CN113584110A

CN113584110A - Construction and application of engineering strain for biosynthesizing mogroside V by taking mogrol as substrate

Info

Publication number: CN113584110A
Application number: CN202110919424.8A
Authority: CN
Inventors: 赵云现; 舒柔; 杨志彬; 胡江林; 崔金旺; 赵凯
Original assignee: Hebei Weidakang Biotechnology Co ltd
Current assignee: Hebei Weidakang Biotechnology Co ltd
Priority date: 2021-08-11
Filing date: 2021-08-11
Publication date: 2021-11-02

Abstract

The invention relates to construction and application of an engineering strain for biosynthesizing mogroside V by taking mogrol as a substrate. Converting a substrate mogrol by a recombinant cell containing a glycosyltransferase gene or a recombinant cell containing the glycosyltransferase gene and a UDP-glucose pyrophosphorylase gene to obtain the mogroside V, and expressing the glucose glycosyltransferase gene and the UDP-glucose pyrophosphorylase gene by the recombinant cell to produce UGT enzyme and UDP-glucose pyrophosphorylase. According to the invention, exogenous species UGT enzyme gene and UDP-glucose pyrophosphorylase gene are introduced through a gene recombination technology and are transformed into genetic engineering bacteria for expression, a new biosynthesis path of the momordica grosvenori glycoside V is reconstructed, and the momordica grosvenori glycoside V is biosynthesized by taking momordica grosvenori alcohol as a substrate through overexpression of the UGT enzyme gene and the UDP-glucose pyrophosphorylase gene, so that the synthesis yield is high.

Description

Construction and application of engineering strain for biosynthesizing mogroside V by taking mogrol as substrate

Technical Field

The invention belongs to the technical field of biology, and particularly relates to construction and application of an engineering strain for biosynthesis of momordica grosvenori glucoside V by taking momordica grosvenori alcohol as a substrate.

Background

Sweet foods are a kind of living seasonings, and the unique taste of the sweet foods endows the people with endless fun in life. The sweet taste is mainly derived from traditional sweeteners: sucrose. The cane sugar can provide certain calorie while meeting the requirement of human sweet taste. However, the intake of sucrose is strictly controlled in patients with obesity, hyperlipidemia, hyperglycemia, etc. A reduction in sugar intake means a reduction in sweet enjoyment. How to meet the sweet taste requirement while reducing sugar intake? Synthetic sweeteners offer one possibility, however recent studies have indicated that synthetic sweeteners may affect the normal intestinal flora and even cause metabolic syndrome and glucose intolerance. Natural sugar-free sweeteners that combine the safety of natural sugars with the non-caloric benefits of synthetic sweeteners have moved into the human eye and the subject of developing natural sugar-free sweeteners to meet the "need" of human beings for sweetness has clearly attracted a great deal of interest to food practitioners.

The mogroside is a natural sweetener extracted directly from Momordica grosvenori (Siraitiagrosvenonori) of Cucurbitaceae family without chemical synthesis. It contains almost no energy, but has about 300 times the sweetness of sucrose, and is an ideal sucrose substitute. Meanwhile, the mogroside also has the functions of regulating blood sugar, resisting oxidation, relieving cough and eliminating phlegm, protecting liver, relaxing bowel, resisting bacteria and diminishing inflammation and the like. The Mogroside is a cucurbitane triterpene glycoside compound and comprises a plurality of components such as Mogroside I (Mogroside I), Mogroside II E (Mogroside II E), Mogroside III (Mogroside III), Mogroside III E (Mogroside III E), Mogroside IV (Mogroside IV), Mogroside V (Mogroside V), 11-Oxo-Mogroside V (11-Oxo-Mogroside V), Neomogroside (Neomogroside) and Siamenoside I (Simenoside I), wherein the Mogroside V (M5) is a main sweet component, and has the advantages of high sweetness, low energy, safety, no toxicity and the like, and has a wide application prospect.

However, the content of mogroside V in momordica grosvenori is only 0.8-1.3% (W/W), and the large-scale production of high-yield and high-purity mogroside V is difficult to realize only by extracting from fruits. In addition, the planting environment and requirements of the momordica grosvenori are harsh, and the research progress in the aspect of improving the momordica grosvenori glucoside V yield through crossbreeding improvement is limited in the traditional method. Therefore, the biosynthesis of the mogroside V is a necessary trend for large-scale production, but at present, few reports of biosynthesis of the mogroside V exist, so that a method for producing the mogroside V by using key enzyme recombination construction engineering bacteria required by the synthesis of natural mogroside V is provided.

Disclosure of Invention

In order to solve the technical problems, the invention aims to provide an engineering strain for biosynthesizing mogroside V by taking mogrol as a substrate and application thereof.

