CN114774299A

CN114774299A - Metabolic engineering method, lanosterol-producing engineering bacterium, construction method and application thereof

Info

Publication number: CN114774299A
Application number: CN202210527268.5A
Authority: CN
Inventors: 李德芳; 安天悦; 王国丽; 林春华; 李明凯; 武振科; 郑秋生
Original assignee: Yantai Yuhuangding Hospital; Binzhou Medical College
Current assignee: Yantai Yuhuangding Hospital; Binzhou Medical College
Priority date: 2022-05-16
Filing date: 2022-05-16
Publication date: 2022-07-22

Abstract

The application is applicable to the technical field of microorganisms, and provides a metabolic engineering method, an engineering bacterium for producing lanosterol, a construction method and application thereof. Wherein the metabolic engineering method based on the m6A methyltransferase IME4 comprises the following steps: the promoter of the yeast endogenous IME4 gene was replaced with the constitutive promoter PGK1 p. The metabolic engineering method based on saccharomyces cerevisiae m6A methyltransferase IME4 can effectively promote the yield of lanosterol of the haploid saccharomyces cerevisiae engineering bacteria.

Description

Metabolic engineering method, lanosterol-producing engineering bacterium, construction method and application thereof

Technical Field

The application belongs to the technical field of microorganisms, and particularly relates to a metabolic engineering method, an engineering bacterium for producing lanosterol, a construction method and application thereof.

Background

At present, the construction of microbial cell factories becomes an effective means for producing various high-value natural products, and along with the development and improvement of metabolic engineering methods, the production of heterologous microorganisms for various natural products and chemical raw materials continuously makes great progress. For example, by introducing a gene related to the synthesis of beta-carotene into an optimized strain of saccharomyces cerevisiae, the yield of beta-carotene can reach 39.5g/L by releasing the balance of substrate inhibition effect and balanced carbon metabolic flow; the biosynthesis pathway of scutellarin is reconstructed in saccharomyces cerevisiae, and the optimization of related metabolic pathways is carried out, so that the yield of scutellarin reaches 108mg/L, and the method has the potential of industrial production preliminarily. Other high-value natural products, such as the taxol diene, the alpha-balsamic alcohol, the phenethyl alcohol and the like, also obtain high-yield engineering strains by a metabolic engineering method, and provide a material basis for the mass preparation and the industrialization of the high-value natural products.

Saccharomyces cerevisiae contains abundant triterpenes including Squalene (Squalene), Lanosterol (Lanosterol) and Ergosterol (Ergosterol), and has important biological activity and economic value. Lanosterol, for example, is a sterol and tetracyclic triterpenoid. Lanosterol is an important precursor of ergosterol and various triterpenoids with biological activities of resisting cancer, resisting oxidation, regulating immunity and the like, and the analogue of the lanosterol is proved to be a safe and effective blood cholesterol reducing preparation. The research shows that the compound has the functions of relieving and inhibiting the development of cataract, and in addition, the compound has a certain chemoprevention function on colon cancer. In addition to the endogenous triterpenoids, the saccharomyces cerevisiae is also an advantageous strain for heterologous production of other high-value triterpenoid natural products, although the heterologous production of the triterpenoid in the yeast reaches the industrial preparation standard, the construction process of the engineering strains mostly uses the existing classical metabolic engineering optimization strategies, such as optimization of key rate-limiting enzymes in metabolic pathways and "compartmentalization" treatment of the metabolic pathways, so that the production capacity of a yeast cell factory still has a certain space for improvement. Therefore, it is very important to explore and develop a new metabolic engineering strategy to further improve the triterpene production potential of the microbial cell factory.

Disclosure of Invention

The application aims to provide a metabolic engineering method based on m6A methyltransferase IME4, and aims to solve the problem that the triterpene production potential of a microbial cell factory needs to be improved by the existing metabolic engineering strategy.

The application is realized by a metabolic engineering method based on m6A methyltransferase IME4, which comprises the following steps:

the promoter of the yeast endogenous IME4 gene was replaced with the constitutive promoter PGK1 p.

The application also aims at application of the metabolic engineering method based on the m6A methyltransferase IME4 in construction of saccharomyces cerevisiae engineering bacteria.

Another objective of the present application is to provide an engineering bacterium for producing lanosterol, which uses saccharomyces cerevisiae as an original strain and overexpresses IME4, thhmgr, UPC2-1 and ERG9 genes; wherein the content of the first and second substances,

the promoter for replacing the endogenous IME4 gene of the yeast is a constitutive promoter PGK1 p;

integrating tHMG1 and UPC2-1 genes into a site of Saccharomyces cerevisiae chromosome NDT 80;

the promoter for replacing the yeast endogenous ERG9 gene is a constitutive promoter TEF1 p;

the UPC2-1 gene is a UPC2-1 gene obtained by mutating 888 th glycine of the UPC2 gene into aspartic acid.

Another objective of the present application is to provide a method for constructing lanosterol-producing engineering bacteria, comprising the following steps:

overlapping a promoter PGK1p and a screening marker MET to construct an expression module MET-PGK1 p;

overlapping the gene tHMGR, the promoter TDH2p and the terminator ADH1t to construct a gene expression module TDH2p-tHMER-ADH1 t;

overlapping gene UPC2-1, promoter TPI1p and terminator CYC1t to construct gene expression module TPI1p-UPC2-1-CYC1 t; the UPC2-1 gene is a UPC2-1 gene obtained by mutating 888 th glycine of the UPC2 gene into aspartic acid;

overlapping the gene expression module TDH2p-tHMER-ADH1t, the gene expression module TPI1p-UPC2-1-CYC1t and a URA screening marker to construct a gene expression module URA-TDH2p-tHMER-ADH1t-TPI1p-UPC2-1-CYC1 t;

overlapping a promoter TEF1p with a screening marker LEU to construct an expression module LEU-TEF1 p;

transforming the expression module MET-PGK1p into a yeast strain, and screening and culturing SD-MET by using a yeast defect type culture medium to obtain a strain ST 01;

transforming the gene expression module URA-TDH2p-tHMER-ADH1t-TPI1p-UPC2-1-CYC1t into the strain ST01, and screening and culturing by using a yeast defective culture medium SD-MET-URA to obtain a strain ST 02;

and (3) transforming the expression module LEU-TEF1p into the strain ST02, and screening and culturing by using a yeast defect type culture medium SD-MET-URA-LEU to obtain the lanosterol-producing engineering bacteria.

Another objective of the embodiments of the present application is to provide an application of the above engineering bacteria for producing lanosterol or the engineering bacteria for producing lanosterol constructed by the above construction method for producing lanosterol in the production of lanosterol.

The metabolic engineering method based on saccharomyces cerevisiae m6A methyltransferase IME4 can effectively promote the yield of lanosterol of haploid saccharomyces cerevisiae engineering bacteria.

Drawings

FIG. 1 shows the GC-MS detection results of lanosterol in engineered Saccharomyces cerevisiae strains constructed in the embodiments of the present application;

FIG. 2 is a mass spectrum of lanosterol in engineered Saccharomyces cerevisiae constructed in the examples of the present application;

FIG. 3 is a bar graph of the production of lanosterol in engineered strains of Saccharomyces cerevisiae constructed in the examples of the present application.

Detailed Description

In order to make the objects, technical solutions and advantages of the present application more apparent, the present application is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the present application and are not intended to limit the present application.

The application provides a metabolic engineering method based on m6A methyltransferase IME4, which comprises the following steps:

The application provides application of the metabolic engineering method based on m6A methyltransferase IME4 in construction of saccharomyces cerevisiae engineering bacteria.

Specifically, the application provides an engineering bacterium for producing lanosterol, which takes saccharomyces cerevisiae as an initial strain and overexpresses IME4, tHMGR, UPC2-1 and ERG9 genes; wherein, the first and the second end of the pipe are connected with each other,

integrating tHMG1 and UPC2-1 genes into a saccharomyces cerevisiae chromosome NDT80 site;

the tHMGR gene is obtained by removing a transmembrane region of an HMGR gene coding protein;

the preferred strain of the saccharomyces cerevisiae is BY 4741.

The tHMGR and UPC2 genes can be obtained BY cloning from a Saccharomyces cerevisiae BY4741 genome, and the nucleotide sequences of the tHMGR and UPC2 genes are respectively shown as SEQ ID NO. 1-2.

The UPC2-1 gene is a UPC2-1 gene obtained by mutating 888 th glycine of UPC2 gene to aspartic acid, and the nucleotide sequence of the UPC2-1 gene is shown as SEQ ID NO. 3.

