CN113817692B

CN113817692B - Diosgenin synthesis related protein, coding gene and application thereof

Info

Publication number: CN113817692B
Application number: CN202110996101.9A
Authority: CN
Inventors: 章焰生; 李长福; 周晨; 杨喻惠
Original assignee: University of Shanghai for Science and Technology
Current assignee: University of Shanghai for Science and Technology
Priority date: 2021-08-27
Filing date: 2021-08-27
Publication date: 2023-01-20
Anticipated expiration: 2041-08-27
Also published as: CN113817692A

Abstract

The invention relates to the technical field of biosynthesis of diosgenin, in particular to a diosgenin synthesis related protein, a coding gene and application thereof. The diosgenin synthesis related protein provided by the invention has an amino acid sequence shown in any one of SEQ ID NO. 1-3. The protein and the coding gene thereof participate in the synthesis of diosgenin, and provide necessary gene resources for realizing the biosynthesis of the diosgenin. The invention takes the saccharomyces cerevisiae for producing cholesterol as host bacteria, constructs the recombinant engineering bacteria containing three genes of a dioscin synthetic pathway from dioscorea zingiberensis plant, can oxidize the cholesterol to generate the dioscin, realizes the heterologous biosynthesis of the dioscin in yeast cells, provides an effective method for solving the problems of high production cost, large environmental pollution and the like in the production of the dioscin by a plant extraction method, and has better application prospect.

Description

Diosgenin synthesis related protein, coding gene and application thereof

Technical Field

The invention relates to the technical field of biosynthesis of diosgenin, in particular to a protein related to diosgenin synthesis, a coding gene and application thereof.

Background

Diosgenin is a precursor of more than 200 hormone drugs in the world, the world needs about 2000 tons per year, the price of each ton of diosgenin is about 53 ten thousand RMB, and China is the main export country of diosgenin in the world (about 67% of diosgenin is exported in China). Dioscoreaceae is the main synthetic plant of diosgenin, and Dioscorea zingiberensis is the main source of diosgenin in China, and its production method comprises extracting and purifying from the plant. Such conventional phytochemical extraction methods cause severe acidic contamination.

The peltate yam is the main source plant of dioscin in China, and the digging of the relevant gene for dioscin synthesis has important significance for the biosynthesis of dioscin.

Disclosure of Invention

The first objective of the invention is to provide diosgenin synthesis related protein and a coding gene thereof.

It is a second object of the present invention to provide a recombinant microorganism capable of synthesizing diosgenin.

The third purpose of the invention is to provide the diosgenin synthesis related protein, the coding gene thereof and the application of the recombinant microorganism.

The fourth object of the present invention is to provide a method for preparing diosgenin.

Specifically, the invention provides the following technical scheme:

in a first aspect, the present invention provides a diosgenin synthesis-associated protein, which is a protein represented by any one of the following (1) to (4):

(1) Has an amino acid sequence as shown in any one of SEQ ID NO. 1-3;

(2) The protein with the same function is obtained by substituting, deleting or adding one or more amino acids in the amino acid sequence shown in any one of SEQ ID NO. 1-3;

(3) A protein having at least 99%, at least 98%, at least 95%, at least 85% or at least 80% homology to the amino acid sequence as set forth in any of SEQ ID No.1-3 and having the same function;

(4) And (3) a fusion protein obtained by connecting a label to the N terminal and/or the C terminal of the protein shown in any one of (1) to (3).

The three diosgenin synthesis related proteins with amino acid sequences shown in SEQ ID NO.1-3 are three P450 enzymes derived from diosgenin synthesis pathway of Dioscorea zingiberensis, which are respectively named as CYP90B71, CYP90G6, and CYP94N8.

In a second aspect, the present invention provides a composition of diosgenin synthesis-related proteins, comprising three diosgenin synthesis-related proteins having amino acid sequences shown in SEQ ID nos. 1-3.

The sequences of the three diosgenin synthesis related proteins are respectively shown in SEQ ID No.1-3, and the coding genes of the three proteins are simultaneously introduced into the microorganisms, so that the microorganisms can synthesize the diosgenin by taking cholesterol as a substrate.

