Recombinant strain for producing squalene as well as construction method and application of recombinant strain

Abstract

The invention discloses a recombinant strain for producing squalene as well as a construction method and application of the recombinant strain. The construction method comprises the following steps: heterologously expressing cDNA (complementary deoxyribonucleic acid) of each gene in a squalene synthesis route from filamentous fungi in yeast; the squalene synthetic pathway genes comprise a mevalonic acid-5-phosphate kinase gene, a squalene synthase gene, an acetoacetyl coenzyme A thiolase gene, a mevalonic acid kinase gene, a hydroxymethyl glutaryl coenzyme a synthetase gene, a mevalonic acid pyrophosphate decarboxylase gene, a farnesyl pyrophosphate synthetase gene and isopentenyl pyrophosphate. A dimethallylpyrophosphate isomerase gene and a truncated hydroxymethyl glutaryl coenzyme a reductase gene; the culture medium is characterized in that the filamentous fungi are Saroocladium oryzae or Stibella sp. 1056, and the culture medium is a culture medium. The excavated squalene synthesis route can complete efficient de novo synthesis of squalene, and the squalene yield is increased.

Description

Technical Field

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

[0002] Squalene (Squalene, 2,6,10,15,19,23-hexamethyl-2,6,10,14,18,22-tetracosahexene, C 30 H 50 ) is an all-trans triterpenoid compound. It is not only the precursor of many active sterols or triterpenoid compounds such as lanosterol, ergosterol, fumagillic acid, but also widely used in food, medicine, cosmetics and other fields due to its functional properties in regulating immunity, promoting cardiovascular health, anti-cancer, anti-oxidation, anti-bacteria, etc., such as health products, vaccine adjuvants, moisturizers, 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 are trying to use microbial cell factories to efficiently produce squalene in order to reduce pressure on the environment and promote sustainable development.

[0004] In the related art, for example, document [1] discloses the following: the squalene biosynthesis pathway was reconstructed in the peroxisome of Saccharomyces cerevisiae by using an efficient and controllable expression system of endogenous exogenous genes. Enterococcus faecalis of EfmvaE and EfmvaS , overexpression of endogenous genes in Saccharomyces cerevisiae ERG8 , ERG9 , ERG12 , ERG19 , ERG20 and IDI1 , which increased squalene production by 900 times to 1090.41 ± 80.92 mg / L.

[0005] Therefore, further development of 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 and a construction method and application thereof, so as to increase the yield of squalene.

[0007] The technical solution adopted by the present invention is: First, The present invention provides a method for constructing a recombinant strain for producing squalene, the method comprising: heterologously expressing cDNAs of genes of the squalene synthesis pathway from filamentous fungi in yeast, 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; Said Sarocladium oryzae The Genbank accession number of the genome is PRJNA305454; 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; Said Stibella sp. 1056 is deposited under the number CGMCC No.40422; Stibella 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 of sp.1056 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.

[0008] In some embodiments, the yeast is Saccharomyces cerevisiae. Further, in some embodiments, the Saccharomyces cerevisiae is the auxotrophic Saccharomyces cerevisiae CEN.PK2-1D.

[0009] In some embodiments, SOERG8 Gene 、SOERG9 Gene 、SOERG10 Gene 、SOERG12 Gene 、 SOERG13 Gene 、SOERG19 Gene 、SOERG20 Gene 、SOIDI1 Gene, SOtHMGR1 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; IGG The element 5'-CAATCAAAC-3' was ligated. IGG Components are recorded IGG6。

[0010] In some embodiments, 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; IGGThe element 5'-CAATCAAAC-3' was ligated. IGG Components are recorded IGG6。

[0011] In some embodiments, 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. (MDH3 is linked to the carboxyl terminus of the protein so that the nine enzymes are expressed in the peroxisome.) In a more specific embodiment, the peroxisomal localization signal is SKL.

[0012] Second, The present invention also provides a recombinant strain for producing squalene, which is constructed by the above construction method.

[0013] Thirdly, The present invention also provides application of the recombinant strain in producing squalene.

[0014] Fourthly, The present invention also provides a method for producing squalene, comprising fermenting the above-mentioned recombinant strain to obtain squalene from the fermentation liquid.

[0015] In some embodiments, the fermentation medium is YPD medium, and the initial bacterial OD 600 The 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. Preferably, the initial bacterial OD 600 The value was 0.1, the fermentation temperature was 30 ℃, and the culture speed was 200 rpm for 7 days.

[0016] In some embodiments, the components of the YPD medium include: 2% yeast extract, 2% peptone, and 2% glucose.

[0017] In some embodiments, the seed culture medium of the recombinant strain is SC-Ura deficiency medium.

[0018] In some embodiments, the components of the SC-Ura deficiency medium include: 2% glucose, 6.7% amino acid-free yeast nitrogen source, and 0.78 g / L uracil-deficient amino acid mixture.