The two strong promoters PGK1p and TEF1p can be obtained BY cloning from a Saccharomyces cerevisiae BY4741 genome, and the nucleotide sequences of the promoters are respectively shown in SEQ ID NO. 4-5.

The application provides a construction method of the lanosterol-producing engineering bacteria, which comprises the following steps:

(1) the following modules were constructed using overlapping PCRs:

(a) overlapping the promoter PGK1p and MET screening marker to construct an expression module MET-PGK1p, named module I;

(b) overlapping the gene tHMGR, the promoter TDH2p and the terminator ADH1t to construct a gene expression module TDH2p-tHMER-ADH1t which is named module II;

(c) overlapping a gene UPC2-1, a promoter TPI1p and a terminator CYC1t to construct a gene expression module TPI1p-UPC2-1-CYC1t, which is named as a module III;

(d) overlapping module II, module III and URA screening markers to construct a gene expression module URA-TDH2p-tHMER-ADH1t-TPI1p-UPC2-1-CYC1t, which is named module IV;

(e) overlapping the promoter TEF1p with the LEU screening marker to construct an expression module LEU-TEF1p, named module V;

(2) construction of the Strain ST01

Module I is transformed into strain BY4741, and strain ST01 is obtained BY screening and culturing with yeast defective culture medium SD-MET.

(3) Construction of the Strain ST02

Module IV is transformed into strain ST01, and strain ST02 is obtained by screening and culturing with yeast deficient culture medium SD-MET-URA.

(4) Construction of the Strain ST03

And transforming the module V into a strain ST02, and screening and culturing by using a yeast defect type culture medium SD-MET-URA-LEU to obtain a strain ST03, namely the high-yield lanosterol saccharomyces cerevisiae strain.

The tHMGR and UPC2 genes in the step (1) can be obtained BY cloning a saccharomyces cerevisiae BY4741 genome, and the nucleotide sequences of the tHMGR and UPC2 genes are respectively shown as SEQ ID NO. 1-2.

The UPC2-1 gene in the step (1) is UPC2-1 gene obtained by mutating 888 th site glycine of UPC2 gene to aspartic acid, and the nucleotide sequence of the UPC2-1 gene is shown in SEQ ID NO. 3.

PGK1p, TEF1p, TDH2p and TPI1p in the step (1) are promoter sequences which can be obtained BY cloning a saccharomyces cerevisiae BY4741 genome, and the nucleotide sequences of the promoter sequences are respectively shown in SEQ ID NO. 4-7.

ADH1p and CYC1p in the step (1) are terminator sequences, can be obtained BY cloning from a saccharomyces cerevisiae BY4741 genome, and the nucleotide sequences of the terminator sequences are respectively shown in SEQ ID NO. 8-9.

The nucleotide sequence of the screening marker MET in the step (1) is shown as SEQ ID NO. 10.

The nucleotide sequence of the screening marker URA in the step (1) is shown as SEQ ID NO. 11.

The nucleotide sequence of the screening marker LEU in the step (1) is shown as SEQ ID NO. 12.

Converting the yeast in the steps (2) to (4) by using a yeast conversion kit; preferably, the transformation is performed using the SK2400 classical yeast transformation kit (Kulapibo technologies, Inc., Beijing).

The product in the step (4) is lanosterol (C)₃₀H₅₀O) having the formula:

the application also provides application of the lanosterol-producing engineering bacteria or the lanosterol-producing engineering bacteria constructed by the construction method of the lanosterol-producing engineering bacteria in the production of lanosterol.

The application also provides a method for improving yeast lanosterol, which is to activate the yeast engineering bacteria ST03 with high yield of target products and then inoculate the activated yeast engineering bacteria ST03 into a fermentation culture medium for fermentation culture to obtain lanosterol; the method specifically comprises the following steps:

activating the saccharomyces cerevisiae engineering bacteria ST03 for high yield of the lanosterol, and then inoculating the activated saccharomyces cerevisiae engineering bacteria ST03 into a fermentation culture medium for shake flask fermentation culture to obtain the lanosterol.

The shake flask fermentation is realized by the following steps: the method comprises the steps of inoculating the saccharomyces cerevisiae engineering bacteria ST03 capable of highly producing the lanosterol to a SD + MET + URA + LEU solid culture medium, culturing for 48 hours at 30 ℃, selecting a single clone, inoculating to 5mL of SD + MET + URA + LEU liquid culture medium, carrying out shake culture at 220rpm at 30 ℃ until the OD value is 2-3, inoculating 1mL of bacterial liquid to a shake flask of 50mL of YPD liquid culture medium, and carrying out shake fermentation at 220rpm at 30 ℃ for 168 hours.

The present application will be described in further detail with reference to examples, but the embodiments of the present application are not limited thereto. Reagents, methods and apparatus used herein are conventional in the art unless otherwise indicated. Test methods without specifying specific experimental conditions in the following examples are generally performed according to conventional experimental conditions or according to the experimental conditions recommended by the manufacturer. Unless otherwise specified, reagents and starting materials for use herein are commercially available.

The formulations of the media referred to in the examples of the present application are as follows:

(1) YPD medium: peptone 20g/L, yeast extract 10g/L, and glucose 20g/L (20 g/L agar powder was added to the solid YPD medium during preparation).

(2) SD-MET Medium: YNB medium 6.73g/L, MET (methionine) deficient amino acid (100X)10mL/L, glucose 20g/L (20 g/L agar powder was added when preparing solid medium).

(3) SD-MET-URA Medium: YNB medium 6.73g/L, MET (methionine) and URA (uracil) defective amino acid (100X)10mL/L, glucose 20g/L (20 g/L agar powder was added when preparing solid medium).

(4) SD-MET-URA-LEU Medium: YNB medium 6.73g/L, MET (methionine), URA (uracil) and LEU (leucine) defective amino acid (100X)10mL/L, glucose 20g/L (20 g/L agar powder was added when preparing solid medium).

HIS/MET/LEU/URA four-short amino acid mother liquor (100X): 0.12g of arginine, 0.6g of aspartic acid, 0.6g of glutamic acid, 0.18g of lysine, 0.3g of phenylalanine, 2.25g of serine, 1.2g of threonine, 0.24g of tryptophan, 0.18g of tyrosine and 0.9g of valine, wherein the volume is set to 57mL by using distilled water, and the defective amino acid mother liquor (100X) can be prepared without adding any amino acid according to needs. The above raw materials are all purchased from bio-engineering (Shanghai) GmbH.

Methionine (MET)0.12g, Uracil (URA)0.12g, Leucine (LEU)0.36g, Histidine (HIS)0.12g

EXAMPLE 1 cloning of Yeast endogenous genes and promoters

1. Extraction of Yeast genome

(1) The monoclonal Saccharomyces cerevisiae BY4741 was picked up into 5mL YPD liquid medium, cultured overnight at 30 ℃ at 220rpm, and then centrifuged at 3500rpm for 3min for collection.

(2) The collected thalli is extracted by using a yeast genome DNA rapid extraction kit (B518227, biological engineering Co., Ltd.) and the specific operation process is shown in the specification.

2. Cloning of Yeast endogenous genes and expression elements

(1) The yeast genome obtained above was used as a template to clone the following two genes, four constitutive promoters and two terminators, respectively (see Table 1 for amplification primers):

gene tHMG1(tHMG1-F, tHMG1-R), gene UPC2(UPC2-F, UPC2-R), promoter PGK1p (primer PGK1p-F, PGK1p-R), promoter TEF1p (primer TEF1p-F, TEF1p-R), promoter ADH2p (primer ADH2p-F, ADH2p-R), TPI1p promoter (primer TPI1p-F, TPI1p-R), terminator CYC1t (primer CYC1t-F, CYC1t-R), terminator ADH1t (primer ADH1t-F, ADH1 t-R).

And (3) PCR reaction system: PrimeSTAR Max Premix (R045Q, Baobai) 10. mu.L, forward and reverse primers 0.5. mu.L each, ddH₂O8. mu.l, template 1. mu.l, total reaction 20. mu.l.

The PCR amplification reaction conditions are as follows: 1min at 98 ℃; 15s at 98 ℃, 15s at 50-60 ℃, 30s-2min at 72 ℃ and 32 cycles; 7min at 72 ℃.