In a third aspect, the invention provides a nucleic acid encoding the diosgenin synthesis-related protein or the composition of the protein.

Preferably, the nucleic acid encoding the diosgenin synthesis-related protein is a nucleic acid as shown in any one of (1) to (3) below:

(1) A nucleic acid having a nucleotide sequence as set forth in any one of SEQ ID nos. 4 to 6 or a complementary sequence thereof;

(2) A nucleic acid that can hybridize to the nucleic acid shown in (1) under stringent conditions;

(3) A nucleic acid having at least 99%, at least 98%, at least 95%, at least 85% or at least 80% homology to a nucleotide sequence as set forth in any of SEQ ID nos. 4-6.

The stringent conditions described in the present invention are hybridization at 68 ℃ in a solution of 6 XSSC (containing 0.5% SDS), followed by washing once each of the membranes with 2 XSSC (containing 0.1% SDS) and 1 XSSC (containing 0.1% SDS).

The coding gene of the diosgenin synthesis related protein can be obtained by the following method:

using the dioscorea zingiberensis plant cDNA as a template, amplifying a diosgenin synthesis pathway gene CYP90B71 by using an RT-PCR technology, wherein the primer pair is as follows: CYP90B 71-F5 'ATGGCGCCGATGGAGCTTCTTC-3'; CYP90B 71-R5 '-TCAGGCTGAAGCTTTGTGATCAATTGC-3'.

Using the dioscorea zingiberensis plant cDNA as a template, amplifying a diosgenin synthesis pathway gene CYP90G6 by using an RT-PCR technology, wherein the used primer pair is as follows: CYP90G 6-F5-; CYP90G 6-R5.

Using the dioscorea zingiberensis plant cDNA as a template, amplifying a diosgenin synthesis pathway gene CYP94N8 by using an RT-PCR technology, wherein the primer pair is as follows: CYP94N 8-F5; CYP94N 8-R5.

In a fourth aspect, the invention provides a biological material containing said nucleic acid, said biological material comprising an expression cassette, a vector or a host cell.

Wherein, the expression cassette is a recombinant DNA obtained by connecting elements for driving the transcription and translation of the nucleic acid coding the diosgenin synthesis related protein at the upstream or downstream. The vector may be an expression vector or a cloning vector, including but not limited to a plasmid vector, a phage vector, a transposon, and the like. The host cell can be a microbial cell, a transgenic plant cell.

In a fifth aspect, the present invention provides any one of the following applications of the diosgenin synthesis-associated protein, the nucleic acid or the biological material:

(1) Application in preparing diosgenin;

(2) The application in improving the yield of diosgenin;

(3) The application in constructing diosgenin producing strains.

In a sixth aspect, the present invention provides a recombinant microorganism expressing the diosgenin synthesis-associated protein, or expressing three diosgenin synthesis-associated proteins having amino acid sequences as shown in SEQ ID nos. 1 to 3.

In one embodiment of the present invention, the microorganism is yeast. Preferably Saccharomyces cerevisiae, which is capable of synthesizing cholesterol.

The invention introduces three coding genes of diosgenin synthesis related proteins into yeast cells, so that the yeast simultaneously expresses the three diosgenin synthesis related proteins, and the recombinant yeast can oxidize cholesterol to form diosgenin by taking the cholesterol as a substrate.

Specifically, the invention provides a recombinant saccharomyces cerevisiae, which contains a yeast expression vector pESC-URA carrying CYP90B71 and CYP90G6 genes and a yeast expression plasmid pESC-Leu2d-CPR carrying CYP94N8 genes, and the saccharomyces cerevisiae can synthesize cholesterol.

In a seventh aspect, the present invention provides a method for constructing the recombinant microorganism, the method comprising: and introducing the coding gene of the diosgenin synthesis related protein into a microorganism to obtain a recombinant microorganism expressing the diosgenin synthesis related protein.

The method for introducing the coding gene of the diosgenin synthesis related protein into the microorganism can be any one or combination of more than one of the following methods:

(1) By introducing an expression plasmid containing the gene into a microorganism;

(2) By inserting the gene into the chromosome of the microorganism.

In an eighth aspect, the present invention provides an application of the recombinant microorganism in the preparation of diosgenin.

