CN117402763B - Saccharomyces cerevisiae engineering strain for producing squalene, construction method and application thereof - Google Patents

Saccharomyces cerevisiae engineering strain for producing squalene, construction method and application thereof Download PDF

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CN117402763B
CN117402763B CN202311257635.5A CN202311257635A CN117402763B CN 117402763 B CN117402763 B CN 117402763B CN 202311257635 A CN202311257635 A CN 202311257635A CN 117402763 B CN117402763 B CN 117402763B
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靳红磊
王宏斌
林晓娜
安天悦
傅丹妮
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Guangzhou University of Traditional Chinese Medicine
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Abstract

The invention provides a saccharomyces cerevisiae engineering strain for producing squalene, and a construction method and application thereof, and belongs to the technical field of metabolic engineering. A saccharomyces cerevisiae engineering strain XN01 for producing squalene integrates a HIF-1 alpha gene and an ARNT gene in a saccharomyces cerevisiae genome. The present invention also integrates tHMGR and UPC2-1 genes into strain XN01 to form strain XN02, and further uses P based on strain XN02 PGK1 The promoter replaces the promoter of the ERG9 gene in the Saccharomyces cerevisiae genome to form strain XN03. The invention is based on the expression level of two subunit encoding genes HIF-1 alpha and ARNT of the over-expressed tumor cell hypoxia inducible factor HIF-1 complex, over-expresses tHMGR and UPC2-1 and improves the expression of ERG9 genes, and can greatly improve the yield of squalene of saccharomyces cerevisiae.

Description

Saccharomyces cerevisiae engineering strain for producing squalene, construction method and application thereof
Technical Field
The invention belongs to the technical field of metabolic engineering, and particularly relates to a saccharomyces cerevisiae engineering strain for producing squalene, and a construction method and application thereof.
Background
The natural product of triterpenes of medicinal plants has extremely high medicinal value, is an important source for developing new medicines, such as lupeane pentacyclic triterpene lupeol and derivatives thereof (such as betulinic acid and the like) have excellent curative effect in treating cancers (Hsu MJ, pengsF, chueh FS, et al Lupeol suppresses migration and invasion via p/MAPK and PI3K/Akt signaling pathways in humanoXNeosarcoma U-2 OS cells.Bioscience Biotechnology and BiochemiXNry,2019, 83:1729-1739). However, the compounds have low content in plants and are needed to be synthesized by other ways.
For example, squalene is currently sold mainly extracted from shark liver, which is also one of the reasons for excessive fishing of sharks. However, squalene extracted from shark liver cannot completely eliminate sanitary problems, and the shark may be infected by pathogens and may have harmful effects on human body. While microalgae produce squalene under heterotrophic conditions, the quality of the production can meet the demands of food, cosmetics and medical fields in Europe, the microalgae produce squalene and also produce other kinds of lipid substances, such as docosahexaenoic acid (DHA), in large quantities, which undoubtedly brings about tedious operations for the subsequent separation and utilization of squalene.
In view of the above problems, the ability to produce squalene from a microbial point of view, for example, using Saccharomyces cerevisiae, is well known, but the yield is very low, approximately 0.041mg/g biomass. In order to further increase the yield of squalene produced by yeast engineering bacteria, the induction of biosynthesis of squalene and inhibition of the catabolic effects of squalene have been achieved, for example, publication No. WO2010/023551 discloses that there are many genes involved in squalene synthesis, including mevalonate kinase, phosphomevalonate kinase, pyrophosphatase decarboxylase, isopentenyl pyrophosphate isomerase, HMGR and squalene synthase. However, the currently developed saccharomyces cerevisiae engineering bacteria still cannot meet the higher demand of squalene on the market in terms of squalene yield.
Disclosure of Invention
Accordingly, the present invention is directed to a squalene-producing saccharomyces cerevisiae engineering strain, wherein hypoxia inducible factor HIF-1 related genes in human tumor cells are integrated into the saccharomyces cerevisiae engineering strain, and mevalonate pathway is promoted by regulating acetyl-coa production in glycolytic pathway, and meanwhile, squalene synthesis related genes are overexpressed to increase squalene yield.
The invention provides a saccharomyces cerevisiae engineering strain XN01 for producing squalene, wherein a saccharomyces cerevisiae genome is integrated with a HIF-1 alpha gene and an ARNT gene.
The invention provides a saccharomyces cerevisiae engineering strain XN02 for producing squalene, wherein tHMGR and UPC2-1 genes are overexpressed in a genome of the saccharomyces cerevisiae engineering strain XN 01.
The invention provides a saccharomyces cerevisiae engineering strain XN03 for producing squalene, which is based on the saccharomyces cerevisiae engineering strain XN02, and replaces an endogenous promoter of an ERG9 gene with P PGK1 A promoter.
Preferably, the Saccharomyces cerevisiae genome is also integrated with a selectable marker gene;
the screening marker genes include URA, his and MET marker genes:
preferably, the nucleotide sequence of the HIF-1 alpha gene is shown in SEQ ID NO:1 is shown in the specification;
The nucleotide sequence of the ARNT gene is shown as SEQ ID NO:2 is shown in the figure;
the nucleotide sequence of tHMGR is shown as SEQ ID NO:3 is shown in the figure;
the nucleotide sequence of the UPC2-1 gene is shown as SEQ ID NO:5 is shown in the figure;
the P is PGK1 The nucleotide sequence of the promoter is shown in SEQ ID NO:6 is shown in the figure;
preferably, the original strain of the saccharomyces cerevisiae engineering strain XN03 is a saccharomyces cerevisiae BY4741 strain.
The invention provides a construction method of a squalene-producing saccharomyces cerevisiae engineering strain XN03, which comprises the following steps:
construction of Gene expression Module P PGK1 -HIF-1α-T ADH1 And P TEF1 -ARNT-T CYC1 ；
Gene expression Module P PGK1 -HIF-1α-T ADH1 、P TEF1 -ARNT-T CYC1 And first selection marker construction Gene expression Module first selection marker-P PGK1 -HIF-1α-T ADH1 -P TEF1 -ARNT-T CYC1 ；
Construction of Gene expression Module P GAL1 -tHMGR-T ADH1 And P GAL1 0-UPC2-1-T CYC1 ；
Gene expression Module P GAL1 -tHMGR-T ADH1 、P GAL10 -UPC2-1-T CYC1 And second selectable marker construction Gene expression Module second selectable marker-P GAL1 -tHMGR-T ADH1 -P GAL10 -UPC2-1-T CYC1 ；
Promoter P PGK1 And third selectable marker construction Gene expression Module third selectable marker-P PGK1 ；
First screening marker of Gene expression Module-P PGK1 -HIF-1α-T ADH1 -P TEF1 -ARNT-T CYC1 Integrating into a saccharomyces cerevisiae genome to obtain a strain XN01;
second screening marker-P GAL1 -tHMGR-T ADH1 -P GAL10 -UPC2-1-T CYC1 Integration into the genome of strain XN01 gives strain XN02:
labeling the third screening marker L-P PGK1 The saccharomyces cerevisiae engineering strain XN03 is obtained by homologous recombination to replace an endogenous promoter of an ERG9 gene in a genome of the strain XN 02.