After the PCR reaction is finished, carrying out electrophoresis detection on 1% agarose gel, cutting a target strip after the strip size is correct, and carrying out gel recovery by using a DNA gel recovery kit (TSP601, Beijing Optimus department Biotechnology Co., Ltd.), wherein the specific operation process is shown in the specification.

(2) DNA fragment ligation subcloning vector pLB vector

The DNA fragment was ligated with subcloning vector pLB (VT205, Tiangen Biochemical technology Ltd.), and then transformed into competent E.coli DH 5. alpha. according to the instructions of the products.

(3) After overnight incubation at 37 ℃, single colonies were picked for colony PCR.

And (3) PCR reaction system: 2X M5 HiPer Taq HiFi PCR mix (MF002, Mimex) 5. mu.L, forward and reverse primers (Table 1) 0.3. mu.L each, ddH₂O 4.4μL。

The PCR amplification conditions were: 3min at 95 ℃; 15s at 94 ℃, 15s at 55 ℃, 1-3min at 72 ℃ and 30 cycles; 7min at 72 ℃.

And after the PCR reaction is finished, carrying out agarose gel electrophoresis detection, and selecting a positive clone strain for sequencing.

3. Site-directed mutagenesis of UPC2

UPC2 is a positive regulatory factor of mevalonate pathway, and its 888 th glycine is mutated into aspartic acid, which can further improve its regulatory action. The site-directed mutagenesis of UPC2 was performed by PCR using the pLB-UPC2 plasmid as a template (see Table 1 for primer sequences).

(1) And (3) PCR reaction system: PrimeSTAR Max Premix (R045Q, Takara) 10. mu.L, forward and reverse primers 0.5. mu.L each, ddH₂O8. mu.l, template 1. mu.L, total reaction 20. mu.L.

(2) The PCR amplification reaction conditions are as follows: 1min at 98 ℃; 15s at 98 ℃, 15s at 50-60 ℃, 30s-2min at 72 ℃ and 32 cycles; 7min at 72 ℃.

(3) Taking 10 mu L of the PCR product, adding 0.5 mu L of restriction enzyme Dpn I, and reacting for 1h at 37 ℃.

(4) The above reaction system was transformed into competent E.coli DH 5. alpha. and plated and cultured overnight at 37 ℃.

(5) And selecting a single clone, carrying out colony PCR identification, and sequencing a positive colony.

TABLE 1 primer sequences for cloning of the respective genes and expression elements in the examples

Example 2 construction of each expression Module

Construction of Gene expression modules I-V Using overlapping PCRs

(1) First round of overlapping PCR reaction: and (3) PCR reaction system: PrimeSTAR Max Premix (R045Q, Takara) 10. mu.L, forward and reverse primers 0.5. mu.L each, ddH₂O8 mu L, template 1 mu L and total reaction system 20 mu L; the primer sequences are shown in Table 2.

First round of overlap PCR reaction conditions:

the PCR amplification reaction conditions are as follows: 1min at 98 ℃; 15 cycles of 15s at 98 ℃, 15s at 50-60 ℃ and 1-3min at 72 ℃; 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 carrying out reaction system: and (3) PCR reaction system: PrimeSTAR Max Premix (R045Q, Baobai) 10. mu.L, forward and reverse primers 0.5. mu.L each, ddH₂O8 mu L, 1 mu L of template and 20 mu L of total reaction system; the primer sequences are shown in Table 2.

Second round of overlapping PCR reaction conditions: 1min at 98 ℃; 30s at 95 ℃, 30s at 50-60 ℃, 1-4 min at 72 ℃ and 32 cycles; 5min at 72 ℃.

(4) The PCR product was recovered by gel using DNA gel recovery kit (TSP601, Biotech, Inc., Beijing Okagaku), the specific procedure is described in the specification, and the sequence was determined by connecting PLB subcloning vector (VT205, Tiangen Biotechnology, Inc.).

The following 5 modules are constructed according to the steps:

(a) constructing a gene expression module MET-PGK1p by using a MET screening marker and PGK1p, and naming the module as a module I; wherein, the first round of PCR clone MET screening marker primers are I-F1 and I-R1, and the clone PGK1p primers are I-F2 and I-R2; the second round PCR primers are I-F1 and I-R2; primer sequences are shown in Table 2.

(b) TDH2p, tHMG1 and ADH1t were linked to construct a gene expression module TDH2p-tHMG1-ADH1t, which was named module II. Wherein, the primers of the first round of PCR clone PGK1p are II-F1 and II-R1, the primers of clone tHMGR are II-F2 and II-R2, and the primers of clone ADH1t are II-F3 and II-R3; the PCR primers of the second round are II-F1 and II-R3; primer sequences are shown in Table 2.

(c) TPI1p, UPC2-1 and CYC1t are connected to construct a gene expression module TPI1p-UPC2-1-CYC1t, which is named as module III. Wherein, the primers of the first PCR clone TPI1p are III-F1 and III-R1, the primers of the clone UPC2-1 are III-F2 and III-R2, and the primers of the clone CYC1t are III-F3 and III-R3; the PCR primers of the second round are III-F1 and III-R3; the primer sequences are shown in Table 2.

(d) The URA screening marker, module II and module III are connected to construct a gene expression module URA-TDH2p-tHMER-ADH1t-TPI1p-UPC2-1-CYC1t, which is named module IV. Wherein, the primers of the URA screening marker of the first round of PCR cloning are IV-F1 and IV-R1, the primers of the cloning module II are IV-F2 and IV-R2, and the primers of the cloning module III are IV-F3 and IV-R3; the PCR primers of the second round are IV-F1 and IV-R3; the primer sequences are shown in Table 2.

(e) Constructing a gene expression module LEU-TEF1p by using an LEU screening marker and TEF1p, and naming the module as a module V; wherein primers of the LEU screening marker of the first round of PCR cloning are V-F1 and V-R1, and primers of clone TEF1p are V-F2 and V-R2; the second round PCR primers are V-F1 and V-R2; the primer sequences are shown in Table 2.

TABLE 2

Example 3 construction of engineered Yeast

1. Construction of Saccharomyces cerevisiae Strain ST01

(1) Module I was amplified from pLB-MET-PGK1p (i.e., module I was ligated to pLB vector as described above) using primers I-F1 and I-R2, PCR amplification system: 2X M5 HiPer Taq HiFi PCR mix (MF002, Mimex) 25. mu.L, forward and reverse primers (Table 2) 0.5. mu.L each, pLB-MET-PGK1p vector 1. mu.L, ddH₂O23. mu.L. After agarose gel electrophoresis detection, the PCR product is recovered.

(2) And transforming the recovered target PCR fragment into a saccharomyces cerevisiae strain BY4741, replacing a promoter sequence of an IME4 gene in a chromosome of the saccharomyces cerevisiae strain BY4741 with a module I, screening BY using a methionine defective solid medium (SD-MET medium) plate, and verifying BY colony PCR to obtain a saccharomyces cerevisiae engineering strain ST 01.

2. Construction of the ST02 Strain

(1) The module IV is obtained from pLB-URA-TDH2p-tHMER-TPI1p-UPC2-1-CYC1t (namely the module IV is connected with pLB vector) and amplified by using primers IV-F1 and IV-R3 to obtain a PCR amplification system: 2X M5 HiPer Taq HiFi PCR mix (MF002, Mimex) 25. mu.L, forward and reverse primers (Table 2) 0.5. mu.L each, pLB-URA-TDH2p-tHMER-TPI1p-UPC2-1-CYC1t vector 1. mu.L, ddH₂O23. mu.L. After agarose gel electrophoresis detection, the PCR product is recovered.

(2) And transforming the recovered target PCR fragment into a saccharomyces cerevisiae strain ST01, replacing an NDT80 locus in a chromosome of yeast BY4741 with a module IV, screening BY using a methionine and uracil defective solid medium (SD-MET-URA medium) plate, and verifying BY colony PCR to obtain an engineered saccharomyces cerevisiae strain ST 02.

3. Construction of high-yield lanosterol Saccharomyces cerevisiae strain ST03

(1) Module V was amplified from pLB-LEU-TEF1p (i.e., obtained by ligating module V to pLB vector as described above) using primers V-F1 and V-R2, PCR amplification system: 2X M5 HiPer Taq HiFi PCR mix (MF002, Mimex) 25. mu.L, forward and reverse primers (Table 2) 0.5. mu.L each, pLB-LEU-TEF1p vector 1. mu.L, ddH₂O23. mu.L. After agarose gel electrophoresis detection, the PCR product is recovered.