[0019] The present invention has the following advantages and beneficial effects: The squalene synthesis pathway discovered by the present invention can complete the efficient de novo synthesis of squalene and increase the yield of squalene, and has great application value for developing recombinant strains with high squalene yield.

[0020] The present invention utilizes an efficient and controllable expression system of exogenous genes carried by endogenous genes to express in Saccharomyces cerevisiae. Stibella sp. 1056 or Sarocladium oryzae The squalene biosynthesis pathway in the squalene gene (a total of 9 enzymes: mevalonate-5-phosphate kinase 1056ERG8 / SOERG8; squalene synthase 1056ERG9 / SOERG9; acetoacetyl-CoA thiolase 1056ERG10 / SOERG10; mevalonate kinase 1056ERG12 / SOERG12; hydroxymethylglutaryl-CoA synthetase 1056ERG13 / SOERG13; mevalonate pyrophosphate decarboxylase 1056ERG19 / SOERG19; farnesyl pyrophosphate synthase 1056ERG20 / SOERG20; isopentenyl pyrophosphate: dimethylpropylene pyrophosphate isomerase 1056IDI1 / SOIDI1; truncated hydroxymethylglutaryl-CoA reductase 1056tHMGR1 / SOtHMGR1) was heterologously expressed. Sarocladium oryzae The total squalene production obtained after the squalene synthesis pathway was transferred into Saccharomyces cerevisiae was 1241.37 ± 88.02 mg / L, and the squalene production per unit dry weight of the bacteria reached 301.02 ± 43.49 mg / g cdw; Stibella After the squalene synthesis pathway in sp. 1056 was transferred into Saccharomyces cerevisiae, the total squalene production obtained was 986.31 ± 35.77 mg / L, and the squalene production per unit dry weight of the bacteria reached 318.31 ± 66.14 mg / g cdw. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 HPLC profiles of squalene production for different squalene engineering strains; Among them, HCS1 is described in the literature [1], which is a highly efficient and controllable expression system of exogenous genes carried by endogenous genes to reconstruct the squalene biosynthesis pathway in the peroxisome of Saccharomyces cerevisiae. Enterococcus faecalis of EfmvaE and EfmvaS , overexpression of endogenous genes in Saccharomyces cerevisiae ERG8 , ERG9 , ERG12 , ERG19 , ERG20 and IDI1 . DETAILED DESCRIPTION

[0022] In order to make the purpose, technical solution and advantages of the embodiment of the present invention clearer, the technical solution of the embodiment of the present invention will be clearly and completely described below. Obviously, the described embodiment is a part of the embodiment of the present invention, not all the embodiments. Based on the described embodiment of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work belong to the protection scope of the present invention.

[0023] Unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the molecular genetics, nucleic acid chemistry, and immunology laboratory procedures used herein are all conventional procedures widely used in the corresponding fields or are performed according to the conditions recommended by the manufacturers.

[0024] The following is a description of the terminology used in this article: In this document, in the "first aspect", "second aspect", "third aspect", etc., the terms "first", "second", "third", etc. are used only 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 enumeration and description, and it should be understood that they do not constitute a closed limitation on quantity.

[0025] In this document, reference to “preferred” is only to describe an implementation method or example with better effects, and it should be understood that it does not constitute a limitation on the scope of protection of the present invention.

[0026] The documents cited in the present invention are: Literature [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 ofmultigene pathways in fungi. Nat Commun 14(1): 4267 Reference [2]: Xu Yuquan, Yue Qun, Zhang Liwen, Song Kainan, Ai Yutong, A filamentous fungus with high yield of terpene and peptide metabolites, ZL202310601793.1, Authorization announcement date: August 15, 2023 Literature[3]: Hittalmani, S., Mahesh, HB, Mahadevaiah, C. andPrasannakumar, MK (2016). De novo genome assembly and annotation of ricesheath rot fungus Sarocladium oryzae reveals genes involved in helvolic acidand cerulenin biosynthesis pathways. BMC Genomics 17: 271 Embodiments of the present invention Stibella sp. 1056, which was deposited by the General Microbiology Center (CGMCC) of the China Microbiological Culture Collection Administration on November 11, 2022 (the deposit address is: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing), with the deposit number CGMCC No. 40422. It was found to be alive after testing. It is also recorded in the literature [2]. It comes from the Institute of Biotechnology, Chinese Academy of Agricultural Sciences.

[0027] Embodiments of the present invention Sarocladium oryzae This strain is a fungus reported in literature [3], and its genome accession number in Genbank is PRJNA305454.

[0028] Squalene is an important precursor of triterpenoids, and filamentous fungi are an important source of triterpenoids. The filamentous fungi isolated from soil by the inventors Stibella The yield of fumagillic acid by sp. 1056 in rice medium can reach 4.52 g / kg, and the yield of fumagillic acid by sp. 1056 in YES medium can reach 610 mg / L (Reference [2]); Sarocladium oryzae The fumagillic acid produced is the main virulence factor that causes rice sheath rot (reference [3]). Based on this, the inventors speculated that squalene is a precursor for the biosynthesis of fumagillic acid. Stibella sp.1056 、Sarocladium oryzae The biosynthetic pathway in is highly efficient. The embodiment of the present invention introduces the pathway into Saccharomyces cerevisiae to increase the yield of squalene and develop a recombinant strain with a higher yield of squalene.