Preferably, promoter P PGK1 、P TEF1 、P GAL1 And P GAL10 The nucleotide sequence of (2) is shown as SEQ ID NO: 6-SEQ ID NO: shown as 9;
preferably T ADH1 And T CYC1 The nucleotide sequence of the terminator is shown in SEQ ID NO:10 to SEQ IDNO: 11;
preferably, the first, second and third selectable markers are different markers from one of the URA, his and MET marker genes;
preferably, the nucleotide sequence of the URA marker gene is shown in SEQ ID NO: shown at 12;
the nucleotide sequence of the His tag gene is shown in SEQ ID NO: 13;
the nucleotide sequence of the MET marker gene is shown in SEQ ID NO: 14.
Preferably, the gene expression module P PGK1 -HIF-1α-T ADH1 The HIF-1 alpha amplification primer comprises a nucleotide sequence shown in SEQ ID NO:33 and the nucleotide sequence of the forward primer is shown in SEQ ID NO:34, a reverse primer shown in FIG. 34; p (P) PGK1 The amplification primer comprises a nucleotide sequence shown as SEQ ID NO:35 and the nucleotide sequence of the forward primer is shown as SEQ ID NO:36, a reverse primer shown in FIG. 36; t (T) ADH1 The amplification primer comprises a nucleotide sequence shown as SEQ ID NO:21 and the nucleotide sequence of the forward primer is shown as SEQ ID NO:22, a reverse primer shown in FIG. 22; p (P) PGK1 -HIF-1α-T ADH1 The amplification primer comprises a nucleotide sequence shown as SEQ ID NO:35 and the nucleotide sequence of the forward primer is shown as SEQ ID NO:22, a reverse primer shown in FIG. 22;
The gene expression module P TEF1 -ARNT-T CYC1 The ARNT amplification primer comprises a nucleotide sequence shown in SEQ ID NO:37 and the nucleotide sequence of the forward primer is shown in SEQ ID NO:38, a reverse primer shown in seq id no; p (P) TEF1 The amplification primer comprises a nucleotide sequence shown as SEQ ID NO:39 and a reverse primer having a nucleotide sequence shown in SEQ ID NO: 40; t (T) CYC1 The amplification primer comprises a nucleotide sequence shown as SEQ ID NO:29 and the nucleotide sequence of the forward primer is shown as SEQ ID NO:30, a reverse primer shown in FIG. 30; p (P) TEF1 -ARNT-T CYC1 The amplification primer comprises a nucleotide sequence shown as SEQ ID NO:39 and the nucleotide sequence of the forward primer is shown as SEQ ID NO:30, a reverse primer shown in FIG. 30;
the gene expression module P GAL1 -tHMGR-T ADH1 Middle tHMGR amplification primer with nucleotide sequence shown as SEQ IDThe forward primer and the nucleotide sequence shown in SEQ ID NO. 41: 42; p (P) GAL1 The amplification primer comprises a nucleotide sequence shown as SEQ ID NO:21 and the nucleotide sequence of the forward primer is shown as SEQ ID NO:22, a reverse primer shown in FIG. 22; t (T) ADH1 The amplification primer comprises a nucleotide sequence shown as SEQ ID NO:23 and the nucleotide sequence of the forward primer is shown as SEQ ID NO:24, a reverse primer shown in FIG. 24; gene expression module P GAL1 -tHMGR-T ADH1 The amplification primer comprises a nucleotide sequence shown as SEQ ID NO:21 and the nucleotide sequence of the forward primer is shown as SEQ ID NO:24, a reverse primer shown in FIG. 24;
The gene expression module P GAL10 -UPC2-1-T CYC1 The UPC2-1 amplification primer comprises a nucleotide sequence shown in SEQ ID NO:25 and the nucleotide sequence of the forward primer is shown in SEQ ID NO:26, a reverse primer shown in seq id no; p (P) GAL10 The amplification primer comprises a forward primer with a nucleotide sequence shown as SEQ ID NO:27 and a forward primer with a nucleotide sequence shown as SEQ ID NO:28, a reverse primer shown in FIG. 28; t (T) CYC1 The amplification primer comprises a nucleotide sequence shown as SEQ ID NO:29 and the nucleotide sequence of the forward primer is shown as SEQ ID NO:30, a reverse primer shown in FIG. 30; gene expression module P GAL1 0-UPC2-1-T CYC1 The amplification primer comprises a nucleotide sequence shown as SEQ ID NO:27 and the nucleotide sequence of the forward primer is shown in SEQ ID NO:30, a reverse primer shown in FIG. 30;
promoter P PGK1 The amplification primer comprises a nucleotide sequence shown as SEQ ID NO:31 and the nucleotide sequence of the forward primer is shown as SEQ ID NO:32, a reverse primer shown in FIG. 32;
the URA marker gene amplification primer comprises a nucleotide sequence shown as SEQ ID NO:15 and the nucleotide sequence of the forward primer is shown as SEQ ID NO:16, a reverse primer shown in FIG. 16;
the His tag gene amplification primer comprises a nucleotide sequence shown as SEQ ID NO:17 and the nucleotide sequence of the forward primer is shown as SEQ ID NO:18, a reverse primer shown in FIG. 18;
the MET marker gene amplification primer comprises a nucleotide sequence shown as SEQ ID NO:19 and the nucleotide sequence of the forward primer is shown as SEQ ID NO: 20.
Preferably, the gene expression module first selection marker-P PGK1 -HIF-1α-T ADH1 -P TEF1 -ARNT-T CYC1 An NDT80 site integrated into the saccharomyces cerevisiae genome;
second selectable marker-P GAL1 -tHMGR-T ADH1 -P GAL10 -UPC2-1-T CYC1 Integration into the Saccharomyces cerevisiae genome at GAL80 site.
The invention provides an application of the saccharomyces cerevisiae engineering strain XN01, the saccharomyces cerevisiae engineering strain XN02, the saccharomyces cerevisiae engineering strain XN03 or the saccharomyces cerevisiae engineering strain XN03 obtained by the construction method in production of squalene.
Preferably, the method for producing squalene comprises the following steps:
inoculating bacterial liquids of saccharomyces cerevisiae engineering bacteria strains XN01, XN02 and XN03 into a fermentation medium for shake culture;
the rotation speed of the shake culture is 200-250 rpm, and the temperature of the shake culture is 28-32 ℃; the shake culture time is 165-175 hours;
the fermentation medium is YPD liquid medium;
the preparation method of the bacterial liquid of the saccharomyces cerevisiae engineering bacterial strains XN01, XN02 and XN03 comprises the steps of inoculating the saccharomyces cerevisiae engineering bacterial strains XN03 to a screening solid culture medium for activation culture, picking up monoclonal and inoculating to a liquid screening culture medium for seed liquid shake culture;
the temperature of the activation culture and seed liquid shake culture is 28-32 ℃;
The screening solid culture medium is SD+URA+HIS+MET solid culture medium; the liquid screening culture medium is SD+URA+HIS+MET liquid culture medium.
The invention provides a saccharomyces cerevisiae engineering strain XN01 for producing squalene, wherein a saccharomyces cerevisiae genome is integrated with a HIF-1 alpha gene and an ARNT gene. The invention integrates the genes of HIF-1 alpha and ARNT in human tumor cell coded hypoxia inducible factor HIF-1 complex into saccharomyces cerevisiae genome, and improves the generation of acetyl coenzyme A by positively regulating the expression of enzyme genes in glycolysis pathway, and acetyl coenzyme A is used as a raw material for synthesizing mevalonate pathway to promote the synthesis of mevalonate; mevalonic acid is used as a precursor for squalene synthesis, and the content of mevalonic acid is increased to enable metabolic flux to promote the synthesis direction of squalene. Therefore, the invention adjusts the synthesis condition of related intermediate substances in squalene metabolic pathways by utilizing a genetic engineering means, thereby constructing a saccharomyces cerevisiae engineering strain capable of producing squalene in high yield and realizing mass synthesis of squalene. Experiments show that compared with the original strain Saccharomyces cerevisiae BY4741, the strain XN01 provided BY the invention has great progress in the aspect of producing squalene, so that the yield of squalene is improved BY 2.5 times. It can be seen that the strain XN01 provided by the invention provides a basis for the synthesis of triterpenes, as the cancer treatment provides a powerful postshield.
