CN112812983B

CN112812983B - Saccharomyces cerevisiae engineering bacterium for producing campesterol and construction method thereof

Info

Publication number: CN112812983B
Application number: CN202110176165.4A
Authority: CN
Inventors: 饶志明; 周武林; 邵明龙; 杨套伟; 张显; 徐美娟
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2021-02-06
Filing date: 2021-02-06
Publication date: 2022-10-11
Anticipated expiration: 2041-02-06
Also published as: CN112812983A

Abstract

The invention discloses a saccharomyces cerevisiae engineering bacterium for producing campesterol and a construction method thereof, belonging to the field of genetic engineering and biological engineering. According to the invention, an expression cassette element of 7-dehydrocholesterol reductase is introduced into a yeast cell body, a CRISPR/Cas9 gene operation system is integrated into a yeast genome, C-22 sterol desaturase is also knocked out, the competition of an ergosterol branch pathway is relieved, and the yeast synthesis from glucose to campesterol is realized. The method has simple process and environmental protection, and can be used for producing the campesterol by fermentation. The yield of the campesterol prepared BY the saccharomyces cerevisiae engineering strain BY 4742-delta erg5-TEF1p-dhcr7-ADH1t can reach 253.35mg/L in the shaking stage, the horizontal yield of a fermentation tank can reach 916.88mg/L, and is improved BY 2.6 times compared with the yield of the campesterol of the strain in the shaking stage.

Description

Saccharomyces cerevisiae engineering bacterium for producing campesterol and construction method thereof

Technical Field

The invention relates to a saccharomyces cerevisiae engineering bacterium for producing campesterol and a construction method thereof, belonging to the field of genetic engineering and biological engineering.

Background

In recent years, steroid drugs have been highly regarded by the biomedical industry because of their excellent anti-inflammatory, antiallergic, and contraceptive effects, and their market needs have been high, becoming the second largest drug after antibiotics. With the increasing demand of people for steroid hormone drugs, the production process of the steroid hormone drugs in the world is undergoing several changes, including chemical total synthesis, saponin extraction from plants and chemical semi-synthesis, novel microbial synthesis and other stages, and until now, the synthesis process of the steroid hormone drugs is continuously explored and innovated. Campesterol is an important synthetic precursor of steroidal drugs (progesterone, androstenedione, hydrocortisone, etc.) and is also one of the major sterols of plant origin. The structure of the compound is only one more methyl at the C24 position compared with the main sterol (cholesterol) of animal origin, and the compound is different from the main sterol (ergosterol) of microbial origin in that the C7-8 and C22-23 positions are saturated single bonds. Therefore, the method provides a feasible theoretical basis for producing the campesterol by the microbial catalysis method.

At present, steroid drugs are mainly produced by two main ways, namely chemical synthesis and biocatalysis. Since the chemical synthesis method causes serious environmental pollution and the intermediate steps are complicated, the biological catalysis is very concerned because of its environmental protection and simple reaction process. However, the steroid drugs and their intermediates have poor water solubility and are difficult to be used in and out of cells, resulting in low biotransformation rate for the biocatalytic production of steroid drugs. Although methods of adding a cosolvent and adding a substrate by dissolving it in an organic solvent have been proposed to improve the utilization of the substrate by microorganisms, the conversion rate is still not very high. With the development of synthetic biology, the synthesis of precursor compounds of high-value drugs such as campesterol by using microorganisms has become a development trend. The microbial synthesis has the characteristics of short period, high yield, mild conditions, environmental protection, controllable process and the like, so that the microbial synthesis can be used as a novel production mode for producing steroid medicaments.

A natural sterol synthesis way exists in a yeast cell body, so that the yeast cell body can be used as an excellent chassis cell for synthesizing steroid medicines. In the prior art, although there are reports of the production of campesterol by using yeast cells, the activity of the key enzyme 7 dehydrocholesterol reductase DHCR7 in the pathway (shown in figure 3) is not high, thereby limiting the high-efficiency production of the campesterol. Therefore, how to obtain a method for efficiently producing campesterol becomes a hotspot of research.

Disclosure of Invention

Technical problem

The invention aims to solve the technical problems of providing a saccharomyces cerevisiae engineering bacterium for efficiently producing campesterol, a construction method thereof and a method for efficiently producing the campesterol by using the saccharomyces cerevisiae engineering bacterium.

Technical scheme

In order to solve the problems, the invention provides a saccharomyces cerevisiae engineering bacterium, which is knocked out of C-22 sterol desaturase Erg5 and contains 7-dehydrocholesterol reductase derived from Pangasianodon hyphenallus.

In one embodiment of the invention, the amino acid sequence of the 7-dehydrocholesterol reductase is set forth in SEQ ID NO 1.

In one embodiment of the invention, the nucleotide sequence of the 7-dehydrocholesterol reductase is set forth in SEQ ID NO 2.

In one embodiment of the invention, the amino acid sequence of the C-22 sterol desaturase gene is set forth in SEQ ID NO 3.

In one embodiment of the invention, the nucleotide sequence of the C-22 sterol desaturase gene is set forth in SEQ ID NO 4.

In one embodiment of the invention, the saccharomyces cerevisiae engineering bacteria take saccharomyces cerevisiae BY4742 as a host cell.

In one embodiment of the invention, the saccharomyces cerevisiae engineering bacteria further comprise a promoter, wherein the promoter is used for enhancing the expression of 7-dehydrocholesterol reductase; the promoter is one or more of PGK1p, GPM1p, TDH3p, TEF1p, TPI1p, GPD1p, TEF2p, ACT1p, GAL1p and TDH2p.

In one embodiment of the present invention, the nucleotide sequences of the promoters PGK1p, GPM1p, TDH3p, TEF1p, TPI1p, GPD1p, TEF2p, ACT1p, GAL1p and TDH2p are shown in SEQ ID NO 5 to SEQ ID NO 14.

The invention also provides a method for constructing the saccharomyces cerevisiae engineering bacteria, which comprises the following steps:

(1) Construction of the expression cassette: constructing an upstream homology arm of the integration site, a yeast promoter, a 7-dehydrocholesterol reductase gene, a yeast terminator and a downstream homology arm of the integration site to obtain a 7-dehydrocholesterol reductase expression cassette element; introducing the obtained 7-dehydrocholesterol reductase expression cassette element and knockout plasmid containing Cas9 protein into a yeast cell body in a yeast transformation mode, and completing assembly of the element by homologous recombination of yeast to obtain a complete expression cassette;

(2) The expression cassette is inserted into the 1114a site on the chromosome ChrXI of Saccharomyces cerevisiae BY4742 yeast, and C-22 sterol desaturase is knocked out to successfully construct the Saccharomyces cerevisiae engineering bacteria.

In one embodiment of the invention, the yeast promoter is one or more of PGK1p, GPM1p, TDH3p, TEF1p, TPI1p, GPD1p, TEF2p, ACT1p, GAL1p and TDH2p.

In one embodiment of the present invention, the yeast terminator is ADH1t.

The invention also provides a method for producing campesterol, which is to prepare the campesterol by adopting the saccharomyces cerevisiae engineering bacteria.

In one embodiment of the present invention, the method is: inoculating the saccharomyces cerevisiae engineering bacteria into a seed culture medium to prepare a seed solution; transferring the seed solution into a fermentation culture medium, collecting cultured cells, adding a saponification reaction solution, adding n-hexane for extraction after the reaction is finished, and preparing the campesterol.

In one embodiment of the present invention, the saponification reaction liquid is 20% to 30% KOH (or NaOH) and 50% C ₂ H ₅ OH (or CH) ₃ OH)。

In one embodiment of the present invention, 2mL of saponification reaction solution (20% -30% KOH or NaOH and 50% C) is added to the collected cells ₂ H ₅ OH or CH ₃ OH), adding 100 mu L of 1g/L cholesterol as an internal standard, and carrying out warm bath for 2-3h at the temperature of 85-100 ℃.

In one embodiment of the invention, after the reaction is finished, 6mL of n-hexane is added to fully shake and extract, the upper layer of extract is taken out and dried by nitrogen in a nitrogen blowing instrument, and 1mL of chromatographic grade methanol is added to redissolve.