In a ninth aspect, the present invention provides a method for preparing diosgenin, comprising: culturing the recombinant microorganism, and separating and extracting the obtained culture to obtain diosgenin.

The invention has the beneficial effects that: the invention provides a relevant protein for synthesizing diosgenin in a dioscorea zingiberensis plant and a coding gene thereof, wherein the protein and the coding gene thereof participate in the synthesis of the diosgenin, and necessary gene resources are provided for realizing the biosynthesis of the diosgenin.

The invention takes the saccharomyces cerevisiae for producing cholesterol as host bacteria, constructs the recombinant engineering bacteria containing three genes of a dioscin synthetic pathway from dioscorea zingiberensis C.H.Wright, and the recombinant engineering bacteria can oxidize the cholesterol to generate the dioscin, thereby realizing the heterologous biosynthesis of the dioscin in yeast cells, providing an effective method for solving the problems of high production cost, large environmental pollution and the like in the production of the dioscin by a plant extraction method, and having better application prospect.

Drawings

Fig. 1 is a synthesis analysis of dioscin and dioscin from dioscorea zingiberensis in two different developmental stages in example 1 of the present invention: a.5 month seedling age size of Dioscorea zingiberensis plant picture; b.8 month seedling age size peltate leaf dioscorea opposita plant picture; C. analyzing the synthesis amount of diosgenin and dioscin in leaf and tuber tissues, wherein diosgenin and dioscin are diosgenin and dioscin compounds respectively, aug _ L, aug _ R and Nov _ R are leaf and tuber tissues with the age of 5 months and tuber tissues with the age of 8 months respectively

FIG. 2 is an agarose gel electrophoresis test of total RNA of tuber (R) of Dioscorea zingiberensis C.H.Wright in example 2 of the present invention, wherein R1, R2 and R3 represent 3 biological repeats, and M is DNA molecular weight standard.

FIG. 3 shows the result of agarose gel electrophoresis detection of RT-PCR amplification of full-length cDNA of 3 diosgenin synthesis pathway genes in example 2, wherein M is DNA molecular weight standard.

FIG. 4 is a LC-MS detection spectrum of Diosgenin synthesized by transgenic yeast in example 3, wherein Diosgenin is Diosgenin, CK represents empty vector control, and CYP90B71+ CYP90G6+ CYP94N8 represents co-expression of three Diosgenin synthesis genes.

Detailed Description

The following examples are intended to illustrate the invention, but are not intended to limit the scope of the invention.

Example 1 content analysis of diosgenin in Dioscorea zingiberensis leaf and tuber tissues

The wild peltate yam rhizome plant is collected from the Zhuxi county of Hubei province in China, tuber transplanting soil is adopted for plant propagation, 5-month-old-seedling leaves and tubers and 8-month-old-tubers (representative plants are shown as A and B in figure 1) are collected for diosgenin and dioscin extraction, and the extraction method comprises the following steps:

respectively taking about 1g of fresh plant tissues in liquid nitrogen, grinding the samples into powder, ultrasonically extracting the sample powder in 1ml of methanol at 30 ℃ for 20min (3 biological repetitions of each sample), volatilizing the methanol extract to be dry in vacuum, adding sulfuric acid with the final concentration of 1.5M to perform acidolysis at 100 ℃ for 4 hours, extracting residues after the acidolysis by using n-hexane, washing the residues to be neutral, volatilizing the n-hexane extract to be dry, and performing High Performance Liquid Chromatography (HPLC) analysis by fixing the volume in the methanol solvent.

The extraction method of dioscin is basically the same as that of the dioscin (removing the acidolysis step): extracting the sample powder in methanol, filtering the methanol extract to remove impurities, and directly analyzing by HPLC; HPLC analysis adopts LC-20AT instrument model, ODS-SP reverse phase column (250 mm × 4.6mm,5 μm) as chromatographic column, column temperature of 30 deg.C, mobile phase of water and methanol, and flow rate of 0.8ml/min; the liquid phase separation conditions of diosgenin are as follows: separating 90% methanol for 30min, and separating dioscin by gradient flow phase under the conditions of: 0-20min,10-90% methanol; 20-30min,90-10% methanol; 30-40min,10% methanol.