The invention provides a saccharomyces cerevisiae engineering strain XN02 for producing squalene, wherein tHMGR and UPC2-1 genes are overexpressed in a genome of the saccharomyces cerevisiae engineering strain XN 01. Based on the strain XN01, the tHMGR gene and the UPC2-1 modified gene UPC2 are integrated into the saccharomyces cerevisiae genome, so that the synthesis of mevalonate is further improved. The increase in mevalonate content as a precursor for squalene synthesis and the upregulation of squalene synthase genes allow metabolic flux to promote squalene synthesis. Therefore, the invention adjusts the synthesis condition of related intermediate substances and the expression condition of related enzymes in squalene metabolic pathways by utilizing a genetic engineering means, thereby constructing a saccharomyces cerevisiae engineering strain capable of producing squalene in high yield and realizing mass synthesis of squalene. Experiments show that compared with the original strain Saccharomyces cerevisiae BY4741, the strain XN02 provided BY the invention has great progress in the aspect of producing squalene, so that the yield of squalene is improved BY 26.5 times, and compared with the strain XN01, the yield of squalene produced BY the strain XN02 is improved BY 9.8 times. It can be seen that the strain XN02 provided by the invention provides a basis for the synthesis of triterpenes, as cancer treatment provides a powerful postshield.
The invention also provides a saccharomyces cerevisiae engineering strain XN03 for producing squalene, and the endogenous of ERG9 genes is carried out on the basis of the saccharomyces cerevisiae engineering strain XN02Substitution of promoter with P PGK1 A promoter. On the basis of the strain XN02, the expression level of squalene synthase gene (ERG 9 gene) is also improved, and the yield of squalene is further improved. Experiments show that compared with the original strain Saccharomyces cerevisiae BY4741, the strain XN03 provided BY the invention has great progress in the aspect of producing squalene, and the yield of squalene is improved BY 45.2 times. The yield of squalene produced by the strain XN03 is improved by 16.7 times compared with the strain XN01, and the yield of squalene produced by the strain XN03 is improved by 1.7 times compared with the strain XN 02.
Drawings
FIG. 1 shows the GC-MS detection results of squalene in a Saccharomyces cerevisiae engineering strain XN01 constructed by the invention;
FIG. 2 is a mass spectrum diagram of squalene in Saccharomyces cerevisiae engineering strain XN01 constructed by the invention;
FIG. 3 shows the yield of squalene in Saccharomyces cerevisiae engineering strain XN01 constructed according to the present invention;
FIG. 4 shows the yields of squalene in Saccharomyces cerevisiae engineering strains XN02 and XN03 constructed by the invention.
Detailed Description
The invention provides a saccharomyces cerevisiae engineering strain XN01 for producing squalene, wherein a saccharomyces cerevisiae genome is integrated with a HIF-1 alpha gene and an ARNT gene.
In the invention, the HIF-1 alpha and ARNT genes are genes encoding two subunits of the hypoxia inducible factor HIF-1 alpha and ARNT in human tumor cells, and the two genes are integrated into the saccharomyces cerevisiae genome to facilitate the over-expression of the HIF-1, thereby promoting the glycolytic pathway and the generation of acetyl coenzyme A. The acetyl coenzyme A is used as a raw material of a path for synthesizing mevalonate by using a triterpene compound, thereby providing a basis for metabolic path to facilitate the synthesis direction of squalene. The nucleotide sequence of the HIF-1 alpha gene is preferably shown in SEQ ID NO: 1. The nucleotide sequence of the ARNT gene is shown as SEQ ID NO: 2. The HIF-1. Alpha. Gene and the ARNT gene form a fusion gene that is integrated into the NDT80 site of the genome of Saccharomyces cerevisiae.
In the present invention, the s.cerevisiae genome is preferably further integrated with a selectable marker gene; the screening marker gene comprises one of URA, his and MET marker genes. The original strain of the saccharomyces cerevisiae engineering strain XN01 is a saccharomyces cerevisiae BY4741 strain.
The invention provides a saccharomyces cerevisiae engineering strain XN02 for producing squalene, wherein tHMGR and UPC2-1 genes are overexpressed in a genome of the saccharomyces cerevisiae engineering strain XN 01.
In the present invention, tHMGR and UPC2-1 genes are key enzyme coding genes in mevalonate pathway, and the expression of these two genes is improved to increase the activity of the corresponding two enzymes, thereby promoting the synthesis of mevalonate. The nucleotide sequence of tHMGR is shown as SEQ ID NO:3 is shown in the figure; the nucleotide sequence of the UPC2-1 gene is shown as SEQ ID NO: shown at 5. The UPC2-1 gene is obtained by mutating a transcription factor gene UPC2 by a PCR-based site-directed mutagenesis technology, and the nucleotide sequence of the transcription factor gene UPC2 is shown as SEQ ID NO: 4. The present invention integrates tHMGR and UPC2-1 gene into GAL80 locus of Saccharomyces cerevisiae genome. In the present invention, the s.cerevisiae genome is preferably further integrated with a selectable marker gene; the screening marker genes include two of URA, his and MET marker genes.
The invention provides a saccharomyces cerevisiae engineering strain XN03 for producing squalene, which is based on the saccharomyces cerevisiae engineering strain XN02, and replaces an endogenous promoter of an ERG9 gene with P PGK1 A promoter.
In the present invention, a strong promoter P is used PGK1 Replacement of the endogenous promoter of the ERG9 gene is beneficial to improving the expression of the ERG9 gene. The overexpression of the ERG9 gene is beneficial to improving the enzyme activity of squalene synthase and further promoting the synthesis of squalene. The P is PGK1 The nucleotide sequence of the promoter is shown in SEQ ID NO: shown at 6.
In the present invention, the integration in the Saccharomyces cerevisiae genome preferably also incorporates a selectable marker gene. To verify whether each fusion gene or strong promoter was successfully integrated into the s.cerevisiae genome, a selection marker gene was inserted into each of the three exogenous gene fragments. The screening marker genes include URA, his and MET marker genes. The nucleotide sequence of the URA marker gene is preferably as set forth in SEQ ID NO: shown at 12; the nucleotide sequence of the His tag gene is preferably as set forth in SEQ ID NO: 13; the nucleotide sequence of the MET marker gene is preferably set forth in SEQ ID NO:14, screening of positive strains was achieved by culturing corresponding auxotrophs against the recombinant strain at each stage. The starting strain of the Saccharomyces cerevisiae strain is not particularly limited, and starting strains known in the art may be used.