(2) And transforming the recovered target PCR fragment into a saccharomyces cerevisiae strain ST02, replacing an NDT80 locus in a chromosome of yeast BY4741 with a module IV, screening BY using a methionine, uracil and leucine defective solid culture medium (SD-MET-URA-LEU culture medium) plate, and verifying BY colony PCR to obtain a saccharomyces cerevisiae engineering strain ST 03.

EXAMPLE 4 fermentative Synthesis of lanosterol by Strain ST03

1. Strain ST03 fermentation synthesis of lanosterol

(1) Selecting a single colony of the strain ST03 to 5mL SD-URA-MET-LEU culture medium, and carrying out shake culture at 220rpm at the temperature of 30 ℃ until the OD value is 2-3.

(2) 1mL of the culture broth was inoculated into a 250mL shake flask containing 50mL of the medium SD-URA-MET-LEU, and fermented at 30 ℃ with shaking at 220rpm for 120 hours.

2. Detection of fermentation product of Strain ST03

(1) The fermentation broth of ST03 was centrifuged at 3000rpm at 4 ℃ to obtain supernatant and cells, respectively.

(2) 10mL of 20% KOH alkaline lysis solution was added to the cells, and the cells were heated at 95 ℃ for 15min to lyse.

(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 equal volume of ethyl acetate into the conical flask, performing ultrasonic extraction for 20min, and standing for 72 h.

(5) And (3) rotatably evaporating the organic layer which is kept standing and layered, adding the organic layer into a clean liquid phase small bottle, drying the organic layer by using a nitrogen blowing instrument, adding 100 mu L of a silanization reagent MSTFA, and carrying out warm bath at the temperature of 80 ℃ for 30 min.

(6) The lanosterol yield is determined by GC-MS using a GC-MS instrument (preferably Agilent 7890B-5977B). The detection method comprises the following steps: HP-5ms capillary column; the sample volume is 1 mu L, and no flow distribution is carried out; the injection port temperature is 250 ℃, the initial temperature is 50 ℃ and is kept for 3min, then the temperature is increased to 70 ℃ at the speed of 20 ℃/min and is kept for 1min, and then the temperature is increased to 300 ℃ at the speed of 15 ℃/min and is kept for 3 min; electron Ionization ion source, energy intensity 70 eV; the MS solvent delay is set to 12min, the voltage multiplication mode is turned on, and the gain factor is set to 1. The results of the measurements are shown in FIGS. 1-3.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Sequence listing

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tgtttctttt tgacaagccc tgagatttct gaggggaatt cacataaatg ggatcaggtc 720