The results of the HPLC analysis are shown in C of FIG. 1. Through the metabolic analysis, the tuber is the main synthetic tissue of the diosgenin, and the 5-month-old plant synthesizes more diosgenin than the 8-month-old plant. Therefore, the invention adopts the 5-month-sized peltate yam rhizome plant tuber for separating the diosgenin synthesis pathway gene.

Example 2 obtaining of full-Length cDNA sequence of diosgenin Synthesis Gene

1. Taking total RNA of the new-born tuber of the dioscorea zingiberensis C.H. Wright of 5 months (figure 2), the specific method is as follows:

weighing about 100mg of peltate yam rhizome plant tuber, quickly grinding in liquid nitrogen, and extracting total RNA from the ground sample powder by using an EASYspin plus plant RNA quick extraction kit (Edelay).

2. The RNA is reversely transcribed into cDNA by the following specific method:

the following components were added to the RNase-free PCR tube: mu.l RNA (ca. 1.0. Mu.g), 1. Mu.l Oligo (dT) 18 and 1. Mu.l dNTP MIX (10 mM). The mixture was first pre-denatured at 65 ℃ for 5min, immediately placed on ice, and then 4. Mu.l of 5 XTRT buffer, 0.5. Mu.l of RNase inhibitor and 1. Mu.l of RT were added in this order, mixed well and then incubated at 42 ℃ for 1 hour to synthesize first strand cDNA. The synthesized cDNA was stored at-80 ℃ in a refrigerator.

3. The specific method for amplifying the dioscin synthetic pathway gene comprises the following steps:

combining the specificity of dioscin in the dioscorea zingiberensis plant tuber tissue synthesis, obtaining cDNA sequence information of target candidate genes by comparing transcriptomic databases of leaves and tubers, and obtaining dioscin synthesis pathway genes CYP90B71, CYP90G6 and CYP94N8 by adopting the following PCR reaction system amplification.

20 μ l PCR reaction system: mu.l of cDNA template, 1. Mu.l of forward and reverse primers, 10. Mu.l of 2 XPrimerSTAR Max Premix (Takara Co., ltd.), ddH ₂ O6. Mu.l. Wherein CYP90B71-F and CYP90B71-R are used for amplifying CYP90B71; CYP90G6-F and CYP90G6-R are used for amplifying CYP90G6; CYP94N8-F and CYP94N8-R are used to amplify CYP94N8.

The sequences of the primers are as follows:

CYP90B71-F:5'-ATGGCGCCGATGGAGCTTCTTC-3'；

CYP90B71-R:5'-TCAGGCTGAAGCTTTGTGATCAATTGC-3'。

CYP90G6-F:5'-ATGTTTCCTCTAGCTATCATCGTCTTGC-3'；

CYP90G6-R:5'-TCATTCTAGGATGGATAGCTTGCGAACTTTG-3'。

CYP94N8-F:5'-ATGTGTCTTGCAATGGAGTTCACTTGG-3'；

CYP94N8-R:5'-TTAAACAGCTTTGGTACATGGATTTCTCTC-3'。

and (3) PCR reaction conditions: pre-denaturation at 98 ℃ for 2min; the cycle procedure was 95 ℃ denaturation 20s,58 ℃ annealing 20s,72 ℃ extension for 1.5min,30 cycles, and finally 72 ℃ extension for 5min. After the reaction, 2. Mu.l of the reaction product was collected and detected by 1% agarose gel electrophoresis, and the results are shown in FIG. 3. The target strip is recovered by using a common agarose gel DNA recovery kit (purchased from Beijing Edley), and is connected to a pEASY-Blunt Simple vector (Beijing holotype gold biotechnology, inc.), the connecting product transforms escherichia coli DH5 alpha competent cells, then colony PCR is carried out to determine positive clones, plasmids extracted from the positive clones are sent to a sequencing company (Beijing engine department) for sequencing, the obtained deoxyribonucleotide sequences are respectively shown as SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6, and the sequences of the encoded proteins are respectively shown as SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO. 3.