The invention provides a construction method of a squalene-producing saccharomyces cerevisiae engineering strain XN03, which comprises the following steps:
construction of Gene expression Module P PGK1 -HIF-1α-T ADH1 And P TEF1 -ARNT-T CYC1 ；
Gene expression Module P PGK1 -HIF-1α-T ADH1 、P TEF1 -ARNT-T CYC1 And first selection marker construction Gene expression Module first selection marker-P PGK1 -HIF-1α-T ADH1 -P TEF1 -ARNT-T CYC1 ；
Construction of Gene expression Module P GAL1 -tHMGR-T ADH1 And P GAL10 -UPC2-1-T CYC1 ；
Gene expression Module P GAL1 -tHMGR-T ADH1 、P GAL10 -UPC2-1-T CYC1 And second selectable marker construction Gene expression Module second selectable marker-P GAL1 -tHMGR-T ADH1 -P GAL10 -UPC2-1-T CYC1 ；
Promoter P PGK1 And third selectable marker construction Gene expression Module third selectable marker-P PGK1 ；
First screening marker of Gene expression Module-P PGK1 -HIF-1α-T ADH1 -P TEF1 -ARNT-T CYC1 Integrating into a saccharomyces cerevisiae genome to obtain a strain XN01;
second screening marker-P GAL1 -tHMGR-T ADH1 -P GAL10 -UPC2-1-T CYC1 Integration into the genome of strain XN01 gives strain XN02:
third selectable marker-P PGK1 Substitution of the group of Strain XN02 by homologous recombinationThe endogenous promoter of ERG9 gene in the genome is used to obtain Saccharomyces cerevisiae engineering strain XN03.
In the invention, the construction of each gene expression module is realized by adopting an overlap PCR amplification method. The four genes are respectively expressed by adopting four different promoters, and the expression is stopped by adopting two terminators. Promoter P PGK1 、P TEF1 、P GAL1 And P GAL10 Preferably the nucleotide sequence of (a) is as set forth in SEQ ID NO: 6-SEQ ID NO: shown at 9. Terminator T ADH1 And T CYC1 The nucleotide sequence of the terminator is preferably shown in SEQ ID NO:10-SEQ ID NO: 11. The first, second and third selectable markers are preferably one different marker of the URA, his and MET marker genes.
In the present invention, the gene expression module P PGK1 -HIF-1α-T ADH1 The HIF-1. Alpha. Amplification primers preferably comprise a nucleotide sequence as set forth in SEQ ID NO:33 and a reverse primer having a nucleotide sequence shown in SEQ ID NO: 34; p (P) PG The K1 amplification primer preferably comprises a nucleotide sequence as set forth in SEQ ID NO:35 and the nucleotide sequence of the forward primer is shown as SEQ ID NO:36, a reverse primer shown in FIG. 36; t (T) ADH1 The amplification primer preferably comprises a nucleotide sequence as set forth in SEQ ID NO:21 and the nucleotide sequence of the forward primer is shown as SEQ ID NO:22, a reverse primer shown in FIG. 22; p (P) PGK1 -HIF-1α-T ADH1 The amplification primer preferably comprises a nucleotide sequence as set forth in SEQ ID NO:35 and the nucleotide sequence of the forward primer is shown as SEQ ID NO: 22.
In the present invention, the gene expression module P TEF1 -ARNT-T CYC1 The ARNT amplification primers preferably include a forward primer having a nucleotide sequence set forth in SEQ ID NO: 37 and a forward primer having a nucleotide sequence set forth in SEQ ID NO:38, a reverse primer shown in seq id no; p (P) TEF1 The amplification primer preferably comprises a nucleotide sequence as set forth in SEQ ID NO:39 and the nucleotide sequence of the forward primer is shown as SEQ ID NO: 40. T (T) CYC1 The amplification primer preferably comprises a nucleotide sequence as set forth in SEQ ID NO:29 and the nucleotide sequence of the forward primer is shown as SEQ ID NO:30, a reverse primer shown in FIG. 30; p (P) TEF1 -ARNT-T CYC 1 spreadThe amplification primer preferably comprises a nucleotide sequence as set forth in SEQ ID NO:39 and the nucleotide sequence of the forward primer is shown as SEQ ID NO: 30.
In the present invention, the gene expression module P GAL1 -tHMGR-T ADH1 The primer for amplifying the medium tHMGR comprises a nucleotide sequence shown in SEQ ID NO:41 and the nucleotide sequence of the forward primer is shown as SEQ ID NO: 42; p (P) GAL1 The amplification primer comprises a nucleotide sequence shown as SEQ ID NO:21 and a reverse primer with a nucleotide sequence shown as SEQ ID NO: 22; t (T) ADH1 The amplification primer comprises a nucleotide sequence shown as SEQ ID NO:23 and the nucleotide sequence of the forward primer is shown as SEQ ID NO:24, a reverse primer shown in FIG. 24; gene expression module P GAL1 -tHMGR-T ADH1 The amplification primer comprises a nucleotide sequence shown as SEQ ID NO:21 and the nucleotide sequence of the forward primer is shown as SEQ ID NO:24, a reverse primer shown in FIG. 24;
in the present invention, the gene expression module P GAL10 -UPC2-1-T CYC1 The UPC2-1 amplification primer comprises a forward primer with a nucleotide sequence shown as SEQ ID NO: 25 and a reverse primer with a nucleotide sequence shown as SEQ ID NO: 26; p (P) GAL10 The amplification primer comprises a nucleotide sequence shown as SEQ ID NO:27 and the nucleotide sequence of the forward primer is shown in SEQ ID NO:28, a reverse primer shown in FIG. 28; t (T) CYC1 The amplification primer comprises a nucleotide sequence shown as SEQ ID NO:29 and the nucleotide sequence of the forward primer is shown as SEQ ID NO:30, a reverse primer shown in FIG. 30; gene expression module P GAL10 -UPC2-1-T CYC1 The amplification primer comprises a nucleotide sequence shown as SEQ ID NO:27 and the nucleotide sequence of the forward primer is shown in SEQ ID NO:30, a reverse primer shown in FIG. 30;
in the present invention, the gene expression module is a third selectable marker-P PGK1 Promoter P in (E) PGK1 The amplification primer comprises a nucleotide sequence shown as SEQ ID NO:31 and the nucleotide sequence of the forward primer is shown as SEQ ID NO: 32. The URA marker gene amplification primer comprises a nucleotide sequence shown as SEQ ID NO:15 and the nucleotide sequence of the forward primer is shown as SEQ ID NO: 16. His-tagged gene amplification primersComprising nucleotide sequences shown in SEQ ID NO:17 and the nucleotide sequence of the forward primer is shown as SEQ ID NO: 18. The MET marker gene amplification primer comprises a nucleotide sequence shown as SEQ ID NO:19 and the nucleotide sequence of the forward primer is shown as SEQ ID NO: 20.
In the invention, a first screening marker-PPGK 1-HIF-1 alpha-TADH 1-PTEF1-ARNT-TCYC1 of a gene expression module is integrated into a saccharomyces cerevisiae genome to obtain a strain XN01; integrating a second screening marker-PGAL 1-tHMGR-TADH1-PGAL10-UPC2-1-TCYC1 into the genome of the strain XN01 to obtain a strain XN02; and replacing the endogenous promoter of the ERG9 gene in the genome of the strain XN02 by homologous recombination to obtain the saccharomyces cerevisiae engineering strain XN03 by a third screening marker PPGK 1. The method of integration is not particularly limited by the present invention and can be accomplished by any of the methods of integration known in the art, such as lithium acetate conversion.
The invention provides an application of the saccharomyces cerevisiae engineering strain XN03 or the saccharomyces cerevisiae engineering strain XN03 obtained by the construction method in production of squalene.
In the present invention, the process for producing squalene preferably comprises the steps of:
inoculating the bacterial liquid of the saccharomyces cerevisiae engineering bacterial strain XN03 into a fermentation culture medium for shake culture.