The invention also provides application of the saccharomyces cerevisiae engineering bacteria in preparation of campesterol and a product containing the campesterol.

Advantageous effects

(1) The invention utilizes metabolic engineering and synthetic biology technology, constructs the synthetic route of campesterol by introducing exogenous 7-dehydrocholesterol reductase in saccharomyces cerevisiae, and removes the competitive route by knocking out C-22 sterol desaturase, thereby realizing high-efficiency biosynthesis of campesterol. The whole production process is convenient and quick, has no pollution, and has very wide application prospect. The engineering strain for producing the campesterol saccharomyces cerevisiae provides a feasible method for the microbial production of steroid drugs.

(2) The yield of the campesterol prepared BY the engineering strain BY 4742-delta erg5-TEF1p-dhcr7-ADH1t for producing the campesterol can reach 253.35mg/L in the shaking stage, and can reach 916.88mg/L in the 5-L fermentation tank level, which is 2.6 times higher than the yield of the campesterol of the strain in the shaking stage.

(3) The 7-dehydrocholesterol reductase derived from Pangasianodon hyphenalimus in the invention shows the highest campesterol yield of 216.93 +/-9.42 mg/L, which is 22.6% higher than the campesterol yield (167.91 +/-6.00 mg/L) of the 7-dehydrocholesterol reductase derived from Danio reio reported in the literature.

Drawings

FIG. 1: the component analysis total ion chromatogram of the campesterol produced by fermenting the saccharomyces cerevisiae engineering bacteria.

FIG. 2: and (3) producing the campesterol by fermenting the saccharomyces cerevisiae engineering bacteria.

FIG. 3: a synthetic route diagram of campesterol in saccharomyces cerevisiae.

FIG. 4: and (3) preparing the yield of the campesterol by using the saccharomyces cerevisiae engineering bacteria containing different promoters in the shake flask stage.

FIG. 5: and (3) fed-batch fermentation results of a saccharomyces cerevisiae engineering bacterium 5L fermentation tank.

Detailed Description

The adjustment of the various aspects of the present invention will be illustrated in detail by the preferred examples of the synthesis of campesterol in conjunction with the drawings. It will be understood by those skilled in the art that the specific examples are for purposes of illustration only and are not intended to limit the scope of the present invention. Those skilled in the art may make modifications to the various aspects of the invention without being limited by the scope of the claims Li Quanli, but such modifications are within the scope of the invention. For example, the replacement of the promoter or terminator used in the present experimental example with other promoters or terminators commonly used in the art can be understood and implemented by other persons skilled in the art.

In addition, it should be noted that the various materials and reagents used in the following specific examples are those commonly used in the art, unless otherwise specified. Are available from normal commercial sources; the methods employed are all conventional methods for the skilled worker.

The media involved in the following examples are as follows:

YPD solid Medium: 20g/L glucose, 20g/L peptone and 10g/L yeast extract, and 2% agar powder is added.

Liquid YPD medium: 20g/L glucose, 20g/L peptone and 10g/L yeast extract.

Liquid YPG medium: galactose 20g/L, peptone 20g/L, yeast extract 10g/L.

Fermentation medium in fermentation in a fermenter: 50g/L glucose, 20g/L peptone and 10g/L yeast extract.

The detection methods referred to in the following examples are as follows:

and (3) detecting the glucose content: measuring by using a biosensor analyzer SBA-40E;

the galactose content was measured by HPLC method: and (4) observing a detector RID, wherein a chromatographic column is a cyanogen column, the column temperature is 80 ℃, a mobile phase is ultrapure water, the flow rate is 0.6mL/min, and the detection temperature is 55 ℃.

Method for detecting campesterol content

The product was filtered through a 0.22 μm membrane and analyzed by ThermoFisher HPLC using a Diamonsil C18,5 μm,250 mm. Times.4.6 mm column, UV detector. Campesterol and cholesterol are detected at 205nm, and the mobile phase is composed of pure methanol; the flow rate was 1mL/min and the column temperature was 30 ℃.

Gas chromatography-mass spectrometer (GC-MS) determination: chromatographic column DB-5MS; 1 μ L of sample introduction is carried out at a split ratio of 10; heating at 80 deg.C for 1min, heating to 300 deg.C at 20 deg.C/min, and maintaining for 20min; the mass spectrum range is 50-700m/z.

Example 1: acquisition of related genes in campesterol synthetic pathway

(1) Obtaining of 7-dehydrocholesterol reductase Gene

According to the amino acid sequence of the 7-dehydrocholesterol reductase DHCR7 (Genbank registration serial number is XP _ 026786162) from Pangasianodon hyphenallus, the codons of the 7-dehydrocholesterol reductase gene have yeast preference by saccharomyces cerevisiae codon optimization, and the generated optimized gene sequence, namely the nucleotide sequence of the gene DHCR7 for coding the 7-dehydrocholesterol reductase, is shown as SEQ ID NO 2.

(2) Acquisition of upstream and downstream homology arms of erg5 Gene locus

Designing primers 5'-TGGGAATACTGTACCAGATAATCAAACAT-3' and 5'-CAAAGTTCTGTTTTTCCCCATTTGTTAAAAGGTATTTATTGTCTATTGGAATAGC-3' according to sequences of an upstream homologous arm and a downstream homologous arm of an erg5 gene site in a saccharomyces cerevisiae genome, and amplifying the upstream homologous arm of the erg5 gene site; primers 5'-ATAAATACCTTTTAACAAATGGGGAAAAACAGAACTTTGTCCAGAC-3' and 5'-TGACAGTGACGAACGCTTCAG-3' are designed, downstream homology arms of erg5 gene sites are amplified, a BY4742 genome is used as a template, and the Extaq enzyme is used for PCR amplification.

(3) Acquisition of homology arms upstream and downstream of integration site

Designing a primer fragment according to an online website CASDesigner, selecting a 1114a gene integration site on yeast ChrXI, inputting a corresponding gene sequence and a promoter, and automatically generating a primer sequence capable of cloning upstream and downstream homology arms, wherein an upstream primer 5'-GAGAAATGTTGGGATCCAGAAGAATGA-3' and a downstream primer 5'-TATACGCTATTATCAGCCAAATAGTAAATAGGCTTCGTCTTTATAAGATATATACAGC-3' are used for amplifying the upstream homology arm of the 1114a integration site, an upstream primer 5'-TTCACCCAATTGTAGATATGCTAACTCCCATCATCTAACATCGTGAAACGAATCAG-3' and a downstream primer 5'-AGATAAGAAGTGGGAAGGTAAAATCGAATAC-3' are used for amplifying the downstream homology arm of the 1114a integration site, and an Extaq enzyme is used for PCR amplification BY taking a BY4742 genome as a template.

(4) The chemically synthesized nucleotide sequences are shown as promoters PGK1p, GPM1p, TDH3p, TEF1p, TPI1p, GPD1p, TEF2p, ACT1p, GAL1p and TDH2p shown in SEQ ID NO 5-SEQ ID NO 14.

Example 2: obtaining of saccharomyces cerevisiae engineering strain for producing campesterol bacteria

The method comprises the following specific steps:

(1) Construction of the expression cassette: constructing an expression cassette element of the 7-dehydrocholesterol reductase by using the upstream homology arm of the integration site, the yeast promoter, the 7-dehydrocholesterol reductase gene, the yeast terminator and the downstream homology arm of the integration site, which are prepared in the example 1; introducing the obtained 7-dehydrocholesterol reductase expression cassette element and knockout plasmid containing Cas9 protein into a saccharomyces cerevisiae BY4742 cell body in a yeast transformation mode, and completing assembly of the element BY homologous recombination of yeast to obtain a complete expression cassette;

the genetic manipulation methods involved are as follows:

donor DNA fragments were PCR amplified by using primers generated by CASdesigner to construct an integrated expression cassette (typically containing two 1-kb flanking homology regions, a promoter, a gene sequence and a terminator) containing the 1-kb flanking homology region targeted to the selected genomic site, which was then co-transformed with a Cas9-gRNA plasmid (pCut) targeting the gene into yeast cells. In addition, the primers designed by CASDesigner provide 30-60bp homology arms between fragments, so that 1-5 separate fragments in yeast can be subjected to homologous recombination and self-assembly. The pCut plasmid targeting the genomic locus was assembled in vivo from a linear backbone and a linear PCR fragment containing the new gRNA sequence. New sgrnas were generated by online sgRNA design tools.