[0029] The present invention provides two recombinant strains for producing squalene, wherein: 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 .

[0030] 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 .

[0031] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from conventional biochemical reagent stores unless otherwise specified. The quantitative tests in the following examples are repeated three times, and the results are averaged.

[0032] Vector pJET1.2 / blunt Cloning Vector: purchased from Thermo Fisher Scientific; Plasmids pCAS, pScURA3 and psgtRNA are described in reference [1].

[0033] Saccharomyces cerevisiae CEN.PK2-1D: MATα; ura3-52; trp1-289; leu2-3,112; his3Δ1; MAL2-8C; SUC2.

[0034] Enzymes and kits: High-fidelity DNA amplification MIX and seamless cloning kit were purchased from Novozymes; DNA loading buffer and DNA marker were purchased from Kangwei Century Company; DNA purification gel recovery kit was purchased from Thermo Fisher Scientific; RNA extraction reagent Trizol and kit PureLink TM RNA Mini Kit was purchased from Thermo Fisher Scientific; The reverse transcription kit PrimeScript reagent kit was purchased from TaKaRa; NEBridge ® Golden Gate Assembly Kit (BsaI-HF ® v2) was purchased from NEB.

[0035] Escherichia coli DH5α competent bacteria were purchased from Kangwei Century Company.

[0036] The analytically pure n-hexane solvent used in the experiment was produced by Sinopharm Chemical Reagent Beijing Co., Ltd. Chromatographically pure acetonitrile and methanol were produced by Fisher Chemical.

[0037] Culture medium: SC-Ura-deficient medium (2% glucose, 6.7% yeast nitrogen base without amino acids, 0.78 g / L uracil-deficient amino acid mixture); YPD medium (2% yeast extract, 2% Peptone peptone, 2% glucose); YES medium (2% yeast extract, 0.05% magnesium sulfate heptahydrate, 15% sucrose, 0.001% zinc sulfate heptahydrate, 0.0005% copper sulfate pentahydrate).

[0038] If making solid culture medium, add 2% agar powder.

[0039] In the embodiment of the present invention, SOHMGR1 The 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.

[0040] Example 1 Cloning of cDNA Sequences of Genes in Squalene Biosynthesis Pathway 1.1. Purpose of the experiment exist Stibella sp. 1056 to produce fumagillic acid, the total RNA was extracted, and after reverse transcription, the nine enzymes in the squalene synthesis pathway were amplified and cloned using gene-specific primers to obtain 1056ERG12 , 1056ERG8 , 1056ERG19 , 1056IDI1 , 1056ERG20 , 1056ERG9 , 1056ERG10 , 1056ERG13 and 1056HMGR1 's transcript.

[0041] Through bioinformatics analysis Sarocladium oryzae The genome (Genbank accession number is PRJNA305454) SOERG12 , SOERG8 , SOERG19 , SOIDI1 , SOERG20 , SOERG9 , SOERG10 , SOERG13 , SOHMGR1 The transcripts were predicted and gene synthesis was performed.

[0042] 1.2 Experimental methods 1.2.1 Stibella sp. 1056 Squalene Synthesis Pathway (1) Stibella sp. 1056 total cDNA The YES medium cultured for 5 days was collected by filtration. Stibella sp. 1056 bacteria, total RNA was extracted according to the instructions of the RNA extraction kit, and reverse transcription was performed according to the instructions of the reverse transcription kit to obtain total cDNA.

[0043] (2) Stibella Cloning of cDNA of squalene biosynthesis pathway gene from sp. 1056 Total Stibella sp. 1056 cDNA as template, according to 1056ERG12 , 1056ERG8 , 1056ERG19 , 1056IDI1 , 1056ERG20 , 1056ERG9 , 1056ERG10 , 1056ERG13 and 1056HMGR1 The gene sequence was obtained, and primers were designed to perform PCR amplification using the total cDNA obtained as a template to obtain cDNA from the start codon of the gene to the base before the stop codon, which were then ligated to the pJET1.2 vector. After transformation, positive clones were picked for sequencing.