attcatttac gttgtgtgca aatatgattt aaagatacaa cctttgcaga gagcatgctt 780

tcctaagggt aggcacgtgg aggactaagg gtaaagcatt cttcaagatc agttaatcaa 840

gaaaggtgct ctttgcattc tgaaatgccc ttgttgcaaa tattggttat attgattaaa 900

tttacactta atggaaacaa cctttaactt acagatgaac aaacccacaa aagcaaaaaa 960

tcaaaagccc tacctatgat ttcatatttt ctgtgtaact ggattaaagg attcctgctt 1020

gcttttgggc ataaatgata atggaatatt tccaggtatt gtttaaaatg agggcccatc 1080

tacaaattct tagcaatact ttggataatt ctaaaattca gctggacatt gtctaattgt 1140

tttttatata catctttgct agaatttcaa attttaagta tgtgaattta gttaattagc 1200

tgtgctgatc aattcaaaaa cattactttc ctaaatttta gactatgaag gtcataaatt 1260

caacaaatat atctacacat acaattatag attgtttttc attataatgt cttcatctta 1320

acagaattgt ctttgtgatt gtttttagaa aactgagagt tttaattcat aattacttga 1380

tcaaaaaatt gtgggaacaa tccagcatta attgtatgtg attgttttta tgtacataag 1440

gagtcttaag cttggtgcct tgaagtcttt tgtacttagt cccatgttta aaattactac 1500

tttatatcta aagcatttat gtttttcaat tcaatttaca tgatgctaat tatggcaatt 1560

ataacaaata ttaaagattt cgaaatagaa 1590

<210> 2

<211> 1365

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

atggattttt ttcgggtagt ggaaaaccag cagcctcccg cgacgatgcc cctcaacgtt 60

agcttcacca acaggaacta tgacctcgac tacgactcgg tgcagccgta tttctactgc 120

gacgaggagg agaacttcta ccagcagcag cagcagagcg agctgcagcc cccggcgccc 180

agcgaggata tctggaagaa attcgagctg ctgcccaccc cgcccctgtc ccctagccgc 240

cgctccgggc tctgctcgcc ctcctacgtt gcggtcacac ccttctccct tcggggagac 300

aacgacggcg gtggcgggag cttctccacg gccgaccagc tggagatggt gaccgagctg 360

ctgggaggag acatggtgaa ccagagtttc atctgcgacc cggacgacga gaccttcatc 420

aaaaacatca tcatccagga ctgtatgtgg agcggcttct cggccgccgc caagctcgtc 480

tcagagaagc tggcctccta ccaggctgcg cgcaaagaca gcggcagccc gaaccccgcc 540

cgcggccaca gcgtctgctc cacctccagc ttgtacctgc aggatctgag cgccgccgcc 600

tcagagtgca tcgacccctc ggtggtcttc ccctaccctc tcaacgacag cagctcgccc 660

aagtcctgcg cctcgcaaga ctccagcgcc ttctctccgt cctcggattc tctgctctcc 720

tcgacggagt cctccccgca gggcagcccc gagcccctgg tgctccatga ggagacaccg 780

cccaccacca gcagcgactc tgaggaggaa caagaagatg aggaagaaat cgatgttgtt 840

tctgtggaaa agaggcaggc tcctggcaaa aggtcagagt ctggatcacc ttctgctgga 900

ggccacagca aacctcctca cagcccactg gtcctcaaga ggtgccacgt ctccacacat 960

cagcacaact acgcagcgcc tccctccact cggaaggact atcctgctgc caagagggtc 1020

aagttggaca gtgtcagagt cctgagacag atcagcaaca accgaaaatg caccagcccc 1080

aggtcctcgg acaccgagga gaatgtcaag aggcgaacac acaacgtctt ggagcgccag 1140

aggaggaacg agctaaaacg gagctttttt gccctgcgtg accagatccc ggagttggaa 1200

aacaatgaaa aggcccccaa ggtagttatc cttaaaaaag ccacagcata catcctgtcc 1260

gtccaagcag aggagcaaaa gctcatttct gaagaggact tgttgcggaa acgacgagaa 1320

cagttgaaac acaaacttga acagctacgg aactcttgtg cgtaa 1365

<210> 3

<211> 1584

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

atggctgcag accaattggt gaaaactgaa gtcaccaaga agtcttttac tgctcctgta 60

caaaaggctt ctacaccagt tttaaccaat aaaacagtca tttctggatc gaaagtcaaa 120

agtttatcat ctgcgcaatc gagctcatca ggaccttcat catctagtga ggaagatgat 180

tcccgcgata ttgaaagctt ggataagaaa atacgtcctt tagaagaatt agaagcatta 240

ttaagtagtg gaaatacaaa acaattgaag aacaaagagg tcgctgcctt ggttattcac 300

ggtaagttac ctttgtacgc tttggagaaa aaattaggtg atactacgag agcggttgcg 360

gtacgtagga aggctctttc aattttggca gaagctcctg tattagcatc tgatcgttta 420

ccatataaaa attatgacta cgaccgcgta tttggcgctt gttgtgaaaa tgttataggt 480

tacatgcctt tgcccgttgg tgttataggc cccttggtta tcgatggtac atcttatcat 540

ataccaatgg caactacaga gggttgtttg gtagcttctg ccatgcgtgg ctgtaaggca 600

atcaatgctg gcggtggtgc aacaactgtt ttaactaagg atggtatgac aagaggccca 660

gtagtccgtt tcccaacttt gaaaagatct ggtgcctgta agatatggtt agactcagaa 720

gagggacaaa acgcaattaa aaaagctttt aactctacat caagatttgc acgtctgcaa 780

catattcaaa cttgtctagc aggagattta ctcttcatga gatttagaac aactactggt 840

gacgcaatgg gtatgaatat gatttctaaa ggtgtcgaat actcattaaa gcaaatggta 900

gaagagtatg gctgggaaga tatggaggtt gtctccgttt ctggtaacta ctgtaccgac 960

aaaaaaccag ctgccatcaa ctggatcgaa ggtcgtggta agagtgtcgt cgcagaagct 1020

actattcctg gtgatgttgt cagaaaagtg ttaaaaagtg atgtttccgc attggttgag 1080

ttgaacattg ctaagaattt ggttggatct gcaatggctg ggtctgttgg tggatttaac 1140

gcacatgcag ctaatttagt gacagctgtt ttcttggcat taggacaaga tcctgcacaa 1200

aatgttgaaa gttccaactg tataacattg atgaaagaag tggacggtga tttgagaatt 1260

tccgtatcca tgccatccat cgaagtaggt accatcggtg gtggtactgt tctagaacca 1320

caaggtgcca tgttggactt attaggtgta agaggcccgc atgctaccgc tcctggtacc 1380

aacgcacgtc aattagcaag aatagttgcc tgtgccgtct tggcaggtga attatcctta 1440

tgtgctgccc tagcagccgg ccatttggtt caaagtcata tgacccacaa caggaaacct 1500

gctgaaccaa caaaacctaa caatttggac gccactgata taaatcgttt gaaagatggg 1560

tccgtcacct gcattaaatc ctaa 1584

<210> 4

<211> 2742

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

atgagcgaag tcggtataca gaatcacaag aaagcggtga caaaacccag aagaagagaa 60

aaagtcatcg agctaattga agtggacggc aaaaaggtga gtacgacttc aaccggtaaa 120

cgtaaattcc ataacaaatc aaagaatggg tgcgataact gtaaaagaag aagagttaag 180

tgtgatgaag ggaagccagc ctgtaggaag tgcacaaata tgaagttgga atgtcagtat 240

acaccaatcc atttaaggaa aggtagagga gcaacagtag tgaagtatgt cacgagaaag 300

gcagacggta gcgtggagtc tgattcatcg gtagatttac ctcctacgat caagaaggag 360

cagacaccgt tcaatgatat ccaatcagcg gtaaaagctt caggctcatc caatgattcc 420

tttccatcaa gcgcctctac aactaagagt gagagcgagg aaaagtcatc ggcccctata 480

gaggacaaaa acaatatgac tcctctaagt atgggcctcc agggtaccat caataagaaa 540

gatatgatga ataacttttt ctctcaaaat ggcactattg gttttggttc tcctgaaaga 600

ttgaattcag gtatcgatgg cttactatta ccgccattgc cttctggaaa tatgggtgcg 660

ttccaacttc agcaacagca gcaagtgcag cagcaatctc aaccacagac ccaagcgcag 720

caagcaagtg gaactccaaa cgagagatat ggttcattcg atcttgcggg tagtcctgca 780

ttgcaatcca cgggaatgag cttatcaaat agtctaagcg ggatgttact atgtaacagg 840

attccttccg gccaaaacta cactcaacaa caattacaat atcaattaca ccagcagctg 900

caattgcaac agcatcagca agttcagctg cagcagtatc aacaattacg tcaggaacaa 960

caccaacaag ttcagcaaca acaacaggaa caactccagc aataccaaca acattttttg 1020

caacagcagc aacaagtact gcttcagcaa gagcaacaac ctaacgatga ggaaggtggc 1080

gttcaggaag aaaacagcaa aaaggtaaag gaagggcctt tacaatcaca aacaagcgaa 1140

actactttaa acagcgatgc tgctacatta caagctgatg cattatctca gttaagtaag 1200

atggggctaa gcctaaagtc gttaagtacc tttccaacag ctggtattgg tggtgtttcc 1260

tatgactttc aggaactgtt aggtattaag tttccaataa ataacggcaa ttcaagagct 1320

actaaggcca gcaacgcaga ggaagctttg gccaatatgc aagagcatca tgaacgtgca 1380

gctgcttctg taaaggagaa tgatggtcag ctctctgata cgaagagtcc agcgccatcg 1440

aataacgccc aagggggaag tgctagtatt atggaacctc aggcggctga tgcggtttcg 1500

acaatggcgc ctatatcaat gattgaaaga aacatgaaca gaaacagcaa catttctcca 1560

tcaacgccct ctgcagtgtt gaatgatagg caagagatgc aagattctat aagttctcta 1620

ggaaatctga caaaagcagc cttggagaac aacgaaccaa cgataagttt acaaacatca 1680

cagacagaga atgaagacga tgcatcgcgg caagacatga cctcaaaaat taataacgaa 1740

gctgaccgaa gttctgtttc tgctggtacc agtaacatcg ctaagctttt agatctttct 1800

accaaaggca atctgaacct gatagacatg aaactgtttc atcattattg cacaaaggtc 1860

tggcctacga ttacagcggc caaagtttct gggcctgaaa tatggaggga ctacataccg 1920