The specific transformation method of yeast by using the lithium acetate method is as follows:

fresh overnight-cultured yeast cells were inoculated into 2 XYPD medium to an OD600nm of 0.2, and cultured at 30 ℃ and 200rpm to an OD600nm of 1.0. Then, 5mL of the culture broth was collected and centrifuged at 8000rpm for 5min, and washed twice with 2.5mL of H2O. The cell pellet was resuspended in 50. Mu.L of an aqueous solution containing the donor DNA fragment (2. Mu.g) and pCUT plasmid (0.25 ng), and the resulting suspension was added to the transformation reaction solution (260. Mu.L 50% PEG3350, 36. Mu.L 1M LiOAc, 10. Mu.L ssDNA, 4. Mu. L H2O) and mixed. The mixture was incubated at 42 ℃ for 40 minutes and the precipitate was collected by centrifugation at 6000rpm for 1 minute. Cell pellets were resuspended in 500. Mu. L H ₂ O, then taking 100 mu L H ₂ O was spread evenly on selective agar plates (SC-U). The results of the integration were verified by sequencing and the correct colonies were picked for downstream experimental manipulation after plasmid elimination.

(2) Inserting the expression cassette into 1114a site on chromosome ChrXI of Saccharomyces cerevisiae BY4742 yeast, completing the assembly of elements BY homologous recombination of yeast itself, obtaining a yeast cell BY4742-GAL1p-dhcr7-ADH1t integrated with 7-dehydrocholesterol reductase;

(3) Cloning upstream and downstream homologous arms of C-22 sterol desaturase erg5 gene BY PCR to obtain gene fragments, utilizing CRISPR/Cas9 gene editing technology, designing new sgRNA in the step (1) to be capable of positioning to chromosome erg5 gene, introducing the upstream and downstream homologous arms and knockout plasmid containing Cas9 protein and new sgRNA into yeast cells BY the transformation method shown in the step (1), and finally replacing erg5 gene in a yeast homologous recombination manner to realize knockout of erg5 gene in the yeast cell BY4742-GAL1p-dhcr7-ADH1t integrated with 7-dehydrocholesterol reductase obtained in the step (2) to obtain a recombinant strain BY 4742-delta erg5-GAL1p-dhcr7-ADH1t;

(4) Designing sgRNA capable of being positioned to GAL1p promoter BY using CRISPR/Cas9 gene editing technology and according to the mode of the step (1), introducing a promoter fragment with a corresponding homologous arm and a knockout plasmid containing Cas9 protein and new sgRNA into a yeast cell BY the transformation method shown in the step (1), and finally replacing the promoter of the saccharomyces cerevisiae engineering strain BY4742 delta erg5-GAL1p-dhcr7-ADH1t constructed in the step (3) from GAL1p to PGK1p, GPM1p, TDH3p, TEF1p, TPI1p, GPD1p, TEF2p, ACT1p and TDH2p respectively in a mode of homologous recombination of yeast to obtain the saccharomyces cerevisiae engineering strain: BY 4742-DELTA erg5-PGK1p-dhcr7-ADH1t, BY 4742-DELTA erg5-GPM1p-dhcr7-ADH1t, BY 4742-DELTA erg5-TDH3p-dhcr7-ADH1t, BY 4742-DELTA erg5-TEF1p-dhcr7-ADH1t, BY 4742-DELTA erg5-TPI1p-dhcr7-ADH1t, BY 4742-DELTA erg5-GPD1p-dhcr7-ADH1t, BY 4742-DELTA erg 5-DELTA erg 2p-dhcr7-ADH1t, BY 4742-DELTA erg5-TEF2p-dhcr7-ADH1t, BY 4742-DELTA erg 5-DELTA erg 1p-dhcr7-ADH1t, ADH 4742-DELTA erg 5-dhcr 2p-dhcr7-ADH1t.

Example 3: fermentation of saccharomyces cerevisiae engineering strain for producing campesterol bacteria

The method comprises the following specific steps:

(1) And (3) shake flask stage fermentation:

respectively culturing engineered Saccharomyces cerevisiae BY 4742-delta erg5-GAL1p-dhcr7-ADH1t, BY 4742-delta erg5-PGK1p-dhcr7-ADH1t, BY 4742-delta erg5-GPM1p-dhcr7-ADH1t, BY 4742-delta erg5-TDH3p-dhcr7-ADH1t, BY 4742-delta erg5-TEF1p-dhcr7-ADH1t, BY 4742-delta erg5-TPI1p-dhcr7-ADH1t, BY 4742-delta erg5-GPD1p-dhcr7-ADH1t, BY 4742-delta erg5-TEF2p-dhcr 1t, BY 4742-delta erg5-ACT1p-dhcr7-ADH1t, and active solid on a streaking medium, after 48 hours of growth, a single colony is obtained;

single colonies were picked up and inoculated into 10mL/50mL vials of liquid YPD medium and cultured at 30 ℃ and 200rpm for 24 hours to obtain seed solutions. The seed liquid obtained by the preparation was inoculated into 50mL/250mL of liquid YPD medium at an inoculum size of 2% (v/v), fermented at 30 ℃ and 200rpm for 48 hours, and then yeast cells cultured in the YPD liquid medium were placed in a 50mL sterile centrifuge tube, centrifuged at 8000rpm for 5 minutes, the supernatant was removed, and the cells were collected and transferred to 50mL/250mL of liquid YPG medium for further fermentation for 96 hours. Collecting cells, and extracting and detecting campesterol.

(2) Fermentation in 5L fermenter stage

Selecting the single colony obtained in the step (1) to be inoculated in 10mL of liquid YPD medium, and culturing at 30 ℃ and 200rpm for 24h to obtain first-stage seed liquid; inoculating the primary seed solution into 100mL of liquid YPD medium with an inoculum size of 5% (v/v), and culturing at 30 ℃ and 200rpm for 24h to obtain a secondary seed solution; the secondary seed solution was inoculated at an inoculum size of 10% (v/v) into 2L of fermentation medium.

Fermentation parameters in a 5L fermentation tank were controlled to control temperature, pH, dissolved oxygen and aeration at 30 deg.C, 5.5, >30% and 2vvm, respectively. And (3) monitoring the residual sugar amount in the fermentation liquor, beginning to supplement galactose when the glucose in the culture medium is completely consumed, immediately supplementing the galactose to 40g/L when the galactose is quickly consumed, taking 10mL of the fermentation liquor into a 50mL centrifuge tube until the fermentation is finished, centrifuging at 8000rpm for 5min, removing the supernatant, collecting thalli, and extracting and detecting the campesterol.

Example 4: extraction and detection of fermentation product of saccharomyces cerevisiae engineering bacteria for producing campesterol

The method comprises the following specific steps:

(1) Measuring the OD600 of the fermentation broth, keeping the amount of bacteria less than 10OD600 per mL of liquid (if exceeding, diluting);

(2) 1mL of the diluted cells obtained in example 3 in the shake flask stage and the fermentor stage in step (1) was taken and 2mL of the saponification reaction solution (20% KOH-50% by weight C) ₂ H ₅ OH), 100. Mu.L of 1g/L cholesterol was added as an internal standard, and the mixture was incubated at 85 ℃ for 2 hours.