The preparation method of the bacterial liquid of the saccharomyces cerevisiae engineering bacterial strain XN03 comprises the steps of inoculating the saccharomyces cerevisiae engineering bacterial strain XN03 to a screening solid culture medium for activation culture, and selecting a monoclonal to be inoculated to a liquid screening culture medium for seed liquid shake culture. The temperature of the activation culture and the seed liquid shake culture is preferably 28 to 32 ℃, more preferably 30 ℃. The screening solid culture medium is SD+URA+HIS+MET solid culture medium; the liquid screening culture medium is SD+URA+HIS+MET liquid culture medium. The SD+URA+HIS+MET solid culture medium or the SD+URA+HIS+MET liquid culture medium is a uracil, histidine and methionine defect culture medium which does not contain agar or contains agar.
In the present invention, the rotational speed of the shake culture is preferably 200 to 250rpm, more preferably 220rpm. The temperature of the shake culture is preferably 28 to 32℃and more preferably 30 ℃. The time of the shake culture is preferably 165 to 175 hours, more preferably 168 hours. The fermentation medium is YPD liquid medium.
In the embodiment of the invention, the GC-MS is utilized to detect the content of the synthesized squalene, and the result shows that compared with the original strain Saccharomyces cerevisiae BY4741, the strain XN03 provided BY the invention has great progress in the aspect of producing squalene, so that the yield of squalene is improved BY 45.2 times.
The invention provides a squalene-producing saccharomyces cerevisiae engineering strain, a construction method and application thereof, which are described in detail below with reference to examples, but are not to be construed as limiting the scope of the invention.
The formula of the culture medium related in the embodiment of the invention is as follows:
(1) YPD medium: peptone 20g/L, yeast extract 10g/L, glucose 20g/L (solid YPD medium was added with 20g/L agar powder at the time of preparation).
(2) SD-URA medium: HIS20mg/L, MET 20mg/L and LEU 60mg/L (20 g/L agar powder was added in the preparation of solid medium) were added on the basis of SD medium.
TABLE 1SD medium
Wherein the 100×tetramissing amino acid mother liquor is as follows: arginine 0.12g, aspartic acid 0.6g, glutamic acid 0.6g, lysine 0.18g, phenylalanine 0.3g, serine 2.25g, threonine 1.2g, tryptophan 0.24g, tyrosine 0.18g, valine 0.9g, distilled water to 57mL, optionally adding any amino acid to prepare defective amino acid mother liquor (100X). The above raw materials were purchased from the division of biological engineering (Shanghai).
The preparation method comprises the following steps:
YNB, ammonium sulfate and 100X tetra-amino acid (10 mL) are mixed and then fixed in volume to 900mL, each conical flask is divided into 45mL, 1mL of each conical flask is added with 50X H/L/M, 5mL of glucose is added according to the requirement, and water is added to 50mL;
glucose and galactose were sterilized separately and 100mL was prepared.
(3) SD-URA-HIS medium: MET 20mg/L and LEU 60mg/L (20 g/L agar powder was added at the time of preparation of solid medium) were added on the basis of SD medium.
(4) SD-URA-HIS-MET medium: MET 20mg/L and LEU 60mg/L (20 g/L agar powder when solid medium is prepared) were added to the SD medium (20 g/L agar powder when solid medium is prepared).
The primers and sequences thereof involved in the examples of the present invention are shown in tables 2 and 3.
TABLE 2 primer sequences for cloning of genes and expression elements in the present invention
Example 1
Cloning of Saccharomyces cerevisiae Gene and regulatory elements
1. Extraction of Yeast genome
(1) The monoclonal Saccharomyces cerevisiae BY4741 was picked up in 5mL YPD liquid medium, cultured overnight at 30℃at 220rpm, and then centrifuged at 3000rpm for 2min for bacterial collection.
(2) The collected thalli are subjected to extraction of genome DNA by using a yeast genome DNA rapid extraction kit (B518227, manufactured and bioengineered Co., ltd.) and the specific operation process is shown in the specification.
2. Cloning of Yeast endogenous genes and expression elements
(1) Cloning of tHMG1 and UPC2 genes, P, respectively, using the yeast genome obtained as described above as a template PGK1 、P TEF1 、P GAL10 And P GAL1 Four promoters and T ADH1 And T CYC1 Two terminators, the primers for cloning each element were as follows:
gene tHMG1 (tHMG 1-F, tHMG 1-R), gene UPC2 (UPC 2-F, UPC 2-R), promoter P PGK1 (primer P) PGK1 -F、P PGK1 -R), promoter P TEF1 (primer P) TEF1 -F、P TEF1 -R), promoter P GAL10 (primer P) GAL10 -F、P GAL10 -R)、P GAL1 Promoter (primer P) GAL1 -F、P GAL1 -R) terminator T ADH1 (primer T) ADH1 -F、T ADH1 -R) terminator T CYC1 (primer T) CYC1 -F、T CYC1 -R), the specific sequences of the primers are shown in Table 2.
PCR reaction system: primeSTAR Max (R045Q, takara Shuzo) 10. Mu.L, forward and reverse primers each 0.5. Mu.L, ddH 2 O8. Mu.L, template 1. Mu.L, total reaction system 20. Mu.L.
The PCR amplification reaction conditions were: 98 ℃ for 2min;98℃15s,60℃15s,72℃30s-2min,32 cycles; 7min at 72 ℃.
After the PCR reaction is finished, 1% agarose gel electrophoresis is used for detection, after the size of the band is correct, a target band is cut off, and a DNA gel recovery kit (DP 209-03, tiangen Biochemical technology Co., ltd.) is used for gel recovery, and the specific operation process is shown in the specification.
(2) The DNA fragment was ligated to subclone vector pEASY vector.
The DNA fragment was ligated with subclone vector pEASY (CB 111-01, beijing full gold Biotechnology Co., ltd.) and then competent E.coli DH 5. Alpha. Was transformed, see the product specification for specific procedures.
(3) After overnight incubation at 37℃single colonies were selected for colony PCR.
PCR reaction system: green Taq Mix (P131-01, vazyme) 5. Mu.L, 100mM forward and reverse primers each 0.3. Mu.L, ddH 2 O4.4μL。
The PCR amplification conditions were: 94 ℃ for 2min; 15s at 94 ℃, 15s at 50-60 ℃, 1-3 min at 72 ℃ and 32 cycles; 7min at 72 ℃.
After the PCR reaction is finished, agarose gel electrophoresis detection is carried out, and positive clone strains are selected for sequencing.
3. Site-directed mutagenesis of UPC2
UPC2 is a forward regulator of mevalonate pathway, and mutation of glycine at 888 to aspartic acid can further improve its regulation. The PCR method is used, the pEASY-UPC2 plasmid is used as a template, the site-directed mutagenesis of UPC2 is carried out, and the amplification primer sequences are shown in Table 2.
(1) PCR reaction system: primeSTAR Max (R045Q, takara Shuzo) 10. Mu.L, forward and reverse primers each 0.5. Mu.L, ddH 2 O8. Mu.L, template 1. Mu.L, total reaction system 20. Mu.L.
(2) The PCR amplification reaction conditions were: 98 ℃ for 2min; circulating at 98 ℃ for 15s,60 ℃ for 15s,72 ℃ for 30s-2min and 32; 7min at 72 ℃.
(3) 10. Mu.L of the above PCR product was taken, and 0.5. Mu.L of restriction enzyme Dpn I was added thereto for reaction at 37℃for 1 hour.