gagttagcat ttgactatcc atttttaatg cacgctttgt tggcattcag tgccacccat 1980

ctttcgagga ctgaaactgg actggagcaa tacgtttcat ctcaccgcct agacgctctg 2040

agattattaa gagaagctgt tttagaaata tctgagaata acaccgatgc gctagttgcc 2100

agcgccctga tactaatcat ggactcgtta gcaaatgcta gtggtaacgg cactgtagga 2160

aaccaaagtt tgaatagcat gtcaccaagc gcttggatct ttcatgtcaa aggtgctgca 2220

acaattttaa ccgctgtgtg gcctttgagt gaaagatcta aatttcataa cattatatct 2280

gttgatctta gcgatttagg cgatgtcatt aaccctgatg ttggaacaat tactgaattg 2340

gtatgttttg atgaaagtat tgccgatttg tatcctgtcg gcttagattc gccatatttg 2400

ataacactag cttatttaga taaattgcac cgtgaaaaaa accagggtga ttttattctg 2460

cgggtattta catttccagc attgctagac aagacattcc tggcattact gatgacaggt 2520

gatttaggtg caatgagaat tatgagatca tattataaac tacttcgagg atttgccaca 2580

gaggtcaagg ataaagtctg gtttctcgaa ggagtcacgc aggtgctgcc tcaagatgtt 2640

gacgaataca gtggaggtgg tgatatgcat atgatgctag atttcctcgg tggcggatta 2700

ccatcgatga caacaacaaa tttctctgat ttttcgttat ga 2742

<210> 5

<211> 2742

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

atgagcgaag tcggtataca gaatcacaag aaagcggtga caaaacccag aagaagagaa 60

aaagtcatcg agctaattga agtggacggc aaaaaggtga gtacgacttc aaccggtaaa 120

cgtaaattcc ataacaaatc aaagaatggg tgcgataact gtaaaagaag aagagttaag 180

tgtgatgaag ggaagccagc ctgtaggaag tgcacaaata tgaagttgga atgtcagtat 240

acaccaatcc atttaaggaa aggtagagga gcaacagtag tgaagtatgt cacgagaaag 300

gcagacggta gcgtggagtc tgattcatcg gtagatttac ctcctacgat caagaaggag 360

cagacaccgt tcaatgatat ccaatcagcg gtaaaagctt caggctcatc caatgattcc 420

tttccatcaa gcgcctctac aactaagagt gagagcgagg aaaagtcatc ggcccctata 480

gaggacaaaa acaatatgac tcctctaagt atgggcctcc agggtaccat caataagaaa 540

gatatgatga ataacttttt ctctcaaaat ggcactattg gttttggttc tcctgaaaga 600

ttgaattcag gtatcgatgg cttactatta ccgccattgc cttctggaaa tatgggtgcg 660

ttccaacttc agcaacagca gcaagtgcag cagcaatctc aaccacagac ccaagcgcag 720

caagcaagtg gaactccaaa cgagagatat ggttcattcg atcttgcggg tagtcctgca 780

ttgcaatcca cgggaatgag cttatcaaat agtctaagcg ggatgttact atgtaacagg 840

attccttccg gccaaaacta cactcaacaa caattacaat atcaattaca ccagcagctg 900

caattgcaac agcatcagca agttcagctg cagcagtatc aacaattacg tcaggaacaa 960

caccaacaag ttcagcaaca acaacaggaa caactccagc aataccaaca acattttttg 1020

caacagcagc aacaagtact gcttcagcaa gagcaacaac ctaacgatga ggaaggtggc 1080

gttcaggaag aaaacagcaa aaaggtaaag gaagggcctt tacaatcaca aacaagcgaa 1140

actactttaa acagcgatgc tgctacatta caagctgatg cattatctca gttaagtaag 1200

atggggctaa gcctaaagtc gttaagtacc tttccaacag ctggtattgg tggtgtttcc 1260

tatgactttc aggaactgtt aggtattaag tttccaataa ataacggcaa ttcaagagct 1320

actaaggcca gcaacgcaga ggaagctttg gccaatatgc aagagcatca tgaacgtgca 1380

gctgcttctg taaaggagaa tgatggtcag ctctctgata cgaagagtcc agcgccatcg 1440

aataacgccc aagggggaag tgctagtatt atggaacctc aggcggctga tgcggtttcg 1500

acaatggcgc ctatatcaat gattgaaaga aacatgaaca gaaacagcaa catttctcca 1560

tcaacgccct ctgcagtgtt gaatgatagg caagagatgc aagattctat aagttctcta 1620

ggaaatctga caaaagcagc cttggagaac aacgaaccaa cgataagttt acaaacatca 1680

cagacagaga atgaagacga tgcatcgcgg caagacatga cctcaaaaat taataacgaa 1740

gctgaccgaa gttctgtttc tgctggtacc agtaacatcg ctaagctttt agatctttct 1800

accaaaggca atctgaacct gatagacatg aaactgtttc atcattattg cacaaaggtc 1860

tggcctacga ttacagcggc caaagtttct gggcctgaaa tatggaggga ctacataccg 1920

gagttagcat ttgactatcc atttttaatg cacgctttgt tggcattcag tgccacccat 1980

ctttcgagga ctgaaactgg actggagcaa tacgtttcat ctcaccgcct agacgctctg 2040

agattattaa gagaagctgt tttagaaata tctgagaata acaccgatgc gctagttgcc 2100

agcgccctga tactaatcat ggactcgtta gcaaatgcta gtggtaacgg cactgtagga 2160

aaccaaagtt tgaatagcat gtcaccaagc gcttggatct ttcatgtcaa aggtgctgca 2220

acaattttaa ccgctgtgtg gcctttgagt gaaagatcta aatttcataa cattatatct 2280

gttgatctta gcgatttagg cgatgtcatt aaccctgatg ttggaacaat tactgaattg 2340

gtatgttttg atgaaagtat tgccgatttg tatcctgtcg gcttagattc gccatatttg 2400

ataacactag cttatttaga taaattgcac cgtgaaaaaa accagggtga ttttattctg 2460

cgggtattta catttccagc attgctagac aagacattcc tggcattact gatgacaggt 2520

gatttaggtg caatgagaat tatgagatca tattataaac tacttcgagg atttgccaca 2580

gaggtcaagg ataaagtctg gtttctcgaa ggagtcacgc aggtgctgcc tcaagatgtt 2640

gacgaataca gtggaggtgg tggtatgcat atgatgctag atttcctcgg tggcggatta 2700

ccatcgatga caacaacaaa tttctctgat ttttcgttat ga 2742

<210> 6

<211> 926

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

tcttcaagaa ttggggatct acgtatggtc attcttcttc agattccctc atggagaagt 60

gcggcagatg tatatgacag agtcgccagt ttccaagaga ctttattcag gcacttccat 120

gataggcaag agagaagacc cagagatgtt gttgtcctag ttacacatgg tatttattcc 180

agagtattcc tgatgaaatg gtttagatgg acatacgaag agtttgaatc gtttaccaat 240

gttcctaacg ggagcgtaat ggtgatggaa ctggacgaat ccatcaatag atacgtcctg 300

aggaccgtgc tacccaaatg gactgattgt gagggagacc taactacata gtgtttaaag 360

attacggata tttaacttac ttagaataat gccatttttt tgagttataa taatcctacg 420

ttagtgtgag cgggatttaa actgtgagga cctcaataca ttcagacact tctgacggta 480

tcaccctact tattcccttc gagattatat ctaggaaccc atcaggttgg tggaagatta 540

cccgttctaa gacttttcag cttcctctat tgatgttaca ctcggacacc ccttttctgg 600

catccagttt ttaatcttca gtggcatgtg agattctccg aaattaatta aagcaatcac 660

acaattctct cggataccac ctcggttgaa actgacaggt ggtttgttac gcatgctaat 720

gcaaaggagc ctatatacct ttggctcggc tgctgtaaca gggaatataa agggcagcat 780

aatttaggag tttagtgaac ttgcaacatt tactattttc ccttcttacg taaatatttt 840

tctttttaat tctaaatcaa tctttttcaa ttttttgttt gtattctttt cttgcttaaa 900

tctataacta caaaaaacac atacag 926

<210> 7

<211> 1123

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

tgcaggtctc atctggaata taattccccc ctcctgaagc aaatttttcc tttgagccgg 60

aatttttgat attccgagtt ctttttttcc attcgcggag gttattccat tcctaaacga 120

gtggccacaa tgaaacttca attcatatcg accgactatt tttctccgaa ccaaaaaaat 180

agcagggcga gattggagct gcggaaaaaa gaggaaaaaa ttttttcgta gttttcttgt 240

gcaaattagg gtgtaaggtt tctagggctt attggttcaa gcagaagaga caacaattgt 300

aggtcctaaa ttcaaggcgg atgtaaggag tattggtttc gaaagttttt ccgaagcggc 360

atggcaggga ctacttgcgc atgcgctcgg attatcttca tttttgcttg caaaaacgta 420

gaatcatggt aaattacatg aagaattctc tttttttttt tttttttttt ttttttacct 480

ctaaagagtg ttgaccaact gaaaaaaccc ttcttcaaga gagttaaact aagactaacc 540

atcataactt ccaaggaatt aatcgatatc ttgcactcct gatttttctt caaagagaca 600

gcgcaaagga ttatgacact gttgcattga gtcaaaagtt tttccgaagt gacccagtgc 660

tctttttttt tttccgtgaa ggactgacaa atatgcgcac aagatccaat acgtaatgga 720

aattcggaaa aactaggaag aaatgctgca gggcattgcc gtgccgatct tttgtctttc 780

agatatatga gaaaaagaat attcatcaag tgctgataga agaataccac tcatatgacg 840

tgggcagaag acagcaaacg taaacatgag ctgctgcgac atttgatggc ttttatccga 900

caagccagga aactccacca ttatctaatg tagcaaaata tttcttaaca cccgaagttg 960

cgtgtccccc tcacgttttt aatcatttga attagtatat tgaaattata tataaaggca 1020

acaatgtccc cataatcaat tccatctggg gtctcatgtt ctttccccac cttaaaatct 1080

ataaagatat cataatcgtc aactagttga tatacgtaaa atc 1123

<210> 8

<211> 984

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

ggaagtacct tcaaagaatg gggtcttatc ttgttttgca agtaccactg agcaggataa 60