EXAMPLE 3 application of diosgenin Synthesis Gene

The CYP90B71 and CYP90G6 genes are cloned into the same yeast expression vector pESC-URA to obtain a recombinant vector pESC-URA-CYP90B71-CYP90G6, and the CYP94N8 gene is cloned into a yeast expression plasmid pESC-Leu2d-CPR to obtain a recombinant vector pESC-Leu2d-CPR-CYP94N8. The recombinant plasmid pESC-URA-CYP90B71-CYP90G6 and pESC-Leu2d-CPR-CYP94N8 are transferred into a cholesterol-producing Saccharomyces cerevisiae RH6829 strain (the strain is disclosed in Souza, C.M.et al.A stable yeast strain effective production cholesterol engineering of experimental yeast, but not yet welded acid resistance, metal. Eng.13,555-569 (2011), which can be provided by Shanghai chapter flame group), the control is the co-transformation of empty vector pESC-URA and SC-Leu2d-CPR, the transgenic yeast is screened on SD-URA-LEU culture medium, the co-transformed CYP90B71, CYP90G6 and CYP94N8 gene-producing deficient strain is obtained by storing the glycerol-producing strain 1-80 ml.

Pick muchSingle colony of transgenic yeast was cultured in 5ml of SD-URA-LEU deficient medium (containing 2% glucose) at 30 ℃ at 250 rpm for 48 hours, and the cells were collected by centrifugation at 5000 rpm, washed 3 times with double distilled water, and then resuspended in 30ml of SD-URA-LEU medium (OD after resuspension) containing 2% galactose ₆₀₀ Controlled between 0.4 and 0.6), and inducing and culturing for 48 hours at 30 ℃. The cultured cells were collected by centrifugation, resuspended in a methanol solvent containing 2% KOH, and then packed with 0.45mm acid-washed glass beads to carry out yeast cell disruption by shaking, the disrupted solution was cultured at 22 ℃ for 1 hour to carry out saponification reaction, and then extracted with n-hexane, and the n-hexane extract was evaporated to dryness and dissolved in methanol for LC-MS analysis.

The LC-MS test result is shown in FIG. 4, the yam saponin is not produced in the control yeast containing empty vector, and the yam saponin is synthesized by the yeast containing CYP90B71, CYP90G6 and CYP94N8 genes, and the yield is about 3.0mg/L.

Although the invention has been described in detail with respect to the general description and the specific embodiments thereof, it will be apparent to those skilled in the art that modifications and improvements can be made based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

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atcctcagcg cctggcgcca tggctccacc ttctctgcac aagaggaggg caagaagttc 540

