal localization signal MDH3.

4. A recombinant strain for producing squalene, characterized in that: The method is constructed by any one of claims 1 to 3.

5. Use of the recombinant strain according to claim 4 in the production of squalene.

6. A method for producing squalene, characterized in that: The method comprises fermenting the recombinant strain according to claim 5 to obtain squalene from the fermentation liquid.

7. A method for producing squalene according to claim 6, characterized in that: The seed culture medium of the recombinant strain is SC-Ura defective culture medium, the fermentation culture medium is YPD culture medium, the initial bacterial OD value is 0.08-0.12; the fermentation temperature is 28-32°C, and the culture is carried out at 150-250 rpm for 7-10 days.

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