After the reaction is finished, 6mL of normal hexane is added for full oscillation and extraction, the upper layer of extract is taken out and dried by nitrogen in a nitrogen blowing instrument, and 1mL of chromatographic grade methanol is added for redissolution. The contents of the prepared campesterol were measured (results are shown in fig. 1 to 2, and fig. 4 to 5), respectively, as shown in tables 1 and 2:

table 1: the content of campesterol prepared by saccharomyces cerevisiae engineering bacteria containing different promoters in the shake flask stage

Table 2: the content of campesterol prepared by saccharomyces cerevisiae engineering bacteria containing different promoters in the fermentation tank stage

As can be seen from Table 1, when the Saccharomyces cerevisiae engineering bacteria constructed by using different promoters to carry the gene dhcr7 for encoding 7-dehydrocholesterol reductase are adopted, the yield difference of campesterol is very obvious, and the initial promoter GAL1p shows higher yield of the campesterol 216.93mg/L, but the promoter TEF1p shows the highest yield of the campesterol 253.35mg/L, which is 18.74 percent higher than that of the initial promoter.

As can be seen from Table 2, when the engineering strain is subjected to high-density fermentation in a 5L fermentor BY adopting a fed-batch fermentation strategy, the yield of the campesterol can be further increased, and the highest yield of the campesterol of 916.88mg/L can be finally achieved BY adopting the recombinant strain BY 4742-delta erg5-TEF1p-dhcr7-ADH1t, which is 2.6 times higher than the yield of the campesterol of the strain in the shake flask stage.

Comparative example 1

The present invention is different from examples 1 to 4 in that 7-dehydrocholesterol reductase derived from Pangasolondon hyphenalumus 7-dehydrocholesterol reductase of the present application is regulated to 7-dehydrocholesterol reductase derived from Labeo rohita (whose amino acid sequence is shown in SEQ ID NO.15 and whose GENBANK registration number is RXN 17635.1), triprophysa tibetana (whose amino acid sequence is shown in SEQ ID NO.16 and whose GENBANK registration number is KAA 0723749.1), carassius auratus (whose amino acid sequence is shown in SEQ ID NO.17 and whose GENBANK registration number is NP 026124274.1), and Danio rerio amino acid sequence is shown in SEQ ID NO.18 (whose GENBANK registration number is NP-958487.2), respectively (7-dehydrocholesterol reductase derived from Danio is shown in Biotech mountain test paper), and the results are shown in Biotech et 7-cholesterol reductase 7-dehydrocholesterol reductase 1033, wa7-dehydrocholesterol reductase found in Biotech et 9:

table 3: the content of campesterol is obtained by using saccharomyces cerevisiae engineering bacteria containing 7-dehydrocholesterol reductase from different sources in the shake flask stage

As can be seen from Table 3, the Pangasianon hyphenalimus-derived 7-dehydrocholesterol reductase of the present application showed the highest campesterol production of 216.93. + -. 9.42mg/L, which is 22.6% higher than the reported campesterol production (167.91. + -. 6.00 mg/L) of Danio rerio-derived 7-dehydrocholesterol reductase.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by one skilled in the art without departing from the spirit and scope of the invention as defined by the appended claims.

SEQUENCE LISTING

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tgggttgcta atgcttatca ttttcattgg ttttctccaa ccattatttt tgataattgg 540