taatagaaat gataatatac tatagtagag ataacgtcga tgacttccca tactgtaatt 120

gcttttagtt gtgtattttt agtgtgcaag tttctgtaaa tcgattaatt tttttttctt 180

tcctcttttt attaacctta atttttattt tagattcctg acttcaactc aagacgcaca 240

gatattataa catctgcata ataggcattt gcaagaatta ctcgtgagta aggaaagagt 300

gaggaactat cgcatacctg catttaaaga tgccgatttg ggcgcgaatc ctttattttg 360

gcttcaccct catactatta tcagggccag aaaaaggaag tgtttccctc cttcttgaat 420

tgatgttacc ctcataaagc acgtggcctc ttatcgagaa agaaattacc gtcgctcgtg 480

atttgtttgc aaaaagaaca aaactgaaaa aacccagaca cgctcgactt cctgtcttcc 540

tattgattgc agcttccaat ttcgtcacac aacaaggtcc tagcgacggc tcacaggttt 600

tgtaacaagc aatcgaaggt tctggaatgg cgggaaaggg tttagtacca catgctatga 660

tgcccactgt gatctccaga gcaaagttcg ttcgatcgta ctgttactct ctctctttca 720

aacagaattg tccgaatcgt gtgacaacaa cagcctgttc tcacacactc ttttcttcta 780

accaaggggg tggtttagtt tagtagaacc tcgtgaaact tacatttaca tatatataaa 840

cttgcataaa ttggtcaatg caagaaatac atatttggtc ttttctaatt cgtagttttt 900

caagttctta gatgctttct ttttctcttt tttacagatc atcaaggaag taattatcta 960

ctttttacaa caaatataaa acaa 984

<210> 9

<211> 420

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

gcacacacca tagcttcaaa atgtttctac tcctttttta ctcttccaga ttttctcgga 60

ctccgcgcat cgccgtacca cttcaaaaca cccaagcaca gcatactaaa tttcccctct 120

ttcttcctct agggtgtcgt taattacccg tactaaaggt ttggaaaaga aaaaagagac 180

cgcctcgttt ctttttcttc gtcgaaaaag gcaataaaaa tttttatcac gtttcttttt 240

cttgaaaatt tttttttttg atttttttct ctttcgatga cctcccattg atatttaagt 300

taataaacgg tcttcaattt ctcaagtttc agtttcattt ttcttgttct attacaactt 360

tttttacttc ttgctcatta gaaagaaagc atagcaatct aatctaagtt ttaattacaa 420

<210> 10

<211> 455

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

agtacggatt agaagccgcc gagcgggtga cagccctccg aaggaagact ctcctccgtg 60

cgtcctcgtc ttcaccggtc gcgttcctga aacgcagatg tgcctcgcgc cgcactgctc 120

cgaacaataa agattctaca atactagctt ttatggttat gaagaggaaa aattggcagt 180

aacctggccc cacaaacctt caaatgaacg aatcaaatta acaaccatag gatgataatg 240

cgattagttt tttagcctta tttctggggt aattaatcag cgaagcgatg atttttgatc 300

tattaacaga tatataaatg caaaaactgc ataaccactt taactaatac tttcaacatt 360

ttcggtttgt attacttctt attcaaatgt aataaaagta tcaacaaaaa attgttaata 420

tacctctata ctttaacgtc aaggagaaaa aaccc 455

<210> 11

<211> 212

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

tcaatatagc aatgagcagt taagcgtatt actgaaagtt ccaaagagaa ggttttttta 60

ggctaagata atggggctct ttacatttcc acaacatata agtaagatta gatatggata 120

tgtatatgga tatgtatatg gtggtaatgc catgtaatat gattattaaa cttctttgcg 180

tccatccaaa aaaaaagtaa gaatttttga aa 212

<210> 12

<211> 190

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

atccgctcta accgaaaagg aaggagttag acaacctgaa gtctaggtcc ctatttattt 60

ttttatagtt atgttagtat taagaacgtt atttatattt caaatttttc ttttttttct 120

gtacagacgc gtgtacgcat gtaacattat actgaaaacc ttgcttgaga aggttttggg 180

acgctcgaag 190

<210> 13

<211> 165

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

cgaatttctt atgatttatg atttttatta ttaaataagt tataaaaaaa ataagtgtat 60

acaaatttta aagtgactct taggttttaa aacgaaaatt cttattcttg agtaactctt 120

tcctgtaggt caggttgctt tctcaggtat agcatgaggt cgctc 165

<210> 14

<211> 1165

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

cttaactatg cggcatcaga gcagattgta ctgagagtgc accataaatt cccgttttaa 60

gagcttggtg agcgctagga gtcactgcca ggtatcgttt gaacacggca ttagtcaggg 120

aagtcataac acagtccttt cccgcaattt tctttttcta ttactcttgg cctcctctag 180

tacactctat atttttttat gcctcggtaa tgattttcat tttttttttt cccctagcgg 240

atgactcttt ttttttctta gcgattggca ttatcacata atgaattata cattatataa 300

agtaatgtga tttcttcgaa gaatatacta aaaaatgagc aggcaagata aacgaaggca 360

aagatgacag agcagaaagc cctagtaaag cgtattacaa atgaaaccaa gattcagatt 420

gcgatctctt taaagggtgg tcccctagcg atagagcact cgatcttccc agaaaaagag 480

gcagaagcag tagcagaaca ggccacacaa tcgcaagtga ttaacgtcca cacaggtata 540

gggtttctgg accatatgat acatgctctg gccaagcatt ccggctggtc gctaatcgtt 600

gagtgcattg gtgacttaca catagacgac catcacacca ctgaagactg cgggattgct 660

ctcggtcaag cttttaaaga ggccctactg gcgcgtggag taaaaaggtt tggatcagga 720

tttgcgcctt tggatgaggc actttccaga gcggtggtag atctttcgaa caggccgtac 780

gcagttgtcg aacttggttt gcaaagggag aaagtaggag atctctcttg cgagatgatc 840

ccgcattttc ttgaaagctt tgcagaggct agcagaatta ccctccacgt tgattgtctg 900

cgaggcaaga atgatcatca ccgtagtgag agtgcgttca aggctcttgc ggttgccata 960

agagaagcca cctcgcccaa tggtaccaac gatgttccct ccaccaaagg tgttcttatg 1020

tagtgacacc gattatttaa agctgcagca tacgatatat atacatgtgt atatatgtat 1080

acctatgaat gtcagtaagt atgtatacga acagtatgat actgaagatg acaaggtaat 1140

gcatcattct atacgtgtca ttctg 1165

<210> 15

<211> 2228

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

tcgaggagaa cttctagtat atccacatac ctaatattat tgccttatta aaaatggaat 60

cccaacaatt acatcaaaat ccacattctc ttcaaaatca attgtcctgt acttccttgt 120

tcatgtgtgt tcaaaaacgt tatatttata ggataattat actctatttc tcaacaagta 180

attggttgtt tggccgagcg gtctaaggcg cctgattcaa gaaatatctt gaccgcagtt 240

aactgtggga atactcaggt atcgtaagat gcaagagttc gaatctctta gcaaccatta 300

tttttttcct caacataacg agaacacaca ggggcgctat cgcacagaat caaattcgat 360

gactggaaat tttttgttaa tttcagaggt cgcctgacgc atataccttt ttcaactgaa 420

aaattgggag aaaaaggaaa ggtgagaggc cggaaccggc ttttcatata gaatagagaa 480

gcgttcatga ctaaatgctt gcatcacaat acttgaagtt gacaatatta tttaaggacc 540

tattgttttt tccaataggt ggttagcaat cgtcttactt tctaactttt cttacctttt 600

acatttcagc aatatatata tatatttcaa ggatatacca ttctaatgtc tgcccctatg 660

tctgccccta agaagatcgt cgttttgcca ggtgaccacg ttggtcaaga aatcacagcc 720

gaagccatta aggttcttaa agctatttct gatgttcgtt ccaatgtcaa gttcgatttc 780

gaaaatcatt taattggtgg tgctgctatc gatgctacag gtgtcccact tccagatgag 840

gcgctggaag cctccaagaa ggttgatgcc gttttgttag gtgctgtggc tggtcctaaa 900

tggggtaccg gtagtgttag acctgaacaa ggtttactaa aaatccgtaa agaacttcaa 960

ttgtacgcca acttaagacc atgtaacttt gcatccgact ctcttttaga cttatctcca 1020

atcaagccac aatttgctaa aggtactgac ttcgttgttg tcagagaatt agtgggaggt 1080

atttactttg gtaagagaaa ggaagacgat ggtgatggtg tcgcttggga tagtgaacaa 1140

tacaccgttc cagaagtgca aagaatcaca agaatggccg ctttcatggc cctacaacat 1200

gagccaccat tgcctatttg gtccttggat aaagctaatc ttttggcctc ttcaagatta 1260

tggagaaaaa ctgtggagga aaccatcaag aacgaattcc ctacattgaa ggttcaacat 1320

caattgattg attctgccgc catgatccta gttaagaacc caacccacct aaatggtatt 1380

ataatcacca gcaacatgtt tggtgatatc atctccgatg aagcctccgt tatcccaggt 1440

tccttgggtt tgttgccatc tgcgtccttg gcctctttgc cagacaagaa caccgcattt 1500

ggtttgtacg aaccatgcca cggttctgct ccagatttgc caaagaataa ggttgaccct 1560

atcgccacta tcttgtctgc tgcaatgatg ttgaaattgt cattgaactt gcctgaagaa 1620

ggtaaggcca ttgaagatgc agttaaaaag gttttggatg caggtatcag aactggtgat 1680

ttaggtggtt ccaacagtac caccgaagtc ggtgatgctg tcgccgaaga agttaagaaa 1740

atccttgctt aaaaagattc tcttttttta tgatatttgt acataaactt tataaatgaa 1800

attcataata gaaacgacac gaaattacaa aatggaatat gttcataggg tagacgaaac 1860

tatatacgca atctacatac atttatcaag aaggagaaaa aggaggatag taaaggaata 1920