[0044] In the above method, primers 1056ERG8-F (shown in SEQ ID NO.21) and primers 1056ERG8-R (shown in SEQ ID NO.22) were used to amplify the gene 1056ERG8 , primers 1056IDI1-F (shown in SEQ ID NO.23) and primers 1056IDI1-R (shown in SEQ ID NO.24) were used to amplify the gene 1056IDI1 , primers 1056ERG9-F (shown in SEQ ID NO.25) and primers 1056ERG9-R (shown in SEQ ID NO.26) were used to amplify the gene 1056ERG9 , primers 1056ERG20-F (shown in SEQ ID NO.28) and primers 1056ERG20-R (shown in SEQ ID NO.27) were used to amplify the gene 1056ERG20 , primers 1056ERG12-F (shown in SEQ ID NO.29) and primers 1056ERG12-R (shown in SEQ ID NO.30) were used to amplify the gene 1056ERG12 , primers 1056ERG19-F (shown in SEQ ID NO.31) and primers 1056ERG19-R (shown in SEQ ID NO.32) were used to amplify the gene 1056ERG19 , primers 1056ERG10-F (shown in SEQ ID NO.33) and primers 1056ERG10-R (shown in SEQ ID NO.34) were used to amplify the gene 1056ERG10 , primers 1056ERG13-F (shown in SEQ ID NO.35) and primers 1056ERG13-R (shown in SEQ ID NO.36) were used to amplify the gene 1056ERG13 , primers 1056HMGR1-F (shown in SEQ ID NO.37) and primers 1056HMGR1-R (shown in SEQ ID NO.38) were used to amplify the gene 1056HMGR1 .

[0045] 1.2.2 Sarocladium oryzae Squalene synthesis pathway Sarocladium oryzae Prediction and synthesis of cDNA of genes involved in squalene biosynthesis pathway Using the auxotrophic Saccharomyces cerevisiae CEN.PK2-1D ERG12 , ERG8 , ERG19 , IDI1 , ERG20 , ERG9 , ERG10 , ERG13 , HMGR1 Gene pair Sarocladium oryzae The genome (Genbank accession number is PRJNA305454) was aligned by tblastn to predict SOERG12 , SOERG8 , SOERG19 , SOIDI1 , SOERG20 , SOERG9 , SOERG10 , SOERG13 and SOHMGR1 The open reading frames were predicted and gene synthesized by FGENESH to obtain positive clones containing cDNA fragments of these genes.

[0046] 1.3. Experimental results and analysis Use YES medium Stibella sp. 1056, it was found that all 9 enzymes in the squalene biosynthesis pathway were expressed, and the correct cDNAs of these 9 genes were obtained by sequencing.

[0047] Obtained through bioinformatics analysis prediction and gene synthesis Sarocladium oryzae Squalene biosynthesis pathway gene cDNA.

[0048] Example 2 Reconstruction of the squalene synthesis pathway 2.1. Purpose of the experiment Using the auxotrophic Saccharomyces cerevisiae CEN.PK2-1D as the chassis cell, select RPS10A , CYS3 , RPS25A , PFY1 , RPS0A , RPL38 , TDH2 , TIF1 , TDH3 As Sarocladium oryzae Squalene synthesis pathway: SOERG12 , SOERG8 , SOERG19 , SOIDI1 , SOERG20 , SOERG9 , SOERG10 , SOERG13 , SOHMGR1 (or SOtHMGR1 );or Stibella sp. 1056 Squalene Synthesis Pathway: 1056ERG12 , 1056ERG8 , 1056ERG19 , 1056IDI1 , 1056ERG20 , 1056ERG9 , 1056ERG10 , 1056ERG13 , 1056HMGR1 (or 1056tHMGR1 ) and the corresponding endogenous gene of Saccharomyces cerevisiae was used as a control. IGG6 The element 5'-CAATCAAAC-3' acts as a linker and adds a peroxisomal localization signal for each target gene MDH3 , the target gene was transferred into yeast using CRISPR gene editing technology, and the squalene produced in the positive transformants was detected by HPLC.

[0049] 2.2 Experimental methods: (1) Obtaining plasmids and donor DNA for CRISPR gene editing According to the endogenous target gene, target gene, IGG element( IGG6 , 5'-CAATCAAAC-3'), MDH3 Design primers based on the sequence of the target gene and add the homologous fragment of the target gene to the 5' of the forward primer. IGG6 sequence, add a homologous fragment of the target gene to the 5' end of the reverse primer and MDH3 Sequence, PCR amplification of donor DNA was performed using the positive cDNA clone of squalene synthesis pathway gene as template.