attccattgt tgtggtgcac caatattttg ggatacaccg tttctatatt tgtgttcatt 600

aaggcttatt tgtttccaac caatcccgaa gattgcaagt ttactggtaa cctcttttac 660

gactttatga tgggaataga gtttaatcca aggattagaa agtggtttga ttttaaattg 720

ttttttaatg gtagaccagg tattgttgct tggaccttga ttaatttgtc ttatgctgct 780

aaacaacaag aattgtatgg tcatgttacg aacagtatga ttctcgtgaa cgtcttgcaa 840

gctatttatg tattggactt tttctggaat gaggcttggt atttgaaaac cattgatatt 900

tgtcatgatc attttggttg gtatttgggt tggggtgatt gtgtttggtt gccatttttg 960

tataccttgc aaggtttgta tttggtttat aatccaattc aattgtctac cccatatgct 1020

tgggctgttt tggttttggg tttggttggt tattatattt ttagatctgc taatcatcaa 1080

aaagatttgt ttagaagaac ccaaggtgaa tgtaaaattt ggggttctaa accaaccttt 1140

attgaatgtt cttatagatc tgctgatggt ggtattcata aatctaaatt gttgacctct 1200

ggtttttggg gtgctgctag acatttgaat tataccggtg atttgttggg tgctttggct 1260

tattgtatgg cttgttcgca tggacatctg ctgccatatt tttatatcgt ttatatgacg 1320

atattgttgt tgcatagatg tattagagat gaacatagat gttcttctaa atatggtaaa 1380

gattggaaaa gatataccgc taccgttaga tatagattgt tgccaggtat ttttta 1436

<210> 3

<211> 538

<212> PRT

<213> Artificial sequence

<400> 3

Met Ser Ser Val Ala Glu Asn Ile Ile Gln His Ala Thr His Asn Ser

1 5 10 15

Thr Leu His Gln Leu Ala Lys Asp Gln Pro Ser Val Gly Val Thr Thr

20 25 30

Ala Phe Ser Ile Leu Asp Thr Leu Lys Ser Met Ser Tyr Leu Lys Ile

35 40 45

Phe Ala Thr Leu Ile Cys Ile Leu Leu Val Trp Asp Gln Val Ala Tyr

50 55 60

Gln Ile Lys Lys Gly Ser Ile Ala Gly Pro Lys Phe Lys Phe Trp Pro

65 70 75 80

Ile Ile Gly Pro Phe Leu Glu Ser Leu Asp Pro Lys Phe Glu Glu Tyr

85 90 95

Lys Ala Lys Trp Ala Ser Gly Pro Leu Ser Cys Val Ser Ile Phe His

100 105 110

Lys Phe Val Val Ile Ala Ser Thr Arg Asp Leu Ala Arg Lys Ile Leu

115 120 125

Gln Ser Ser Lys Phe Val Lys Pro Cys Val Val Asp Val Ala Val Lys

130 135 140

Ile Leu Arg Pro Cys Asn Trp Val Phe Leu Asp Gly Lys Ala His Thr

145 150 155 160

Asp Tyr Arg Lys Ser Leu Asn Gly Leu Phe Thr Lys Gln Ala Leu Ala

165 170 175

Gln Tyr Leu Pro Ser Leu Glu Gln Ile Met Asp Lys Tyr Met Asp Lys

180 185 190

Phe Val Arg Leu Ser Lys Glu Asn Asn Tyr Glu Pro Gln Val Phe Phe

195 200 205

His Glu Met Arg Glu Ile Leu Cys Ala Leu Ser Leu Asn Ser Phe Cys

210 215 220

Gly Asn Tyr Ile Thr Glu Asp Gln Val Arg Lys Ile Ala Asp Asp Tyr

225 230 235 240

Tyr Leu Val Thr Ala Ala Leu Glu Leu Val Asn Phe Pro Ile Ile Ile

245 250 255

Pro Tyr Thr Lys Thr Trp Tyr Gly Lys Lys Thr Ala Asp Met Ala Met

260 265 270

Lys Ile Phe Glu Asn Cys Ala Gln Met Ala Lys Asp His Ile Ala Ala

275 280 285

Gly Gly Lys Pro Val Cys Val Met Asp Ala Trp Cys Lys Leu Met His

290 295 300

Asp Ala Lys Asn Ser Asn Asp Asp Asp Ser Arg Ile Tyr His Arg Glu

305 310 315 320

Phe Thr Asn Lys Glu Ile Ser Glu Ala Val Phe Thr Phe Leu Phe Ala

325 330 335

Ser Gln Asp Ala Ser Ser Ser Leu Ala Cys Trp Leu Phe Gln Ile Val

340 345 350

Ala Asp Arg Pro Asp Val Leu Ala Lys Ile Arg Glu Glu Gln Leu Ala

355 360 365

Val Arg Asn Asn Asp Met Ser Thr Glu Leu Asn Leu Asp Leu Ile Glu

370 375 380

Lys Met Lys Tyr Thr Asn Met Val Ile Lys Glu Thr Leu Arg Tyr Arg

385 390 395 400

Pro Pro Val Leu Met Val Pro Tyr Val Val Lys Lys Asn Phe Pro Val

405 410 415

Ser Pro Asn Tyr Thr Ala Pro Lys Gly Ala Met Leu Ile Pro Thr Leu

420 425 430

Tyr Pro Ala Leu His Asp Pro Glu Val Tyr Glu Asn Pro Asp Glu Phe

435 440 445

Ile Pro Glu Arg Trp Val Glu Gly Ser Lys Ala Ser Glu Ala Lys Lys

450 455 460

Asn Trp Leu Val Phe Gly Cys Gly Pro His Val Cys Leu Gly Gln Thr

465 470 475 480

Tyr Val Met Ile Thr Phe Ala Ala Leu Leu Gly Lys Phe Ala Leu Tyr

485 490 495

Thr Asp Phe His His Thr Val Thr Pro Leu Ser Glu Lys Ile Lys Val

500 505 510

Phe Ala Thr Ile Phe Pro Lys Asp Asp Leu Leu Leu Thr Phe Lys Lys

515 520 525

Arg Asp Pro Ile Thr Gly Glu Val Phe Glu

530 535

<210> 4

<211> 1617

<212> DNA

<213> Artificial sequence

<400> 4

atgagttctg tcgcagaaaa tataatacaa catgccactc ataattctac gctacaccaa 60

ttggctaaag accagccctc tgtaggcgtc actactgcct tcagtatcct ggatacactt 120

aagtctatgt catatttgaa aatatttgct actttaatct gtattctttt ggtttgggac 180

caagttgcat atcaaatcaa gaaaggttcc atcgcaggtc caaagtttaa gttctggccc 240

atcatcggtc catttttgga atccttagat ccaaagtttg aagaatataa ggctaagtgg 300

gcatccggtc cactttcatg tgtttctatt ttccataaat ttgttgttat cgcatctact 360

agagacttgg caagaaagat cttgcaatct tccaaattcg tcaaaccttg cgttgtcgat 420

gttgctgtga agatcttaag accttgcaat tgggtttttt tggacggtaa agctcatact 480

gattacagaa aatcattaaa cggtcttttc actaaacaag ctttggctca atacttacct 540

tcattggaac aaatcatgga taagtacatg gataagtttg ttcgtttatc taaggagaat 600

aactacgagc cccaggtctt tttccatgaa atgagagaaa ttctttgcgc cttatcattg 660

aactctttct gtggtaacta tattaccgaa gatcaagtca gaaagattgc tgatgattac 720

tatttggtta cagcagcatt ggaattagtc aacttcccaa ttattatccc ttacactaaa 780

acatggtatg gtaagaaaac tgcagacatg gccatgaaga ttttcgaaaa ctgtgctcaa 840

atggctaagg atcatattgc tgcaggtggt aagccagttt gtgttatgga tgcttggtgt 900

aagttgatgc acgatgcaaa gaatagtaac gatgatgatt ctagaatcta ccacagagag 960

tttactaaca aggaaatctc cgaagctgtt ttcactttct tatttgcttc tcaagatgcc 1020

tcttcttctt tagcttgttg gttgttccaa attgttgctg accgtccaga tgtcttagct 1080

aagatcagag aagaacaatt ggctgttcgt aacaatgaca tgtctaccga attgaacttg 1140

gatttgattg agaaaatgaa gtacaccaat atggtcataa aagaaacttt gcgttacaga 1200

cctcctgtct tgatggttcc atatgttgtt aagaagaatt tcccagtttc ccctaactat 1260

accgcaccaa agggcgctat gttaattcca accttatacc cagctttaca tgatcctgaa 1320

gtttacgaaa atcctgatga gttcatccct gaaagatggg tagaaggctc taaggctagt 1380

gaagcaaaga agaattggtt ggtttttggt tgtggtccac acgtttgctt aggtcaaaca 1440

tatgtcatga ttaccttcgc cgctttgttg ggtaaatttg cactatatac tgatttccat 1500

catacagtga ctccattaag tgaaaaaatc aaggttttcg ctacaatttt cccaaaagat 1560

gatttgttac tgactttcaa aaagagagac ccaattactg gagaagtctt cgaataa 1617

<210> 5

<211> 502

<212> DNA

<213> Artificial sequence

<400> 5

gtttgcaaaa agaacaaaac tgaaaaaacc cagacacgct cgacttcctg tcttcctatt 60

gattgcagct tccaatttcg tcacacaaca aggtcctagc gacggctcac aggttttgta 120