(4) The reaction system was transformed into competent E.coli DH 5. Alpha. And plated on overnight at 37 ℃.
(5) Monoclonal was selected for colony PCR identification and positive colonies were sequenced.
Example 2
Construction method of gene expression module
Constructing a gene expression module I to a gene expression module V by utilizing overlapping PCR:
(1) First round overlapping PCR reaction system: PCR reaction system: primeSTARMax (R045Q, takara Shuzo) 10. Mu.L, forward and reverse primers each 0.5. Mu.L, ddH 2 O8. Mu.L, template 1. Mu.L, total reaction system 20. Mu.L; the specific sequences of the primers involved are shown in Table 3.
First round overlapping PCR reaction conditions:
the PCR amplification reaction conditions were: 98 ℃ for 2min; 15s at 98 ℃,15 s at 60 ℃, 1-3 min at 72 ℃,32 cycles: 7min at 72 ℃.
(2) Taking 1 mu L of the product of the first round of overlapping PCR reaction as a template of the second round of overlapping PCR reaction, and reacting the reaction system: PCR reaction system: primeSTAR Max (R045Q, takara Shuzo) 10. Mu.L, forward and reverse primers each 0.5. Mu.L, ddH 2 O8. Mu.L, template 1. Mu.L, total reaction system 20. Mu.L; the primer sequences are shown in Table 3.
Second round overlapping PCR reaction conditions: 98 ℃ for 2min; 15s at 98 ℃,15 s at 50-60 ℃ and 1-4 min at 72 ℃ for 15 cycles; and at 72℃for 5min.
(4) The PCR product was gel recovered using a DNA gel recovery kit (DP 209-03, tiangen Biochemical technologies Co., ltd.) and the specific procedure was as described in the specification, and was sequenced in connection with pEASY subclone vector (CB 111-01, beijing full gold Biotechnology Co., ltd.).
The following 5 modules were constructed in total according to the above steps:
(a) Gene HIF-1 alpha and promoter P PGK1 And terminator T ADH1 Ligation, construction of Gene expression Module P PGK1 -HIF-1α-T ADH1 Named module I; wherein, the first round of PCR respectively amplifies HIF-1 alpha and promoter P PGK1 And terminator T ADH1 And (3) a gene. Clone P PGK1 The primers of the gene are I-F1 and I-R1, the primers of the cloned HIF-1 alpha are I-F2 and I-R2, and the cloned T ADH1 The primers of (1) are I-F3 and I-R3; the three fragments of the clone are overlapped and spliced into a DNA sequence through the second PCR amplification, and the primers for amplification are I-F1 and I-R3; the primer sequences are shown in Table 3.
(b) Gene ARNT and promoter P TEF1 And terminator T CYC1 Ligation, construction of Gene expression Module P TEF1 -ARNT-T CYC1 Named module II. Wherein, the first round of PCR amplifies ARNT and promoter P respectively TEF1 And terminator T CYC1 And (3) a gene. Clone P TEF1 The primers of (a) are II-F1 and II-R1, the primers of the cloned ARNT are II-F2 and II-R2, and the cloned T CYC1 The primers of (2) are II-F3 and II-R3; the three fragments obtained by the second round of PCR are overlapped and spliced into a DNA sequence, and the primers for amplification are II-F1 and II-R3; the primer sequences are shown in Table 3.
(c) And (3) connecting the module I, the module 2 and the URA screening marker to construct a gene expression module URA-I-II, which is named as a module III. Wherein the first round of PCR amplifies module I, module 2 and URA, respectively. The primers of the clone selection marker URA are III-F1 and III-R1, the primers of the clone module I are III-F2 and III-R2, and the primers of the clone module II are III-F3 and III-R3; the three fragments obtained by the second round of PCR are overlapped and spliced into a DNA sequence, and primers for amplification are III-F1 and III-R3; the primer sequences are shown in Table 3.
(d) Gene tHMGR and promoter P GAL1 And terminator T ADH1 Ligation, construction of Gene expression Module P GAL1 -tHMGR-T ADH1 Named module IV; wherein the first round of PCR separately amplifies tHMGR and promoter P GAL1 And terminator T ADH1 And (3) a gene. Clone P GAL1 The primers of (a) are IV-F1 and IV-R1, the primers of cloning tHMGR are IV-F2 and IV-R2, and cloning T ADH1 The primers of (a) are IV-F3 and IV-R3; the three fragments obtained by the second round of PCR are overlapped and spliced into a DNA sequence, and the primers for amplification are IV-F1 and IV-R3; the primer sequences are shown in Table 3.
(e) Gene UPC2-1 and promoter P GAL10 And terminator T CYC1 Ligation, construction of Gene expression Module P GAL10 -UPC2-1-T CYC1 Designated as module V. Wherein, the first round of PCR respectively amplifies the gene UPC2-1 and the promoter P GAL10 And terminator T CYC1 . Clone P GAL10 The primers of (a) are V-F1 and V-R1, the primers of cloning UPC2-1 are V-F2 and V-R2, and cloning T CYC1 The primers of (2) are V-F3 and V-R3; the three fragments obtained by the second round of PCR are overlapped and spliced into a DNA sequence, and the primers for amplification are V-F1 and V-R3; the primer sequences are shown in Table 3.
(f) Connecting the module IV, the module V and the HIS screening marker to construct a gene expression module HIS-P GAL1 -tHMGR-T ADH1 -P GAL10 -UPC2-1-T CYC1 Named module VI. Wherein, the first round of PCR amplifies module IV, module V and HIS screening markers, respectively. The primers of the cloning screening mark HIS are VI-F1 and VI-R1, the primers of the cloning module IV are VI-F2 and VI-R2, and the primers of the cloning module V are VI-F3 and VI-R3; the three fragments obtained by the second round of PCR are overlapped and spliced into a DNA sequence, and the primers for amplification are VI-F1 and VI-R3; the primer sequences are shown in Table 3.
(g) MET selection markers and P PGK1 Construction of the Gene expression Module MET-P PGK1 Named module VII; wherein, the first round of PCR amplifies MET screening mark and P respectively PGK1 . Cloning MET screening marker primers VII-F1 and VII-R1, cloning P PGK1 The primer is VII-F2 or VII-R2; the 2 fragments obtained by the second round of PCR are overlapped and spliced into a DNA sequence, and the primers for amplification are VII-F1 and VII-R2; the primer sequences are shown in Table 3.
Example 3
Construction method of saccharomyces cerevisiae engineering bacteria strain
1. Construction of Saccharomyces cerevisiae Strain XN01
(1) The primers III-F1 and III-R3 were used to remove the module III from pEASY-URA-P PGK1 -HIF-1α-T ADH1 -P TEF1 -ARNT-T CYC1 (i.e., the above module III was ligated to pEASY vector) and PCR amplification system: primeSTAR Max (R045Q, takara Shuzo) 25. Mu.L, each of the forward and reverse primers (Table 2) 0.5. Mu.L, pEASY-URA-P PGK1 -HIF-1α-T ADH1 -P TEF1 -ARNT-T CYC1 Vector 1. Mu.L, ddH 2 O23. Mu.L. And detecting the size by agarose gel electrophoresis, and recovering the target PCR product.
(2) The recovered target PCR fragment is transformed into a saccharomyces cerevisiae strain BY4741 BY a lithium acetate transformation method, a module III is integrated into an NDT80 site of a chromosome of the saccharomyces cerevisiae BY4741, a methionine-deficient solid culture medium (SD-URA culture medium) plate is used for screening, and colony PCR verification is carried out to obtain a saccharomyces cerevisiae engineering strain XN01.