[0050] In the above method: Refactoring Sarocladium oryzae Primers related to the squalene synthesis pathway: Primers SO-TDH2-ERG10-F (shown in SEQ ID NO.39) and primers SO-TDH2-ERG10-R (shown in SEQ ID NO.40) were used to amplify the donor DNA TDH2- IGG6-SOERG10 , using primers SO-RPS10A-ERG12-F (shown in SEQ ID NO.41) and primers SO-RPS10A-ERG12-R (shown in SEQ ID NO.42) to amplify the donor DNA RPS10A-IGG6-SOERG12 , using primers SO-TIF1-ERG13-F (shown in SEQ ID NO.43) and primers SO-TIF1-ERG13-R (shown in SEQ ID NO.44) to amplify the donor DNA TIF1-IGG6-SOERG13 , using primers SO-RPS0A-ERG20-F (shown in SEQ ID NO.45) and primers SO-RPS0A-ERG20-R (shown in SEQ ID NO.46) to amplify the donor DNA RPS0A-IGG6-SOERG20 , using primers SO-CYS3-ERG8-F (shown in SEQ ID NO.47) and primers SO-CYS3-ERG8-R (shown in SEQ ID NO.48) to amplify the donor DNA CYS3-IGG6-SOERG8 , using primers SO-RPS25A-ERG19-F (shown in SEQ ID NO.49) and primers SO-RPS25A-ERG19-R (shown in SEQ ID NO.50) to amplify the donor DNA RPS25A-IGG6-SOERG19 , using primers SO-PFY1-IDI1-F (shown in SEQ ID NO.51) and primers SO-PFY1-IDI1-R (shown in SEQ ID NO.52) to amplify the donor DNA PFY1-IGG6-SOIDI1 , using primers SO-RPL38-ERG9-F (shown in SEQ ID NO.53) and primers SO-RPL38-ERG9-R (shown in SEQ ID NO.54) to amplify the donor DNA RPL38-IGG6-SOERG9 , using primers SO-TDH3-tHMGR1-F (shown in SEQ ID NO.55) and primers SO-TDH3-tHMGR1-R (shown in SEQ ID NO.56) to amplify the donor DNA TDH3-IGG6-SOtHMGR1 , using primers SO-TDH3-HMGR1-F (shown in SEQ ID NO.57) and primers SO-TDH3-HMGR1-R (shown in SEQ ID NO.58) to amplify the donor DNA TDH3-IGG6-SOHMGR1 .

[0051] Refactoring Stibella sp. 1056 Primers related to the squalene synthesis pathway: Primer 1056-CYS3-ERG8-F (shown in SEQ ID NO.59) and primer 1056-CYS3-ERG8-R (shown in SEQ ID NO.60) were used to amplify the donor DNA CYS3-IGG6-1056ERG8 , using primers 1056-PFY1-IDI1-F (shown in SEQ ID NO.61) and primers 1056-PFY1-IDI1-R (shown in SEQ ID NO.62) to amplify the donor DNA PFY1-IGG6-1056IDI1 , using primers 1056-RPL38-ERG9-F (shown in SEQ ID NO.63) and primers 1056-RPL38-ERG9-R (shown in SEQ ID NO.64) to amplify the donor DNA RPL38-IGG6-1056ERG9 , using primers 1056-RPS0A-ERG20-F (shown in SEQ ID NO.66) and primers 1056-RPS0A-ERG20-R (shown in SEQ ID NO.65) to amplify the donor DNA RPS0A-IGG6-1056ERG20 , using primers 1056-RPS10A-ERG12-F (shown in SEQ ID NO.67) and primers 1056-RPS10A-ERG12-R (shown in SEQ ID NO.68) to amplify the donor DNA RPS10A-IGG6-1056ERG12 , using primers 1056-RPS25A-ERG19-F (shown in SEQ ID NO.69) and primers 1056-RPS25A-ERG19-R (shown in SEQ ID NO.70) to amplify the donor DNA RPS25A- IGG6-1056ERG19 , using primers 1056-TDH2-ERG10-F (shown in SEQ ID NO.71) and primers 1056-TDH2-ERG10-R (shown in SEQ ID NO.72) to amplify the donor DNA TDH2-IGG6-1056ERG10 , using primers 1056-TIF1-ERG13-F (shown in SEQ ID NO.73) and primers 1056-TIF1-ERG13-R (shown in SEQ ID NO.74) to amplify the donor DNA TIF1-IGG6-1056ERG13 , using primers 1056-TDH3-HMGR1-F (shown in SEQ ID NO.75) and primers 1056-TDH3-HMGR1-R (shown in SEQ ID NO.76) to amplify the donor DNA TDH3-IGG6-1056HMGR1 , using primers 1056-TDH3-tHMGR1-F (shown in SEQ ID NO.77) and primers 1056-TDH3-HMGR1-R (shown in SEQ ID NO.76) to amplify the donor DNA TDH3-IGG6-1056tHMGR1 .