acaagcaatc gaaggttctg gaatggcggg aaagggttta gtaccacatg ctatgatgcc 180

cactgtgatc tccagagcaa agttcgttcg atcgtactgt tactctctct ctttcaaaca 240

gaattgtccg aatcgtgtga caacaacagc ctgttctcac acactctttt cttctaacca 300

agggggtggt ttagtttagt agaacctcgt gaaacttaca tttacatata tataaacttg 360

cataaattgg tcaatgcaag aaatacatat ttggtctttt ctaattcgta gtttttcaag 420

ttcttagatg ctttcttttt ctctttttta cagatcatca aggaagtaat tatctacttt 480

ttacaacaaa tataaaacaa tg 502

<210> 6

<211> 502

<212> DNA

<213> Artificial sequence

<400> 6

aagttacata tatatatata tatatatata tatatatata tagccatagt gatgtctaag 60

taacctttat ggtatatttc ttaatgtgga aagatactag cgcgcgcacc cacacacaag 120

cttcgtcttt tcttgaagaa aagaggaagc tcgctaaatg ggattccact ttccgttccc 180

tgccagctga tggaaaaagg ttagtggaac gatgaagaat aaaaagagag atccactgag 240

gtgaaatttc agctgacagc gagtttcatg atcgtgatga acaatggtaa cgagttgtgg 300

ctgttgccag ggagggtggt tctcaacttt taatgtatgg ccaaatcgct acttgggttt 360

gttatataac aaagaagaaa taatgaactg attctcttcc tccttcttgt cctttcttaa 420

ttctgttgta attaccttcc tttgtaattt tttttgtaat tattcttctt aataatccaa 480

acaaacacac atattacaat aa 502

<210> 7

<211> 502

<212> DNA

<213> Artificial sequence

<400> 7

aacagtttat tcctggcatc cactaaatat aatggagccc gctttttaag ctggcatcca 60

gaaaaaaaaa gaatcccagc accaaaatat tgttttcttc accaaccatc agttcatagg 120

tccattctct tagcgcaact acagagaaca ggggcacaaa caggcaaaaa acgggcacaa 180

cctcaatgga gtgatgcaac ctgcctggag taaatgatga cacaaggcaa ttgacccacg 240

catgtatcta tctcattttc ttacaccttc tattaccttc tgctctctct gatttggaaa 300

aagctgaaaa aaaaggttga aaccagttcc ctgaaattat tcccctactt gactaataag 360

tatataaaga cggtaggtat tgattgtaat tctgtaaatc tatttcttaa acttcttaaa 420

ttctactttt atagttagtc ttttttttag ttttaaaaca ccaagaactt agtttcgaat 480

aaacacacat aaacaaacaa aa 502

<210> 8

<211> 502

<212> DNA

<213> Artificial sequence

<400> 8

agcaacaggc gcgttggact tttaattttc gaggaccgcg aatccttaca tcacacccaa 60

tcccccacaa gtgatccccc acacaccata gcttcaaaat gtttctactc cttttttact 120

cttccagatt ttctcggact ccgcgcatcg ccgtaccact tcaaaacacc caagcacagc 180

atactaaatt tcccctcttt cttcctctag ggtgtcgtta attacccgta ctaaaggttt 240

ggaaaagaaa aaagagaccg cctcgtttct ttttcttcgt cgaaaaaggc aataaaaatt 300

tttatcacgt ttctttttct tgaaaatttt tttttttgat ttttttctct ttcgatgacc 360

tcccattgat atttaagtta ataaacggtc ttcaatttct caagtttcag tttcattttt 420

cttgttctat tacaactttt tttacttctt gctcattaga aagaaagcat agcaatctaa 480

tctaagtttt aattacaaaa tg 502

<210> 9

<211> 502

<212> DNA

<213> Artificial sequence

<400> 9

gatttaaact gtgaggacct taatacattc agacacttct gcggtatcac cctacttatt 60

cccttcgaga ttatatctag gaacccatca ggttggtgga agattacccg ttctaagact 120

tttcagcttc ctctattgat gttacacctg gacacccctt ttctggcatc cagtttttaa 180

tcttcagtgg catgtgagat tctccgaaat taattaaagc aatcacacaa ttctctcgga 240

taccacctcg gttgaaactg acaggtggtt tgttacgcat gctaatgcaa aggagcctat 300

atacctttgg ctcggctgct gtaacaggga atataaaggg cagcataatt taggagttta 360

gtgaacttgc aacatttact attttccctt cttacgtaaa tatttttctt tttaattcta 420

aatcaatctt tttcaatttt ttgtttgtat tcttttcttg cttaaatcta taactacaaa 480

aaacacatac ataaactaaa aa 502

<210> 10

<211> 502

<212> DNA

<213> Artificial sequence

<400> 10

aaaaaagaag aaaacagaag gccaagacag ggtcaatgag actgttgtcc tcctactgtc 60

cctatgtctc tggccgatca cgcgccattg tccctcagaa acaaatcaaa cacccacacc 120

ccgggcaccc aaagtcccca cccacaccac caatacgtaa acggggcgcc ccctgcaggc 180

cctcctgcgc gcggcctccc gccttgcttc tctccccttc cttttctttt tccagttttc 240

cctattttgt ccctttttcc gcacaacaag tatcagaatg ggttcatcaa atctatccaa 300

cctaattcgc acgtagactg gcttggtatt ggcagtttcg tagttatata tatactacca 360

tgagtgaaac tgttacgtta ccttaaattc tttctccctt taattttctt ttatcttact 420

ctcctacata agacatcaag aaacaattgt atattgtaca ccccccccct ccacaaacac 480

aaatattgat aatataaaga tg 502

<210> 11

<211> 502

<212> DNA

<213> Artificial sequence

<400> 11

ggcgccataa ccaaggtatc tatagaccgc caatcagcaa actacctccg tacattcatg 60

ttgcacccac acatttatac acccagaccg cgacaaatta cccataaggt tgtttgtgac 120

ggcgtcgtac aagagaacgt gggaactttt taggctcacc aaaaaagaaa gaaaaaatac 180

gagttgctga cagaagcctc aagaaaaaaa aaattcttct tcgactatgc tggaggcaga 240

gatgatcgag ccggtagtta actatatata gctaaattgg ttccatcacc ttcttttctg 300

gtgtcgctcc ttctagtgct atttctggct tttcctattt ttttttttcc atttttcttt 360

ctctctttct aatatataaa ttctcttgca ttttctattt ttctctctat ctattctact 420

tgtttattcc cttcaaggtt tttttttaag gagtacttgt ttttagaata tacggtcaac 480

gaactataat taactaaaca tg 502

<210> 12

<211> 502

<212> DNA

<213> Artificial sequence

<400> 12

ttaacctaca ttcttcctta tcggatcctc aaaaccctta aaaacatatg cctcacccta 60

acatattttc caattaaccc tcaatatttc tctgtcaccc ggcctctatt ttccattttc 120

ttctttaccc gccacgcgtt tttttctttc aaattttttt cttccttctt ctttttcttc 180

cacgtcctct tgcataaata aataaaccgt tttgaaacca aactcgcctc tctctctcct 240

ttttgaaata tttttgggtt tgtttgatcc tttccttccc aatctctctt gtttaatata 300

tattcattta tatcacgctc tctttttatc ttcctttttt tcctctctct tgtattcttc 360

cttccccttt ctactcaaac caagaagaaa aagaaaaggt caatctttgt taaagaatag 420

gatcttctac tacatcagct tttagatttt tcacgcttac tgcttttttc ttcccaagat 480

cgaaaattta ctgaattaac aa 502

<210> 13

<211> 502

<212> DNA

<213> Artificial sequence

<400> 13

ggaactttca gtaatacgct taactgctca ttgctatatt gaagtacgga ttagaagccg 60

ccgagcgggc gacagccctc cgacggaaga ctctcctccg tgcgtcctcg tcttcaccgg 120

tcgcgttcct gaaacgcaga tgtgcctcgc gccgcactgc tccgaacaat aaagattcta 180

caatactagc ttttatggtt atgaagagga aaaattggca gtaacctggc cccacaaacc 240

ttcaaattaa cgaatcaaat taacaaccat aggatgataa tgcgattagt tttttagcct 300

tatttctggg gtaattaatc agcgaagcga tgatttttga tctattaaca gatatataaa 360

tggaaaagct gcataaccac tttaactaat actttcaaca ttttcagttt gtattacttc 420

ttattcaaat gtcataaaag tatcaacaaa aaattgttaa tatacctcta tactttaacg 480

tcaaggagaa aaaactataa tg 502

<210> 14

<211> 502

<212> DNA

<213> Artificial sequence

<400> 14

ctaattcaat aagtatgtca tgaaatacgt tgtgaagagc atccagaaat aatgaaaaga 60

aacaacgaaa ctgggtcggc ctgttgtttc ttttctttac cacgtgatct gcggcattta 120