2. Construction of Strain XN02
(1) From pEASY-HIS-P, module VI GAL1 -tHMGR-T ADH1 -P GAL10 -UPC2-1-T CYC1 The PCR amplification system was amplified using primers IV-F1 and IV-R3 on the pEASY vector (i.e., module IV described above): primeSTAR Max (R045Q, takara Shuzo) 25. Mu.L, each of the forward and reverse primers (Table 2) 0.5. Mu.L, pEASY-HIS-P GAL1 -tHMGR-T ADH1 -P GAL10 -UPC2-1-T CYC1 Vector 1. Mu.L, ddH 2 O23. Mu.L. And detecting the size by agarose gel electrophoresis, and recovering the target PCR product.
(2) And (3) converting the recovered target PCR fragment into a saccharomyces cerevisiae strain XN01 by using a lithium acetate conversion method, integrating a module VI into a GAL80 site of a genome of the strain XN01, screening by using a uracil and histidine defect type solid medium (SD-URA-HIS medium) plate, and obtaining a saccharomyces cerevisiae engineering strain XN02 through colony PCR verification.
3. Construction of Saccharomyces cerevisiae Strain XN03
(1) From pEASY-MET-P, module VII PGK1 The PCR amplification system was amplified using primers VII-F1 and VII-R2 on the pEASY vector (i.e., obtained by ligating the above module VII): 2x M5 HiPer Taq HiFi PCR mix (MF 002, meta. Polymer) 25. Mu.L, forward and reverse primers (Table 2) 0.5. Mu.L each, pLB-LEU-TEF1p vector 1. Mu.L, ddH 2 O23. Mu.L. And detecting the size by agarose gel electrophoresis, and recovering the target PCR product.
(2) And (3) converting the recovered target PCR fragment into a saccharomyces cerevisiae strain XN02 by using a lithium acetate conversion method, enabling a module VII to replace a promoter of ERG9 in a yeast XN02 chromosome, screening by using a methionine, uracil and histidine-deficient solid medium (SD-MET-URA-HIS medium) plate, and obtaining the saccharomyces cerevisiae engineering strain XN03 through colony PCR verification.
Example 4
Method for synthesizing squalene by fermenting strain XN03
1. Squalene is synthesized by fermenting strain XN01, strain XN02 and strain XN03
(1) Single colonies of the strain XN01, the strain XN02 and the strain XN03 are selected into 5mL of SD-MET-URA-HIS culture medium, and the OD value is adjusted to 2-3 by shaking culture at 220rpm under the condition of 30 ℃.
(2) 1mL of the culture solution is inoculated into a 250mL shaking flask containing 50mL of a culture medium SD-MET-URA-HIS, and the three fermentation products are obtained by shaking fermentation for 120h at 220rpm at 30 ℃.
2. Detection of fermentation products of Strain XN01, strain XN02 and Strain XN03
(1) Taking fermentation liquor of the strain XN01, the strain XN02 and the strain XN03, and centrifuging at 3000rpm at 4 ℃ to obtain supernatant and thalli respectively.
(2) 10mL of 20% KOH alkaline lysate was added to the cells, and the cells were heated at 95℃for 15 minutes to carry out lysis.
(3) After completion of the lysis, the lysed cell mixture and the supernatant obtained in (1) were mixed and added to a 250mL Erlenmeyer flask.
(4) Adding ethyl acetate with equal volume into the conical flask, performing ultrasonic extraction for 20min, and standing for 72h.
(5) The above-mentioned organic layer which was left to stand and delaminated was distilled off, then added to a clean liquid phase vial, blow-dried with a nitrogen blower, added with 100. Mu.L of silylation reagent MSTFA, and incubated at 80℃for 30min.
(6) The lanosterol yield was measured by GC-MS using a gas chromatograph, preferably Agilent 7890B-5977B. The detection method comprises the following steps: HP-5ms capillary column; the sample injection amount is 1 mu L, and the flow is not split; the temperature of the sample inlet is 250 ℃, the initial temperature is 50 ℃ for 3min, then the sample is raised to 70 ℃ at the speed of 20 ℃/min for 1min, and then the sample is raised to 300 ℃ at the speed of 15 ℃/min for 3min; electron Ionization ion source, energy intensity 70eV; the MS solvent delay was set to 12min, the voltage multiplication mode was turned on, and the gain factor was set to 1.
From fig. 1 and 2, a characteristic peak exists at about 9min of retention time, and the fragment ion spectrum of the peak is consistent with the squalene standard quality spectrum, which shows that the detected product is squalene.
Compared with the original strain Saccharomyces cerevisiae BY4741, the strain XN01 provided BY the invention has great progress in the production of squalene, and the yield of squalene is improved BY 2.5 times (see figure 3).
Compared with the original strain Saccharomyces cerevisiae BY4741, the strain XN03 provided BY the invention has great progress in the aspect of producing squalene, so that the yield of squalene is improved BY 45.2 times. The yield of squalene produced by strain XN03 was increased 16.7 times as compared to strain XN01, and the yield of squalene produced by strain XN03 was increased 1.7 times as compared to strain XN02 (see FIG. 4).
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims (10)

1. An application of a saccharomyces cerevisiae engineering strain XN01 in producing squalene, wherein the saccharomyces cerevisiae engineering strain XN01 integrates a human HIF-1 alpha gene and a human ARNT gene in a saccharomyces cerevisiae genome.
2. Use of a saccharomyces cerevisiae engineering strain XN02 for the production of squalene, said saccharomyces cerevisiae engineering strain XN02 over-expressing the hmgr and UPC2-1 genes in the genome of said saccharomyces cerevisiae engineering strain XN01 of claim 1;
the nucleotide sequence of the UPC2-1 gene is shown as SEQ ID NO. 5.
3. Use of a saccharomyces cerevisiae engineering strain XN03 for producing squalene, wherein the saccharomyces cerevisiae engineering strain XN03 is based on the saccharomyces cerevisiae engineering strain XN02 described in claim 2, and the endogenous promoter of ERG9 gene is replaced by P PGK1 A promoter.
4. The use according to claim 3, wherein the saccharomyces cerevisiae engineering strain XN03 further has a selectable marker gene integrated into its genome;
The screening marker genes comprise URA, his and MET marker genes;
the nucleotide sequence of the HIF-1 alpha gene is shown as SEQ ID NO. 1;
the nucleotide sequence of the ARNT gene is shown as SEQ ID NO. 2;
the nucleotide sequence of tHMGR is shown as SEQ ID NO. 3;
the P is PGK1 The nucleotide sequence of the promoter is shown as SEQ ID NO. 6.
5. Use according to claim 3, characterized in that the starting strain of the saccharomyces cerevisiae engineering strain XN03 is the saccharomyces cerevisiae BY4741 strain.