gccttcaatc ttatggtgaa gcatctgatg agcatggacc cgggaatgcc ggagacggag 600

caactgagga aggagtacat taccttcatg aagggcatgg cctccatccc tttgaacttg 660

ccaggaactg cttataggaa agccttgcag tcaagatcca taatcctgaa aataatgggg 720

caaaagctgg atgagagggt ggagaaggtc aagaggggat gtgaaggcct tgaagaagat 780

gaccttcttg catcagttgc tgctcaatca aacatcacaa gagatcaaat tctcgacctg 840

atactcagca tgctctttgc tggccatgag acgtcctctg ccgccatttg cctcgccatc 900

tacttccttg agtcttctcc caaagctctt caacaacttc gagaggagca catcaacata 960

gccaaaatga agaaggagaa aggagagact gggcttacat gggatgacta caaacagatg 1020

gagttcactc actgtgtgat caatgaaact cttaggcttg gcaacattgt gaagttcttg 1080

cacaggaagg ccattaagga tgtccaatac aaaggttatg atattccttg tggatgggaa 1140

gttgtcccta taatctcctc cgcacatttg gatccctcca tttatgacga tccacagtcc 1200

tacaatcctt ggagatggca gacaatctca acagcgacat caaagaacaa caatatcatg 1260

tcattcagcg gcggtcctcg tctgtgccct ggggccgagc tcgcaaagat ggagatggct 1320

gtcttcctgc accaccttgt ccagaagttc aactgggagt tggctgagca tgactaccct 1380

gtatccttcc catttctagg gttccccaag cacttgccaa tcaaagtgca tgcaattgat 1440

cacaaagctt cagcctga 1458

<210> 5

<211> 1467

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

atgtttcctc tagctatcat cgtcttgcta tttcccacac tgctgctcct cttcatagga 60

gtggccctgg gtttgagaag tggagccaat gagagctgga agaagagggg gctcaacatc 120

cccccaggaa gcatgggctg gccgctcctc ggcgagacca tcgccttccg gaagctccat 180

ccctgcacct ctctcggcga gtacatggag gatcgtctcc agaggtatgg aaagatctac 240

cgctcgaact tgttcggcgc gccgacggtg gtttcggcgg atgcagagct gaaccggttc 300

gtgctgatga acgacgggaa gctgttcgag ccgagctggc cgaagagcgt ggcggacata 360

ctgggaaaga cgtcgatgct ggtgctcaca ggggagatgc atcgctacat gaagtccttg 420

tccgtcaact tcatggggat cgctaggctt cggaatcact tccttgggga ctctgagcgc 480

tatatcttgg agaaccttgc gacctggaag gagggcgttc ctttccctgc taaagaagaa 540

gcttgcaaga taaccttcaa tttaatggtg aagaacatac tgagtatgaa tcctggggag 600

ccagagaccg agaggttgcg cattctctac atgtccttca tgaagggagt gatagctatg 660

cctctcaatt tccctggaac tgcatacagg aaagccattc agtctagagc tacaatcctg 720

aaaaccattg agcatttgat ggaggatagg ctggagaaga agaaggcagg cactgataat 780

atcggagaag ctgatcttct aggtttcatt ctagagcagt cgaacttgga tgctgaacaa 840

ttcggagact tgctgttagg tttgcttttt ggtggccatg agacctcctc cactgccatc 900

actctggcta tctacttcct tgaaggatgc cctaaagctg tacaagaact aagggaagag 960

catttgaacc tggtgaggat gaagaagcag agaggagagt ccaaagcact cacttgggaa 1020

gactacaaat ccatggactt tgcacagtgt gtggtgagtg agactctaag gctgggaaac 1080

atcatcaagt ttgtgcacag gaaggctaac actgatgtcc aatttaaagg atatgacata 1140

ccgagtggct ggagtgtgat tccggtgttc gccgcagctc atttagatcc tactgtctat 1200

gacaatcctc agaaatttga tccttggaga tggcagacaa tctcctccag cactgctagg 1260

attgacaatt acatgccatt cggtcagggg ctgcgcaact gtgctggcct tgagctcgcc 1320

aagatggaga tcgccgtgtt ccttcaccac cttgtcctta acttcgactg ggagcttgct 1380

gagccagatc accccctcgc ctacgccttc cctgaattcg aaaagggcct tcctatcaaa 1440

gttcgcaagc tatccatcct agaatga 1467

<210> 6

<211> 1497

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

atgtgtcttg caatggagtt cacttggctt cttctcctcc tcttcatcac caccaccatc 60

ttcctcctcc tccatctaaa cccaactccc acccccaact tcacccctct aaaaccctac 120

ccaatcctcc aaaacctccc ccacttagtc aaaaactccc accgtctcct cttcttcgtc 180

accgagctcg tctcctcctc tccctcctcc acctccaccc tcatcccctt cgtcttcacc 240

tccaacccct ccaacgtcga acacatgctc cgctccaact tccccaacta catcaaaggc 300

tcttccgtca tctccactct gcacgacttc ctcggcgacg gcatcttcaa ctccaacggt 360

cctctctggc gcctccagcg caagaccgcc agcttcgagt tcaacaccaa gtccctccgc 420

tccttcatct tccaccacgt ccgccacgaa tccctccacg ctctcctccc cattttatcg 480

aacacctccc gtgcatccct ccccgtcctc atcgacctcc aggacctcct cgaacgcttc 540

gccttcgaca acgtctgctc cctcgtcttc ggccacgacc cccgctgcct ccacgactcc 600

gccgacggcc tccgcttctt ccacgccttc caggaggcct cccatctcag catcgagcgg 660

atgaaccacg cctttgatct tctctggaag gtcaacaagt ggctcaacgt cggctctgag 720

cgccgtttaa accattcgtt gttgatcgtt cgtgagtacg cttcgagatt cgtgtccttg 780

cggaagaccc aagccggtga cgacctcctc tcccgtttcg ccgcggatga gaccatctcc 840