caggaagtcg cgcgttttgc gcagttgttg caacgcagct acggctaaca aagcctagtg 180

gaactcgact gatgtgttag ggcctaaaac tggtggtgac agctgaagtg aactattcaa 240

tccaatcatg tcatggctgt cacaaagacc ttgcggaccg cacgtacgaa cacatacgta 300

tgctaatatg tgttttgata gtacccagtg atcgcagacc tgcaattttt ttgtaggttt 360

ggaagaatat ataaaggttg cactcattca agatagtttt tttcttgtgt gtctattcat 420

tttattattg tttgtttaaa tgttaaaaaa accaagaact tagtttcaaa ttaaattcat 480

cacacaaaca aacaaaacaa aa 502

<210> 15

<211> 493

<212> PRT

<213> Artificial sequence

<400> 15

Met Gly Arg Val Lys Trp Arg Ser Ile Thr Thr Tyr Asn Tyr Ile Met

1 5 10 15

Thr Thr Gly Glu Ala Val Arg Lys Arg His Lys Gly Ser Ser Asn Gly

20 25 30

Ala Arg Ala Gly Val Lys Asn His Ala Lys Glu Pro Val Gln Trp Gly

35 40 45

Arg Ala Trp Glu Val Asp Trp Phe Ser Leu Thr Gly Val Ile Leu Leu

50 55 60

Leu Cys Phe Ala Pro Phe Ile Val Phe Phe Phe Ile Met Ala Cys Asp

65 70 75 80

Gln Tyr Gln Cys Ser Ile Thr His Pro Leu Leu Asp Leu Tyr Asn Gly

85 90 95

Asp Ala Thr Leu Leu Thr Ile Trp Asn Arg Ala Pro Ser Phe Thr Trp

100 105 110

Ala Ala Ala Lys Ile Tyr Ala Ile Trp Val Thr Phe Gln Val Val Leu

115 120 125

Tyr Met Cys Val Pro Asp Phe Met His Lys Ile Leu Pro Gly Tyr Val

130 135 140

Gly Gly Val Gln Glu Gly Ala Arg Thr Pro Ala Gly Leu Ile Asn Lys

145 150 155 160

Tyr Glu Val Asn Gly Leu Gln Cys Trp Ile Ile Thr His Val Leu Trp

165 170 175

Val Ala Asn Ala Gln Tyr Phe His Trp Phe Ser Pro Thr Ile Ile Ile

180 185 190

Asp Asn Trp Ile Pro Leu Leu Trp Cys Thr Asn Ile Leu Gly Tyr Ala

195 200 205

Val Ser Thr Phe Ala Phe Ile Lys Ala Tyr Leu Phe Pro Thr Asn Pro

210 215 220

Glu Asp Cys Lys Phe Thr Gly Asn Ile Phe Tyr Asn Tyr Met Met Gly

225 230 235 240

Ile Glu Phe Asn Pro Arg Ile Gly Lys Trp Phe Asp Phe Lys Leu Phe

245 250 255

Phe Asn Gly Arg Pro Gly Ile Val Ala Trp Thr Leu Ile Asn Leu Ser

260 265 270

Tyr Ala Ala Lys Gln Gln Glu Leu Tyr Gly His Val Thr Asn Ser Met

275 280 285

Ile Leu Val Asn Val Leu Gln Ala Ile Tyr Val Leu Asp Phe Phe Trp

290 295 300

Asn Glu Ala Trp Tyr Leu Lys Thr Ile Asp Ile Cys His Asp His Phe

305 310 315 320

Gly Trp Tyr Leu Gly Trp Gly Asp Cys Val Trp Leu Pro Phe Leu Tyr

325 330 335

Thr Leu Gln Gly Leu Tyr Leu Val Tyr Asn Pro Val Gln Leu Ala Thr

340 345 350

Pro His Ala Thr Gly Val Leu Ile Leu Gly Leu Ala Gly Tyr Tyr Ile

355 360 365

Phe Arg Ser Gly Asn His Gln Lys Asp Leu Phe Arg Arg Thr Glu Gly

370 375 380

Asn Cys Lys Ile Trp Gly Lys Lys Pro Thr Phe Ile Glu Cys Ser Tyr

385 390 395 400

Arg Ser Ala Asp Gly Arg Ile His Lys Ser Lys Leu Met Thr Ser Gly

405 410 415

Phe Trp Gly Val Ala Arg His Met Asn Tyr Thr Gly Asp Leu Met Gly

420 425 430

Ser Leu Ala Tyr Cys Leu Ala Cys Gly Gly Glu His Leu Leu Pro Tyr

435 440 445

Phe Tyr Ile Val Tyr Met Thr Ile Leu Leu Val His Arg Cys Ile Arg

450 455 460

Asp Glu His Arg Cys Ser Asn Lys Tyr Gly Lys Asp Trp Glu Arg Tyr

465 470 475 480

Thr Ala Ala Val Pro Tyr Arg Leu Leu Pro Asn Ile Phe

485 490

<210> 16

<211> 417

<212> PRT

<213> Artificial sequence

<400> 16

Met Ala Cys Asp Gln Tyr Gln Cys Ser Val Thr His Pro Leu Leu Asp

1 5 10 15

Leu Tyr Asn Gly Asp Ala Thr Leu Leu Thr Ile Trp Asn Arg Ala Pro

20 25 30

Ser Phe Thr Trp Thr Ala Ala Lys Ile Tyr Ala Thr Trp Val Thr Phe

35 40 45

Gln Val Val Leu Tyr Met Phe Ile Pro Asp Ile Leu His Lys Ile Leu

50 55 60

Pro Gly Tyr Val Gly Gly Val Gln Asp Gly Ala Arg Thr Pro Ala Gly

65 70 75 80

Leu Ile Asn Lys Tyr Glu Ile Asn Gly Leu Gln Cys Trp Ile Ile Ser

85 90 95

His Val Leu Trp Val Ala Asn Ala Gln Tyr Phe His Trp Phe Ser Pro

100 105 110

Thr Ile Ile Ile Asp Asn Trp Ile Pro Leu Leu Trp Cys Thr Asn Ile

115 120 125

Leu Gly Tyr Phe Val Ser Thr Phe Val Phe Phe Lys Ala Tyr Leu Phe

130 135 140

Pro Thr Asn Pro Glu Asp Cys Lys Phe Thr Gly Asn Ile Phe Tyr Asn

145 150 155 160

Tyr Met Met Gly Ile Glu Phe Asn Pro Arg Ile Gly Lys Trp Phe Asp

165 170 175

Phe Lys Leu Phe Phe Asn Gly Arg Pro Gly Ile Val Ala Trp Thr Leu

180 185 190

Ile Asn Leu Ser Tyr Ala Ala Lys Gln Gln Glu Leu Tyr Gly Tyr Val

195 200 205

Thr Asn Ser Met Ile Leu Val Asn Val Leu Gln Val Ile Tyr Val Leu

210 215 220

Asp Phe Phe Trp Asn Glu Ala Trp Tyr Leu Lys Thr Ile Asp Ile Cys

225 230 235 240

His Asp His Phe Gly Trp Tyr Leu Gly Trp Gly Asp Cys Val Trp Leu

245 250 255

Pro Phe Leu Tyr Thr Leu Gln Gly Leu Tyr Leu Val Tyr Asn Pro Val

260 265 270

Gln Leu Ser Thr Pro His Ala Ala Gly Val Leu Ile Leu Gly Leu Leu

275 280 285

Gly Tyr Tyr Ile Phe Arg Ala Thr Asn His Gln Lys Asp Leu Phe Arg

290 295 300

Arg Thr Glu Gly Asn Cys Lys Ile Trp Gly Lys Lys Pro Thr Phe Ile

305 310 315 320

Glu Cys Ser Tyr Arg Ser Ser Asp Gly Gly Ile His Lys Ser Lys Leu

325 330 335

Met Thr Ser Ala Phe Trp Gly Met Ala Arg His Met Asn Tyr Thr Gly

340 345 350

Asp Leu Met Gly Ser Leu Ala Tyr Cys Met Ala Cys Gly Gly Ala His

355 360 365

Val Leu Pro Tyr Phe Tyr Ile Ile Tyr Met Thr Ile Leu Leu Val His

370 375 380

Arg Cys Ile Arg Asp Glu His Arg Cys Ser Ser Lys Tyr Ser Lys Asp

385 390 395 400

Trp Glu Arg Tyr Thr Ala Ala Val Pro Tyr Arg Leu Leu Pro Gly Ile

405 410 415

Phe

<210> 17

<211> 478

<212> PRT

<213> Artificial sequence

<400> 17

Met Thr Thr Ala Asp Ala Val Arg Lys Arg His Lys Gly Ser Ser Ser

1 5 10 15

Gly Ala Arg Ala Gly Val Lys Asp Gln Ala Lys Glu Pro Val Gln Trp

20 25 30

Gly Arg Ala Trp Glu Val Asp Trp Phe Ser Leu Thr Gly Val Ile Leu

35 40 45

Leu Leu Cys Phe Ala Pro Phe Ile Val Phe Phe Phe Ile Met Ala Cys

50 55 60

Asp Gln Tyr Gln Cys Ser Ile Ser His Pro Leu Leu Asp Leu Tyr Asn

65 70 75 80

Gly Asp Thr Thr Leu Leu Thr Ile Trp Ser Arg Ala Pro Ser Phe Thr

85 90 95

Trp Ala Ala Ala Lys Ile Tyr Ala Val Trp Val Thr Phe Gln Val Val

100 105 110

Leu Tyr Met Cys Val Pro Asp Ile Met His Lys Ile Leu Pro Gly Tyr

115 120 125

Val Gly Gly Val Gln Asp Gly Ala Arg Thr Pro Ala Gly Leu Ile Asn

130 135 140

Lys Tyr Glu Val Asn Gly Leu Gln Cys Trp Ile Ile Thr His Val Leu

145 150 155 160

Trp Val Ala Asn Ala Gln Tyr Phe His Trp Phe Ser Pro Thr Ile Ile

165 170 175

Ile Asp Asn Trp Ile Pro Leu Leu Trp Cys Thr Asn Ile Leu Gly Tyr