caggtaagca aattgatact aatggctcaa cgtgataagg aaaaagaatt gcactttaac 1980

attaatattg acaaggagga gggcaccaca caaaaagtta ggtgtaacag aaaatcatga 2040

aactacgatt cctaatttga tattggagga ttttctctaa aaaaaaaaaa atacaacaaa 2100

taaaaaacac tcaatgacct gaccatttga tggagtttaa gtcaatacct tcttgaagca 2160

tttcccataa tggtgaaagt tccctcaaga attttactct gtcagaaacg gccttacgac 2220

gtagtcga 2228

<210> 16

<211> 2842

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60

cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120

ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180

accataaatt cccgttttaa gagcttggtg agcgctagga gtcactgcca attatttttt 240

gctttttctc ttgaggtcac atgatcgcaa aatggcaaat ggcacgtgaa gctgtcgata 300

ttggggaact gtggtggttg gcaaatgact aattaagtta gtcaaggcgc catcctcatg 360

aaaactgtgt aacataataa ccgaagtgtc gaaaaggtgg caccttgtcc aattgaacac 420

gctcgatgaa aaaaataaga tatatataag gttaagtaaa gcgtctgtta gaaaggaagt 480

ttttcctttt tcttgctctc ttgtcttttc atctactatt tccttcgtgt aatacagggt 540

cgtcagatac atagatacaa ttctattacc cccatccata caatgccatc tcatttcgat 600

actgttcaac tacacgccgg ccaagagaac cctggtgaca atgctcacag atccagagct 660

gtaccaattt acgccaccac ttcttatgtt ttcgaaaact ctaagcatgg ttcgcaattg 720

tttggtctag aagttccagg ttacgtctat tcccgtttcc aaaacccaac cagtaatgtt 780

ttggaagaaa gaattgctgc tttagaaggt ggtgctgctg ctttggctgt ttcctccggt 840

caagccgctc aaacccttgc catccaaggt ttggcacaca ctggtgacaa catcgtttcc 900

acttcttact tatacggtgg tacttataac cagttcaaaa tctcgttcaa aagatttggt 960

atcgaggcta gatttgttga aggtgacaat ccagaagaat tcgaaaaggt ctttgatgaa 1020

agaaccaagg ctgtttattt ggaaaccatt ggtaatccaa agtacaatgt tccggatttt 1080

gaaaaaattg ttgcaattgc tcacaaacac ggtattccag ttgtcgttga caacacattt 1140

ggtgccggtg gttacttctg tcagccaatt aaatacggtg ctgatattgt aacacattct 1200

gctaccaaat ggattggtgg tcatggtact actatcggtg gtattattgt tgactctggt 1260

aagttcccat ggaaggacta cccagaaaag ttccctcaat tctctcaacc tgccgaagga 1320

tatcacggta ctatctacaa tgaagcctac ggtaacttgg catacatcgt tcatgttaga 1380

actgaactat taagagattt gggtccattg atgaacccat ttgcctcttt cttgctacta 1440

caaggtgttg aaacattatc tttgagagct gaaagacacg gtgaaaatgc attgaagtta 1500

gccaaatggt tagaacaatc cccatacgta tcttgggttt cataccctgg tttagcatct 1560

cattctcatc atgaaaatgc taagaagtat ctatctaacg gtttcggtgg tgtcttatct 1620

ttcggtgtaa aagacttacc aaatgccgac aaggaaactg acccattcaa actttctggt 1680

gctcaagttg ttgacaattt aaagcttgcc tctaacttgg ccaatgttgg tgatgccaag 1740

accttagtca ttgctccata cttcactacc cacaaacaat taaatgacaa agaaaagttg 1800

gcatctggtg ttaccaagga cttaattcgt gtctctgttg gtatcgaatt tattgatgac 1860

attattgcag acttccagca atcttttgaa actgttttcg ctggccaaaa accatgagtg 1920

tgcgtaatga gttgtaaaat tatgtataaa cctactttct ctcacaagta ctatactttt 1980

ataaaacgaa ctttattgaa atgaatatcc tttttttccc ttgttacatg tcgtgactcg 2040

tactttgaac ctaaattgtt ctaacatcaa agaacagtgt taattcgcag tcgagaagaa 2100

aaatatggtg aacaagactc atctacttca tgagactact ttacgcctcc tataaagctg 2160

tcacactgga taaatttatt gtaggaccaa gttacaaaag aggatgatgg aggtttcttt 2220

acaataaaga agcacatgtg tgttaacgtt tttagtattt gcttgttatg taaatcagga 2280

aaacttcgcg ggatttggtt ggatgctact ttccatacaa taaatattat agatctaaaa 2340

agccaaatta caagtaaaga ttagtaaagc tgttggaatt ccatcgttga taaaaatgtt 2400

agttattaaa tataaaagtc agaataggtg aacttggatt taattgttgg catttcgttg 2460

ctgctagagg ccataatatt agatagccag gacatactag ttctcctcgt ggtataggaa 2520

tccataaaat ggaattggtg attctatgtg atatattcac attcttacta cattatcaat 2580

ccttgcactt cagcttcctc taacctcgat gacatcttct cataacttat gtcatcatct 2640

aacgccgtct attataatat attgatagta taagtattag ttgatagaca atagtggatt 2700

tttattccaa cagtgtcttt gttcgtctca gatatagtcg gattgccctt ttaagcaatc 2760

aatagtgttt tatttgcaac aatgtcgtca tagtttaata tgtcctataa gatgttaact 2820

tgctcaacat tcaacaaagt tt 2842

Claims

1. A metabolic engineering method based on m6A methyltransferase IME4, characterized by comprising:

2. Application of the m6A methyltransferase IME 4-based metabolic engineering method according to claim 1 in construction of saccharomyces cerevisiae engineering bacteria.

3. The lanosterol-producing engineering bacteria are characterized in that the lanosterol-producing engineering bacteria take saccharomyces cerevisiae as an initial strain and overexpress IME4, tHMGR, UPC2-1 and ERG9 genes; wherein the content of the first and second substances,

4. The lanosterol-producing engineered bacterium according to claim 3,

the nucleotide sequences of the tHMGR and UPC2 genes are respectively shown in SEQ ID NO. 1-2;

the nucleotide sequence of the UPC2-1 gene is shown in SEQ ID NO. 3;

the nucleotide sequences of the PGK1p promoter and the TEF1p promoter are respectively shown in SEQ ID NO. 4-5.

5. The lanosterol-producing engineered bacterium of claim 3,

the saccharomyces cerevisiae is saccharomyces cerevisiae BY 4741.

6. A construction method of lanosterol-producing engineering bacteria is characterized by comprising the following steps:

overlapping a gene tHMGR, a promoter TDH2p and a terminator ADH1t to construct a gene expression module TDH2p-tHMER-ADH1 t;

overlapping a promoter TEF1p and a screening marker LEU to construct an expression module LEU-TEF1 p;

transforming a yeast strain with the expression module MET-PGK1p, and screening and culturing SD-MET by using a yeast defect type culture medium to obtain a strain ST 01;

7. The method for constructing lanosterol-producing engineering bacteria according to claim 6,

the nucleotide sequences of the tHMGR gene and the UPC2 gene are respectively shown in SEQ ID NO. 1-2;

the nucleotide sequence of the UPC2-1 gene is shown in SEQ ID NO. 3;

the PGK1p, TEF1p, TDH2p and TPI1p are promoter sequences, and corresponding nucleotide sequences are respectively shown in SEQ ID NO. 4-7;

the ADH1t and CYC1t are terminator sequences, and corresponding nucleotide sequences are respectively shown in SEQ ID NO. 8-9;

the nucleotide sequence of the screening marker MET is shown as SEQ ID NO. 10;

the nucleotide sequence of the screening marker URA is shown as SEQ ID NO. 11;

the nucleotide sequence of the screening marker LEU is shown as SEQ ID NO. 12.

8. The construction method of the lanosterol-producing engineering bacteria according to claim 6, wherein the Saccharomyces cerevisiae is Saccharomyces cerevisiae BY 4741.

9. An application of the lanosterol-producing engineering bacteria constructed according to the lanosterol-producing engineering bacteria of any one of claims 3 to 5 or the lanosterol-producing engineering bacteria constructed according to the construction method of any one of claims 6 to 8 in the production of lanosterol.

10. A method for producing lanosterol, which is characterized by comprising the following steps:

activating the lanosterol-producing engineering bacteria of any one of claims 3 to 5 or the lanosterol-producing engineering bacteria constructed by the construction method of any one of claims 6 to 8, inoculating the activated lanosterol-producing engineering bacteria into a fermentation culture medium, and performing fermentation culture to obtain lanosterol.