A method for synthesizing mogroside V comprises the step of converting a substrate mogrol by a recombinant cell containing a glycosyl transferase (UDP-glucosyltransferase, UGT) gene or a recombinant cell containing the glycosyl transferase (UDP-glucosyltransferase, UGT) gene and a UDP-glucose pyrophosphorylase gene to obtain the mogroside V, wherein the recombinant cell expresses the glycosyl transferase (UDP-glucosyltransferase, UGT) gene and the UDP-glucose pyrophosphorylase gene to produce UGT enzyme and UDP-glucose pyrophosphorylase.

According to the scheme, the glycosyltransferase gene sequence is shown as SEQ ID NO.1, SEQ ID NO.3, SEQ ID NO.5, SEQ ID NO.7 or SEQ ID NO. 9; the sequence of the UDP-glucose pyrophosphorylase gene is shown as SEQ ID NO.11 or SEQ ID NO. 13.

According to the scheme, the method comprises the following specific steps: step (1): obtaining a crude enzyme solution by using a recombinant cell containing a glycosyltransferase (UGT) gene and a UDP-glucose pyrophosphorylase gene; step (2): adding crude enzyme solution into mogrol as a substrate, and converting and synthesizing the mogroside V in the presence of UDP glucose.

According to the scheme, the preparation method of the crude enzyme solution comprises the following steps: inoculating a recombinant cell containing a glycosyltransferase (UGT) gene and a UDP-glucose pyrophosphorylase gene into an LB liquid culture medium to obtain a seed solution, and inoculating the seed solution into the LB liquid culture medium, wherein the culture conditions can be as follows: culturing at 37 deg.C and 200rpm until the OD600 of the thallus is 0.4-1.0, adding IPTG, inducing culture, and inducing expression of target protein; after induction, the cells were collected by centrifugation, resuspended, disrupted by ultrasonic/freezing cycles in liquid nitrogen, and then centrifuged, and the supernatant was collected to obtain a crude enzyme solution.

According to the scheme, the reaction conditions in the step (2) of the step (2) are 25-45 ℃, and the reaction time is 1-48 h.

A recombinant vector comprising a glycosyltransferase (UGT) gene, or comprising a UDP-glycosyltransferase (UGT) gene and a UDP-glucose pyrophosphorylase gene.

A preparation method of a recombinant vector comprises the following steps: (1) carrying out PCR amplification by using a specific primer by using a carrier as a template to obtain a linear carrier reverse amplification fragment; (2) taking a glycosyltransferase gene as a template, carrying out PCR amplification to obtain a UGT positive amplification fragment, recovering the UGT positive amplification fragment, and carrying out reverse amplification connection on the recovered UGT positive amplification fragment and the vector to obtain a first recombinant vector; further, performing PCR amplification by using the first recombinant vector as a template to obtain a linear vector reverse amplification fragment; carrying out PCR amplification by taking UDP-glucose pyrophosphorylase gene as a template to obtain an UDPase amplified fragment, and recycling the UDPase amplified fragment and linking the UDPase amplified fragment with the vector reverse-amplified fragment to obtain a second recombinant vector.

A genetically engineered bacterium for biosynthesizing mogroside V by using mogrol as a substrate expresses a UDP-glucosyltransferase (UGT) gene and a UDP-glucose pyrophosphorylase gene to produce UGT enzyme and UDP-glucose pyrophosphorylase.

The construction method of the genetic engineering bacteria comprises the steps of transforming host cells by the recombinant vector, culturing a transformant, and obtaining the engineering strain for biosynthesizing the grosvenor momordica glycoside V by taking the grosvenor momordica alcohol as a substrate.

According to the scheme, the host cell is escherichia coli.

Provides the application of the recombinant vector or the genetic engineering bacteria in biosynthesis of the momordica grosvenori glucoside V.

Mogroside V is a triterpenoid saponin compound whose precursors for synthesis are Isopentenyl pyrophosphate (IPP) and Dimethylallyl pyrophosphate (DMAPP). IPP and DMAPP are produced from acetyl coa via mevalonate pathway (MVA pathway), then catalyzed by Geranyl Pyrophosphate Synthase (PS), Farnesyl pyrophosphate synthase (FPPS), Squalene synthase (SQS), cyclase and Cucurbitadienol synthase (CDS) to synthesize 24, 25-epoxycucurbitadienol, which is then subjected to Epoxide hydrolase (EPH) and cytochrome P450 enzyme (CYP450) to produce mogrol, and finally gradually glycosylated by glycosyltransferase (UDP-glycosyltransferase, UGT) using mogrol as a substrate to synthesize mogroside V. UGT is a decisive enzyme in the process of converting mogrol into mogroside V, and takes mogrol as a substrate and UDP-glucose as a glycosyl donor to catalyze the glycosylation reaction of C24 and C3 position and C3 and C24 position glucose branched chains of the mogrol, and the mogroside V is synthesized by a tetraosylation intermediate. Meanwhile, UDP-glucose is used as a glycosyl donor, the supply amount of UDP-glucose in the catalytic process also influences the glycosylation efficiency of mogrol, and when the glycosyl supply is insufficient, the mogrol glycosylation reaction is limited, so that the UDP-glucose content is increased or the glycosylation efficiency of mogrol can be obviously promoted.