180 185 190

Ala Val Ser Thr Phe Ala Phe Ile Lys Ala His Leu Phe Pro Thr Asn

195 200 205

Pro Glu Asp Cys Lys Phe Thr Gly Asn Ile Phe Tyr Asn Tyr Met Met

210 215 220

Gly Ile Glu Phe Asn Pro Arg Ile Gly Lys Trp Phe Asp Phe Lys Leu

225 230 235 240

Phe Phe Asn Gly Arg Pro Gly Ile Val Ala Trp Thr Leu Ile Asn Leu

245 250 255

Ser Tyr Ala Ala Lys Gln Gln Glu Leu Tyr Gly Tyr Val Thr Asn Ser

260 265 270

Met Ile Leu Val Asn Val Leu Gln Ala Ile Tyr Val Leu Asp Phe Phe

275 280 285

Trp Asn Glu Ala Trp Tyr Leu Lys Thr Ile Asp Ile Cys His Asp His

290 295 300

Phe Gly Trp Tyr Leu Gly Trp Gly Asp Cys Val Trp Leu Pro Phe Leu

305 310 315 320

Tyr Thr Leu Gln Gly Leu Tyr Leu Val Tyr Asn Pro Ile Gln Leu Ser

325 330 335

Thr Pro His Ala Ala Gly Val Leu Ile Leu Gly Leu Val Gly Tyr Tyr

340 345 350

Ile Phe Arg Ser Thr Asn His Gln Lys Asp Leu Phe Arg Arg Thr Glu

355 360 365

Gly Asn Cys Lys Ile Trp Gly Lys Lys Pro Thr Phe Ile Glu Cys Ser

370 375 380

Tyr Arg Ser Ala Asp Gly Gly Ile His Lys Ser Lys Leu Met Thr Ser

385 390 395 400

Gly Phe Trp Gly Val Ala Arg His Met Asn Tyr Thr Gly Asp Leu Met

405 410 415

Gly Ser Leu Ala Tyr Cys Leu Ala Cys Gly Gly Gly His Leu Leu Pro

420 425 430

Tyr Phe Tyr Ile Val Tyr Met Thr Ile Leu Leu Val His Arg Cys Ile

435 440 445

Arg Asp Glu His Arg Cys Ser Asn Lys Tyr Ser Lys Asp Trp Glu Arg

450 455 460

Tyr Thr Ala Ala Val Pro Tyr Arg Leu Leu Pro Asn Ile Phe

465 470 475

<210> 18

<211> 478

<212> PRT

<213> Artificial sequence

<400> 18

Met Met Ala Ser Asp Arg Val Arg Lys Arg His Lys Gly Ser Ala Asn

1 5 10 15

Gly Ala Gln Thr Val Glu Lys Glu Pro Ser Lys Glu Pro Ala Gln Trp

20 25 30

Gly Arg Ala Trp Glu Val Asp Trp Phe Ser Leu Ser Gly Val Ile Leu

35 40 45

Leu Leu Cys Phe Ala Pro Phe Leu Val Ser Phe Phe Ile Met Ala Cys

50 55 60

Asp Gln Tyr Gln Cys Ser Ile Ser His Pro Leu Leu Asp Leu Tyr Asn

65 70 75 80

Gly Asp Ala Thr Leu Phe Thr Ile Trp Asn Arg Ala Pro Ser Phe Thr

85 90 95

Trp Ala Ala Ala Lys Ile Tyr Ala Ile Trp Val Thr Phe Gln Val Val

100 105 110

Leu Tyr Met Cys Val Pro Asp Phe Leu His Lys Ile Leu Pro Gly Tyr

115 120 125

Val Gly Gly Val Gln Asp Gly Ala Arg Thr Pro Ala Gly Leu Ile Asn

130 135 140

Lys Tyr Glu Val Asn Gly Leu Gln Cys Trp Leu Ile Thr His Val Leu

145 150 155 160

Trp Val Leu Asn Ala Gln His Phe His Trp Phe Ser Pro Thr Ile Ile

165 170 175

Ile Asp Asn Trp Ile Pro Leu Leu Trp Cys Thr Asn Ile Leu Gly Tyr

180 185 190

Ala Val Ser Thr Phe Ala Phe Ile Lys Ala Tyr Leu Phe Pro Thr Asn

195 200 205

Pro Glu Asp Cys Lys Phe Thr Gly Asn Met Phe Tyr Asn Tyr Met Met

210 215 220

Gly Ile Glu Phe Asn Pro Arg Ile Gly Lys Trp Phe Asp Phe Lys Leu

225 230 235 240

Phe Phe Asn Gly Arg Pro Gly Ile Val Ala Trp Thr Leu Ile Asn Leu

245 250 255

Ser Tyr Ala Ala Lys Gln Gln Glu Leu Tyr Gly Tyr Val Thr Asn Ser

260 265 270

Met Ile Leu Val Asn Val Leu Gln Ala Val Tyr Val Val Asp Phe Phe

275 280 285

Trp Asn Glu Ala Trp Tyr Leu Lys Thr Ile Asp Ile Cys His Asp His

290 295 300

Phe Gly Trp Tyr Leu Gly Trp Gly Asp Cys Val Trp Leu Pro Phe Leu

305 310 315 320

Tyr Thr Leu Gln Gly Leu Tyr Leu Val Tyr Asn Pro Ile Gln Leu Ser

325 330 335

Thr Pro His Ala Ala Gly Val Leu Ile Leu Gly Leu Val Gly Tyr Tyr

340 345 350

Ile Phe Arg Val Thr Asn His Gln Lys Asp Leu Phe Arg Arg Thr Glu

355 360 365

Gly Asn Cys Ser Ile Trp Gly Lys Lys Pro Thr Phe Ile Glu Cys Ser

370 375 380

Tyr Gln Ser Ala Asp Gly Ala Ile His Lys Ser Lys Leu Met Thr Ser

385 390 395 400

Gly Phe Trp Gly Val Ala Arg His Met Asn Tyr Thr Gly Asp Leu Met

405 410 415

Gly Ser Leu Ala Tyr Cys Leu Ala Cys Gly Gly Asn His Leu Leu Pro

420 425 430

Tyr Phe Tyr Ile Ile Tyr Met Thr Ile Leu Leu Val His Arg Cys Ile

435 440 445

Arg Asp Glu His Arg Cys Ser Asn Lys Tyr Gly Lys Asp Trp Glu Arg

450 455 460

Tyr Thr Ala Ala Val Ser Tyr Arg Leu Leu Pro Asn Ile Phe

465 470 475

Claims

1. The saccharomyces cerevisiae engineering bacteria are characterized in that C-22 sterol desaturase Erg5 is knocked out, and the expression is derived fromPangasianodonhypophthalmus7-dehydrocholesterol reductase.

2. The engineered saccharomyces cerevisiae strain of claim 1, wherein the amino acid sequence of the 7-dehydrocholesterol reductase is shown as SEQ ID NO 1.

3. The engineered saccharomyces cerevisiae strain as claimed in claim 1 or 2, wherein the engineered saccharomyces cerevisiae strain takes saccharomyces cerevisiae BY4742 as a host cell.

4. The engineered saccharomyces cerevisiae strain of claim 3, further comprising a promoter, wherein the promoter is used for enhancing the expression of 7-dehydrocholesterol reductase; the promoter is one or more of PGK1p, GPM1p, TDH3p, TEF1p, TPI1p, GPD1p, TEF2p, ACT1p, GAL1p and TDH2p.

5. The method for constructing engineered saccharomyces cerevisiae 5363 of any one of claims 1~4, comprising the steps of:

(2) The expression cassette is inserted into the 1114a locus on chromosome ChrXI of Saccharomyces cerevisiae BY4742 yeast, and C-22 sterol desaturase is knocked out to successfully construct the Saccharomyces cerevisiae engineering bacteria.

6. The method of claim 5, wherein the yeast promoter is one or more of PGK1p, GPM1p, TDH3p, TEF1p, TPI1p, GPD1p, TEF2p, ACT1p, GAL1p, and TDH2p.

7. The method of claim 5 or 6, wherein the yeast terminator is ADH1t.

8. A method for producing campesterol, characterized in that the saccharomyces cerevisiae engineering bacteria of claim 1~4 is used for preparing campesterol.

9. The method of claim 8, wherein the engineered saccharomyces cerevisiae is inoculated into a seed culture medium to prepare a seed solution; transferring the seed solution into a fermentation culture medium, collecting cultured cells, adding a saponification reaction solution, adding n-hexane for extraction after the reaction is finished, and preparing the campesterol.

10. Use of the engineered strain of saccharomyces cerevisiae of any of claims 1~4 in the preparation of campesterol and campesterol-containing products.