[0052] Primers for reconstructing the endogenous squalene synthesis pathway of Saccharomyces cerevisiae: Primers CEN-TDH2-ERG10-F (shown in SEQ ID NO.78) and primers CEN-TDH2-ERG10-R (shown in SEQ ID NO.79) were used to amplify the donor DNA TDH2-IGG6- CENERG10 , using primers CEN-RPS10A-ERG12-F (shown in SEQ ID NO.80) and primers CEN-RPS10A-ERG12-R (shown in SEQ ID NO.81) to amplify the donor DNA RPS10A-IGG6-CENERG12 , using primers CEN-TIF1-ERG13-F (shown in SEQ ID NO.82) and primers CEN-TIF1-ERG13-R (shown in SEQ ID NO.83) to amplify the donor DNA TIF1- IGG6-CENERG13 , using primers CEN-RPS0A-ERG20-F (shown in SEQ ID NO.84) and primers CEN-RPS0A-ERG20-R (shown in SEQ ID NO.85) to amplify the donor DNA RPS0A-IGG6-CENERG20 , using primers CEN-CYS3-ERG8-F (shown in SEQ ID NO.86) and primers CEN-CYS3-ERG8-R (shown in SEQ ID NO.87) to amplify the donor DNA CYS3-IGG6-CENERG8 , using primers CEN-RPS25A-ERG19-F (shown in SEQ ID NO.88) and primers CEN-RPS25A-ERG19-R (shown in SEQ ID NO.89) to amplify the donor DNA RPS25A-IGG6-CENERG19 , using primers CEN-PFY1-IDI1-F (shown in SEQ ID NO.90) and primers CEN-PFY1-IDI1-R (shown in SEQ ID NO.91) to amplify the donor DNA PFY1-IGG6-CENIDI1 , using primers CEN-RPL38-ERG9-F (shown in SEQ ID NO.92) and primers CEN-RPL38-ERG9-R (shown in SEQ ID NO.93) to amplify the donor DNA RPL38-IGG6-CENERG9 , using primers CEN-TDH3-tHMGR1-F (shown in SEQ ID NO.94) and primers CEN-TDH3-tHMGR1-R (shown in SEQ ID NO.95) to amplify the donor DNA TDH3-IGG6-CENtHMGR1 .

[0053] According to the literature [1] RPS10A , CYS3 , RPS25A , PFY1 , RPS0A , RPL38 , TDH2 , TIF1 , TDH3 The gRNA primers were designed, and pScURA3 and psgtRNA were used as templates to construct the plasmid pCAS-sgRNA according to the method described in the article. TDH2+ TIF1 , pCAS-sgRNA RPS10A + CYS3 + RPS25A , pCAS-sgRNA PFY1 + RPS0A + RPL38 , pCAS-sgRNA TDH3 .

[0054] In the above method, primers GRNA-TDH2-BSAI-F (shown in SEQ ID NO.96) and primers GRNA-TIF1-BSAI-R4 (shown in SEQ ID NO.97) are used to amplify DNA fragments using psgtRNA as a template. TDH2-TIF1 , using primers GRNA-TIF1-BSAI-F4 (shown in SEQ ID NO.98) and primers SGT4R.URA3R (shown in SEQ ID NO.99) to amplify the DNA fragment using pScURA3 as a template TIF1-URA3 , the two DNA fragments were connected to the plasmid pCAS using the Golden Gate assembly kit to obtain pCAS-sgRNA TDH2+TIF1 The DNA fragment was amplified using primers GRNA-RPS0A-BSAI-F (shown in SEQ ID NO.100) and primers GRNA-RPL38-BSAI-R4 (shown in SEQ ID NO.101) with psgtRNA as template. RPS0A-RPL38 , using primers GRNA-RPL38-BSAI-F4 (shown in SEQ ID NO.102) and primers GRNA-PFY1-BSAI-R4 (shown in SEQ ID NO.103) to amplify DNA fragments using psgtRNA as template RPL38-PFY1 , using primers GRNA-PFY1-BSAI-F4 (shown in SEQ ID NO.104) and primers SGT4R.URA3R (shown in SEQ ID NO.99) with pScURA3 as template to amplify the DNA fragment PFY1-URA3 , use the Golden Gate assembly kit to connect these three DNA fragments to the plasmid pCAS to obtain pCAS-sgRNA PFY1 + RPS0A + RPL38 The DNA fragment was amplified using primers GRNA-RPS10A-BSAI-F1 (shown in SEQ ID NO.105) and primers GRNA-RPS25A-BSAI-R (shown in SEQ ID NO.106) with psgtRNA as template. RPS10A-RPS25A , using primers GRNA-RPS25A-BSAI-F (shown in SEQ ID NO.107) and primers GRNA-CYS3-BSAI-R (shown in SEQ ID NO.108) to amplify DNA fragments using psgtRNA as template RPS25A-CYS3 , using primers GRNA-CYS3-BSAI-F (shown in SEQ ID NO.109) and primers SGT4R.URA3R (shown in SEQ ID NO.99) with pScURA3 as template to amplify the DNA fragment CYS3-URA3 , use the Golden Gate assembly kit to connect these three DNA fragments to the plasmid pCAS to obtain pCAS-sgRNA RPS10A + CYS3 + RPS25A The DNA fragment was amplified using primers GRNA-TDH3-BSAI-F1 (shown in SEQ ID NO.110) and primers SGT4R.URA3R (shown in SEQ ID NO.99) with pScURA3 as template. TDH3-URA3 , use the Golden Gate assembly kit to connect this DNA fragment to the plasmid pCAS to obtain pCAS-sgRNA TDH3 .