6. The use according to any one of claims 3 to 5, characterized in that the construction method of the saccharomyces cerevisiae engineering strain XN03 comprises the following steps:
construction of Gene expression Module P PGK1 -HIF-1α-T ADH1 And P TEF1 -ARNT-T CYC1 ；
Gene expression Module P PGK1 -HIF-1α-T ADH1 、P TEF1 -ARNT-T CYC1 And first selection marker construction Gene expression Module first selection marker-P PGK1 -HIF-1α-T ADH1 -P TEF1 -ARNT-T CYC1 ；
Construction of Gene expression Module P GAL1 -tHMGR-T ADH1 And P GAL10 -UPC2-1-T CYC1 ；
Gene expression Module P GAL1 -tHMGR-T ADH1 、P GAL10 -UPC2-1-T CYC1 And second selectable marker construction Gene expression Module second selectable marker-P GAL1 -tHMGR-T ADH1 -P GAL10 -UPC2-1-T CYC1 ；
Promoter P PGK1 And third selectable marker construction Gene expression Module third selectable marker-P PGK1 ；
First screening marker of Gene expression Module-P PGK1 -HIF-1α-T ADH1 -P TEF1 -ARNT-T CYC1 Integrating into a saccharomyces cerevisiae genome to obtain a strain XN01;
second screening marker-P GAL1 -tHMGR-T ADH1 -P GAL10 -UPC2-1-T CYC1 Integrating into the genome of the strain XN01 to obtain a strain XN02;
Third selectable marker-P PGK1 The saccharomyces cerevisiae engineering strain XN03 is obtained by homologous recombination to replace an endogenous promoter of an ERG9 gene in a genome of the strain XN 02.
7. The use according to claim 6, wherein promoter P PGK1 、P TEF1 、P GAL1 And P GAL10 The nucleotide sequence of (2) is shown as SEQ ID NO 6-SEQ ID NO 9;
T ADH1 and T CYC1 The nucleotide sequence of the terminator is shown as SEQ ID NO 10-SEQ ID NO 11;
the first screening marker, the second screening marker and the third screening marker are different markers in URA, his and MET marker genes;
the nucleotide sequence of the URA marker gene is shown as SEQ ID NO. 12;
the nucleotide sequence of the His tag gene is shown as SEQ ID NO. 13;
the nucleotide sequence of the MET marker gene is shown in SEQ ID NO. 14.
8. The use according to claim 6, wherein the gene expression module P PGK1 -HIF-1α-T ADH1 The HIF-1 alpha amplification primer comprises a forward primer with a nucleotide sequence shown as SEQ ID NO. 33 and a reverse primer with a nucleotide sequence shown as SEQ ID NO. 34; p (P) PGK1 The amplification primer comprises a forward primer with a nucleotide sequence shown as SEQ ID NO. 35 and a reverse primer with a nucleotide sequence shown as SEQ ID NO. 36; t (T) ADH1 The amplification primer comprises a forward primer with a nucleotide sequence shown as SEQ ID NO. 21 and a reverse primer with a nucleotide sequence shown as SEQ ID NO. 22; p (P) PGK1 -HIF-1α-T ADH1 The amplification primer comprises a forward primer with a nucleotide sequence shown as SEQ ID NO. 35 and a reverse primer with a nucleotide sequence shown as SEQ ID NO. 22;
the gene expression module P TEF1 -ARNT-T CYC1 The ARNT amplification primer comprises a forward primer with a nucleotide sequence shown as SEQ ID NO. 37 and a reverse primer with a nucleotide sequence shown as SEQ ID NO. 38; p (P) TEF1 The amplification primer comprises a forward primer with a nucleotide sequence shown as SEQ ID NO. 39 and a reverse primer with a nucleotide sequence shown as SEQ ID NO. 40; t (T) CYC1 The amplification primer comprises a forward primer with a nucleotide sequence shown as SEQ ID NO. 29 and a reverse primer with a nucleotide sequence shown as SEQ ID NO. 30; p (P) TEF1 -ARNT-T CYC1 The amplification primer comprises a forward primer with a nucleotide sequence shown as SEQ ID NO. 39 and a reverse primer with a nucleotide sequence shown as SEQ ID NO. 30;
the gene expression module P GAL1 -tHMGR-T ADH1 The middle tHMGR amplification primer comprises a forward primer with a nucleotide sequence shown as SEQ ID NO. 41 and a reverse primer with a nucleotide sequence shown as SEQ ID NO. 42; p (P) GAL1 The amplification primer comprises a forward primer with a nucleotide sequence shown as SEQ ID NO. 21 and a reverse primer with a nucleotide sequence shown as SEQ ID NO. 22; t (T) ADH1 Amplification primersComprises a forward primer with a nucleotide sequence shown as SEQ ID NO. 23 and a reverse primer with a nucleotide sequence shown as SEQ ID NO. 24; gene expression module P GAL1 -tHMGR-T ADH1 The amplification primer comprises a forward primer with a nucleotide sequence shown as SEQ ID NO. 21 and a reverse primer with a nucleotide sequence shown as SEQ ID NO. 24;
the gene expression module P GAL10 -UPC2-1-T CYC1 The UPC2-1 amplifying primer comprises a forward primer with a nucleotide sequence shown as SEQ ID NO. 25 and a reverse primer with a nucleotide sequence shown as SEQ ID NO. 26; p (P) GAL10 The amplification primer comprises a forward primer with a nucleotide sequence shown as SEQ ID NO. 27 and a reverse primer with a nucleotide sequence shown as SEQ ID NO. 28; t (T) CYC1 The amplification primer comprises a forward primer with a nucleotide sequence shown as SEQ ID NO. 29 and a reverse primer with a nucleotide sequence shown as SEQ ID NO. 30; gene expression module P GAL10 -UPC2-1-T CYC1 The amplification primer comprises a forward primer with a nucleotide sequence shown as SEQ ID NO. 27 and a reverse primer with a nucleotide sequence shown as SEQ ID NO. 30;
promoter P PGK1 The amplification primer comprises a forward primer with a nucleotide sequence shown as SEQ ID NO. 31 and a reverse primer with a nucleotide sequence shown as SEQ ID NO. 32;
the URA marker gene amplification primer comprises a forward primer with a nucleotide sequence shown as SEQ ID NO. 15 and a reverse primer with a nucleotide sequence shown as SEQ ID NO. 16;
the His tag gene amplification primer comprises a forward primer with a nucleotide sequence shown as SEQ ID NO. 17 and a reverse primer with a nucleotide sequence shown as SEQ ID NO. 18;
The MET marker gene amplification primer comprises a forward primer with a nucleotide sequence shown as SEQ ID NO. 19 and a reverse primer with a nucleotide sequence shown as SEQ ID NO. 20.
9. The use according to any one of claims 6 to 8, wherein the gene expression module
First selectable marker-P PGK1 -HIF-1α-T ADH1 -P TEF1 -ARNT-T CYC1 Integration into Saccharomyces cerevisiae GeneNDT80 sites of the group;
second selectable marker-P GAL1 -tHMGR-T ADH1 -P GAL10 -UPC2-1-T CYC1 Integration into the Saccharomyces cerevisiae genome at GAL80 site.
10. Use according to claim 9, characterized in that the process for producing squalene comprises the following steps:
inoculating bacterial liquid of saccharomyces cerevisiae engineering bacterial strain XN01, bacterial strain XN02 or bacterial strain XN03 into a fermentation culture medium for shake culture;
the rotation speed of the shake culture is 200-250 rpm, and the temperature of the shake culture is 28-32 ℃; the shake culture time is 165-175 hours;
the fermentation medium is YPD liquid medium;
the preparation method of the bacterial liquid of the saccharomyces cerevisiae engineering bacterial strain XN03 comprises the steps of inoculating the saccharomyces cerevisiae engineering bacterial strain XN03 to a screening solid culture medium for activation culture, and selecting a monoclonal to be inoculated to the liquid screening culture medium for seed liquid shake culture;
the temperature of the activation culture and seed liquid shake culture is 28-32 ℃;
The screening solid culture medium is SD+URA+HIS+MET solid culture medium;
the liquid screening culture medium is SD+URA+HIS+MET liquid culture medium.
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