gatgacctcc ttgtcgacat cctcatttgc ttcgtcctcg ccggccggga caccaccccc 900

gccgcgctct cctggttctt ctggctcctc tcgtcccgcc cggacgtttc gcggaacatc 960

ttggtcgaga ttcagtctat ccgagctcga tccggcgacc gtgatggcga tcggttcttc 1020

agtctggagg agctgagaga gatgaactac ctccacgccg cgctatccga ggcgatgaga 1080

ctctaccctc cggtgccact cctgccgaga tgcgccgccg aggacgacgt cctcccggat 1140

gggaccttcg tgaagaaagg ctggaccttg atgtacaacg cctacgccat ggggaggatg 1200

gagagcattt ggggcaaaga ctgcatggag atgaggccgg agaggtggct gaaggacggc 1260

gtcttcaagc cgacgagccc gttccggtac ccggtgttcc tcggagggcc gaggatgtgc 1320

ttgggcaagg agatggctta catccagatg aaggcggtgg cggcctgcat tctcgagaag 1380

ttcgacatcg acgtcgtcgg cgcctccggc gagcctcagc tctctgtgac gatgacgatg 1440

aagggtggct tgccagtgag gatcaaagag agaaatccat gtaccaaagc tgtttaa 1497

<210> 7

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

atggcgccga tggagcttct tc 22

<210> 8

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

tcaggctgaa gctttgtgat caattgc 27

<210> 9

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

atgtttcctc tagctatcat cgtcttgc 28

<210> 10

<211> 31

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

tcattctagg atggatagct tgcgaacttt g 31

<210> 11

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

atgtgtcttg caatggagtt cacttgg 27

<210> 12

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

ttaaacagct ttggtacatg gatttctctc 30

Claims

1. The diosgenin synthesis related protein is characterized by being a protein shown in any one of the following (1) to (2):

(1) Protein with amino acid sequence shown in SEQ ID NO.1 or 3;

(2) And (2) a fusion protein obtained by connecting a label to the N end and/or the C end of the protein shown in (1).

2. The composition of diosgenin synthesis related proteins is characterized by consisting of three diosgenin synthesis related proteins with amino acid sequences shown in SEQ ID NO. 1-3.

3. A nucleic acid encoding the diosgenin synthesis-associated protein of claim 1 or the composition of claim 2.

4. The nucleic acid of claim 3, wherein the nucleotide sequence of the nucleic acid encoding the composition of diosgenin synthesis-associated proteins is shown in SEQ ID No. 4-6.

5. Biological material comprising a nucleic acid according to claim 3 or 4, wherein the biological material is an expression cassette, a vector or a host cell; the host cell is a microbial cell.

6. Any one of the following uses of the diosgenin synthesis-related protein of claim 1, or the composition of claim 2, or the nucleic acid of claim 3 or 4, or the biological material of claim 5:

(1) Application in preparing diosgenin;

(2) The application in improving the yield of diosgenin;

(3) The application in constructing diosgenin production strains;

the application is to introduce diosgenin synthesis related protein with an amino acid sequence shown in SEQ ID NO.1-3 into saccharomyces cerevisiae capable of synthesizing cholesterol.

7. A recombinant microorganism expressing the diosgenin synthesis-associated protein of claim 1 or the composition of claim 2.

8. The recombinant microorganism according to claim 7, wherein the microorganism is a yeast.

9. The recombinant microorganism according to claim 8, wherein the microorganism is Saccharomyces cerevisiae capable of synthesizing cholesterol, and the recombinant microorganism expresses diosgenin synthesis-related protein with amino acid sequence shown in SEQ ID No. 1-3.

10. The method of constructing recombinant microorganism of any one of claims 7-9, wherein the gene encoding the diosgenin synthesis-associated protein is introduced into a microorganism to obtain a recombinant microorganism expressing the diosgenin synthesis-associated protein.

11. The use of the recombinant microorganism of claim 9 in the preparation of diosgenin.

12. A method for preparing diosgenin, which comprises the following steps: culturing the recombinant microorganism of claim 9, and isolating and extracting diosgenin from the obtained culture.