[0055] (2) Yeast transformation Yeast electroporation and plasmid elimination were performed according to the method in reference [1]. Sarocladium oryzae Taking the squalene synthesis pathway as an example, first, pCAS-sgRNA TDH2+TIF1 and its corresponding donor DNA TDH2-IGG6-SOERG10 and TIF1-IGG6-SOERG13 Transformed into CEN.PK2-1D, and positive transformants S1 were obtained by colony PCR screening; after eliminating the plasmid, it was used as the host and transformed into pCAS-sgRNA RPS10A + CYS3 + RPS25A and its corresponding donor DNA RPS10A-IGG6-SOERG12 , CYS3-IGG6-SOERG8 and RPS25A-IGG6-SOERG19 , positive transformants S2 were obtained by colony PCR screening; after eliminating the plasmid, it was used as a host to transfer into pCAS-sgRNA PFY1 + RPS0A + RPL38 and its corresponding donor DNA PFY1-IGG6-SOIDI1 , RPS0A-IGG6-SOERG20 and RPL38-IGG6-SOERG9 , positive transformants S3 were obtained by colony PCR screening; after eliminating the plasmid, S3 was used as the host and pCAS-sgRNA was respectively transferred into TDH3 and its corresponding donor DNA TDH3-IGG6-SOHMGR1 (or TDH3-IGG6-SOtHMGR1 ), and the positive transformants HPSS1 (containing SOHMGR1 )、HPSS2(include SOtHMGR1 ).

[0056] 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 SOHMGR1 (or SOtHMGR1 ) was integrated correctly, primers TDH2-F (shown in SEQ ID NO.113) and primers TDH2-R (shown in SEQ ID NO.114) were used to detect SOERG10 Primers TIF1-F (shown in SEQ ID NO.115) and TIF1-R (shown in SEQ ID NO.116) were used to detect whether the integration was correct. SOERG13 Primers RPS10A-F (shown in SEQ ID NO.117) and RPS10A-R (shown in SEQ ID NO.118) were used to detect whether the integration was correct. SOERG12 Primers CYS3-F (shown in SEQ ID NO.119) and CYS3-R (shown in SEQ ID NO.120) were used to detect whether the integration was correct. SOERG8 Primers RPS25A-F (shown in SEQ ID NO.121) and RPS25A-R (shown in SEQ ID NO.122) were used to detect whether the integration was correct.SOERG19 Primers RPS0A-F (shown in SEQ ID NO.123) and RPS0A-R (shown in SEQ ID NO.124) were used to detect whether the integration was correct. SOERG20 Primers RPL38-F (shown in SEQ ID NO.125) and RPL38-R (shown in SEQ ID NO.126) were used to detect whether the integration was correct. SOERG9 Primers PFY1-F (shown in SEQ ID NO.127) and PFY1-R (shown in SEQ ID NO.128) were used to detect whether the integration was correct. SOIDI1 Is the integration correct?

[0057] Transfer in the same way Stibella sp. 1056 squalene synthesis pathway. First, pCAS-sgRNA TDH2 + TIF1 and its corresponding donor DNA TDH2 - IGG6 - 1056ERG10 and TIF1 - IGG6 - 1056ERG13 Transformed into CEN.PK2-1D, and positive transformants E1 were obtained by colony PCR screening; after eliminating the plasmid, it was used as the host and transformed into pCAS-sgRNA RPS10A + CYS3 + RPS25A and its corresponding donor DNA RPS10A - IGG6 - 1056ERG12 , CYS3 - IGG6 - 1056ERG8 and RPS25A - IGG6 - 1056ERG19 , positive transformants E2 were obtained by colony PCR screening; after eliminating the plasmid, it was used as a host to transfer into pCAS-sgRNA PFY1 + RPS0A + RPL38 and its corresponding donor DNA PFY1 - IGG6 - 1056IDI1 , RPS0A - IGG6 - 1056ERG20 and RPL38 - IGG6 - 1056ERG9 , positive transformants E3 were obtained by colony PCR screening; after eliminating the plasmid, E3 was used as the host and pCAS-sgRNA was respectively transferred into TDH3 and its corresponding donor DNA TDH3 - IGG6 - 1056HMGR1 (or TDH3 - IGG6 - 1056tHMGR1 ), and screened the positive transformants HPSE1 (containing 1056HMGR1 )、HPSE2(included 1056tHMGR1 ).

[0058] 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 1056HMGR1 (or 1056tHMGR1) was integrated correctly, primers TDH2-F (shown in SEQ ID NO.113) and primers TDH2-R (shown in SEQ ID NO.114) were used to detect 1056ERG10 Primers TIF1-F (shown in SEQ ID NO.115) and TIF1-R (shown in SEQ ID NO.116) were used to detect whether the integration was correct. 1056ERG13 Primers RPS10A-F (shown in SEQ ID NO.117) and RPS10A-R (shown in SEQ ID NO.118) were used to detect whether the integration was correct. 1056ERG12 Primers CYS3-F (shown in SEQ ID NO.119) and CYS3-R (shown in SEQ ID NO.120) were used to detect whether the integration was correct. 1056ERG8 Primers RPS25A-F (shown in SEQ ID NO.121) and RPS25A-R (shown in SEQ ID NO.122) were used to detect whether the integration was correct. 1056ERG19 Primers RPS0A-F (shown in SEQ ID NO.123) and RPS0A-R (shown in SEQ ID NO.124) were used to detect whether the integration was correct. 1056ERG20 Primers RPL38-F (shown in SEQ ID NO.125) and RPL38-R (shown in SEQ ID NO.126) were used to detect whether the integration was correct. 1056ERG9 Primers PFY1-F (shown in SEQ ID NO.127) and PFY1-R (shown in SEQ ID NO.128) were used to detect whether the integration was correct. 1056IDI1 Is the integration correct?

[0059] The same method was used to transfer the endogenous squalene biosynthesis pathway of Saccharomyces cerevisiae to obtain HPSC2 (containing tHMGR1 ).

[0060] The obtained positive HPSS1, HPSS2, HPSE1, HPSE2 and HPSC2 yeast transformants were streaked on new SC-Ura deficient medium after elimination of plasmids and cultured in a 30°C incubator for about 2 days.

[0061] (3) Fermentation culture First, an appropriate amount of HPSS1, HPSS2, HPSE1, HPSE2 and HPSC2 yeast transformants were inoculated into 3 mL SC-Ura defective medium and cultured at 30°C and 200 rpm for about 16 h as seed culture medium. An appropriate amount of seed culture medium was inoculated into 50 mL YPD medium to make its initial bacterial OD value about 0.1, and cultured at 30°C and 200 rpm for 7 days; the fermentation product was extracted with n-hexane, and the crude extract was obtained by nitrogen blowing, and the crude extract was re-dissolved in 1 mL methanol.

[0062] (4) Squalene liquid phase detection 1 mL of the fermentation product was taken and centrifuged at high speed and filtered through a 0.22 µm filter membrane, and then detected by high performance liquid chromatography.

[0063] The liquid chromatography analysis instrument was Agilent 1260 high performance liquid chromatography, the chromatographic column was Krmasil 100-5C18 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 sample injection volume was 5 μL. The results are shown in Figure 1 shown.

[0064] 2.3. Experimental results and analysis The total yield of squalene produced by the starting strain CEN.PK2-1D is reported to be 0.34 ± 0.10 mg / L (Reference [1]). Figure 1 As shown in Table 1, squalene was successfully detected in HPSS1, HPSS2, HPSE1, HPSE2 and HPSC2 by HPLC. The total yield of HPSS1 was 9.79 ± 0.93 mg / L, the total yield of HPSS2 was 1241.37 ± 88.02 mg / L, the total yield of HPSE1 was 12.23 ± 3.18 mg / L, the total yield of HPSE2 was 986.31 ± 35.77 mg / L, and the total yield of HPSC2 was 1061.96 ± 113.77 mg / L. The total yield of squalene in HPSS2 was 13.84% higher than that in HCS1 and 16.89% higher than that in HPSC2, indicating that compared with the endogenous squalene synthesis pathway in yeast, Sarocladium oryzae The introduction of the squalene synthesis pathway can significantly increase the total production of squalene and provide a new idea for the industrial production of squalene cell factories.

[0065] The squalene production per unit dry weight of the cells was calculated as follows: total squalene production per 1 ml culture / 1 ml cell dry weight. The results are shown in Table 1.

[0066] Table 1 Total squalene production and squalene production per unit dry weight of HCS1, HPSS2, HPSE2 and HPSC2

[0067] It can be seen from Table 1 that compared with HPSS2, HCS1 and HPSC2, the total yield of squalene in HPSE2 is lower than that of the other three, but the yield of squalene in HPSE2 per unit dry weight of cells is higher than them, which is 318.31±66.14 mg / g cdw. When the technicians in this field increase the cell concentration of HPSE2 by genetic engineering, Stibella The introduction of squalene synthesis pathway in sp. 1056 will also become an important means to increase the production of squalene in yeast.

[0068] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary 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 yeast, 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.

2. The method for constructing a recombinant strain for producing squalene according to claim 1, characterized in that: 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.

3. The method for constructing a recombinant strain for producing squalene according to claim 2, characterized in that: The yeast is Saccharomyces cerevisiae.

4. The method for constructing a recombinant strain for producing squalene according to claim 3, characterized in that: The brewer's yeast is the auxotrophic brewer's yeast CEN.PK2-1D.

5. The method for constructing a recombinant strain for producing squalene according to claim 2, 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.

6. 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.

7. A recombinant strain for producing squalene, characterized in that: The method is constructed by any one of claims 1 to 6.

8. Use of the recombinant strain according to claim 7 in producing squalene.

9. A method for producing squalene, characterized in that: The method comprises fermenting the recombinant strain according to claim 7 to obtain squalene from the fermentation liquid.

10. A method for producing squalene according to claim 9, 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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