Therefore, UGT and UDPase enzyme genes are selected, a genetic engineering strain with UGT and UDPase co-expression is created, high expression is carried out in a host strain, and mogrol is added as a substrate for synthesizing the mogroside V. The method has the advantages of simple operation, no environmental pollution, high yield, high purity and low cost, and provides reference for industrial production of mogroside V.

Compared with the prior art, the invention has the following beneficial effects:

according to the invention, exogenous species UGT enzyme gene and UDP-glucose pyrophosphorylase gene are introduced through a gene recombination technology and are transformed into genetic engineering bacteria for expression, a new biosynthesis path of the momordica grosvenori glycoside V is reconstructed, and the momordica grosvenori glycoside V is biosynthesized by taking momordica grosvenori alcohol as a substrate through overexpression of the UGT enzyme gene and the UDP-glucose pyrophosphorylase gene, so that the synthesis yield is high.

The method has the advantages of simple operation, no environmental pollution, high yield, high purity and low cost, and provides a basis for industrial production of mogroside V.

Drawings

FIG. 1 is a schematic diagram of construction of engineering strains expressing UGT and UGT-UDPase.

FIG. 2 shows the yield of mogroside V from the engineered strain.

Detailed Description

Example 1 construction of pET28a-UGT plasmid and recombinant E.coli Strain

The pET28a-UGT plasmid construction method:

(1) PCR amplification was performed using a specific primer using pET28a vector as a template. The pET28a vector is a commercial vector purchased from Novagen; the primer sequences are shown in table 1; recovering the PCR product to obtain a linearized vector pET28 a-reverse amplification, wherein the size of the linearized vector fragment is 5369 bp;

(2) UGT protein sequences of 5 organisms, namely salmon sea lice (Lepeophheirussalmonis), Arabidopsis thaliana (Arabidopsis thaliana), cotton (Gossypiumhirsutum), radish (Raphanussativus) and Drosophila melanogaster (Drosophila simulans) published by NCBI (https:// www.ncbi.nlm.nih.gov /), are shown as SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.6, SEQ ID NO.8 or SEQ ID NO.10, are codon optimized according to codon preference of Escherichia coli, and nucleic acid sequences are artificially synthesized, and LsUGT, AtUGT, GhUGT, RsUGT and DsUGT gene sequences are shown as SEQ ID NO.1, SEQ ID NO.3, SEQ ID NO.5, SEQ ID NO.7 or SEQ ID NO.9 respectively, and 6 is designed to amplify specific primers. The UGT amino acid sequence and the nucleic acid sequence are shown in SEQ Listing, and the primer sequence is shown in Table 1.

TABLE 1 primer sequences

(3) The PCR amplification procedure was pre-denaturation at 95 ℃ for 5min, denaturation at 94 ℃ for 45s, annealing at 60 ℃ for 45s, extension at 72 ℃ for 1.5min, 35 cycles, and extension at 72 ℃ for 10 min.

(4) And recovering the UGT positive expansion fragment, performing reverse amplification connection with the recovered pET28a respectively to obtain a recombinant plasmid, and naming the recombinant plasmid as pET28a-UGT, and obtaining the successfully constructed pET28a-UGT plasmid through sequencing verification.

The construction method of the escherichia coli recombinant strain comprises the following steps:

the pET28a-UGT plasmid is transformed into an escherichia coli cloning host by a heat shock transformation method to obtain a positive transformant, then the transformant plasmid with correct sequencing is transformed into an escherichia coli expression host bacterium BL21(DE3) and coated on an LB solid culture medium containing 50 mug/mL kanamycin, an LB plate is cultured at 37 ℃ until a transformant grows out, and the positive transformant is picked to obtain the expected engineering bacterium.

TABLE 25 pET28a-UGT engineering bacteria types

EXAMPLE 2 inducible expression of the protein of interest

5 kinds of engineering bacteria were inoculated into 5mL of LB liquid medium and cultured overnight at 37 ℃ and 200 rpm. Then, 0.8mL of the overnight culture was inoculated into 20mL of LB liquid medium and cultured at 37 ℃ and 200rpm until the OD600 of the cells became 0.6. Then IPTG was added to a final concentration of 1mM, and the mixture was cultured at room temperature for 16 hours to induce the expression of the target protein. After completion of induction, the cells were collected by centrifugation at 10,000 Xg for 5min, resuspended in 1.5mL of a mixture (50mM Tris. HCl (pH 7.0), 15% (vol/vol) glycerol, 0.1mM EDTA and 5 mM. beta. -mercaptoethanol), and disrupted by sonication for 3 min. The E.coli cell disruption solution was then centrifuged at 20,000 Xg for 10min, and the supernatant was collected to obtain a crude enzyme solution for enzyme characterization. The crude enzyme solution was stored at-20 ℃ until further analysis.

Example 3 determination of mogroside V content

Mogrol was dissolved to 20mM with 50% DMSO. A reaction system prepared by adding 50mM Tris-HCl (pH 7.0, containing 5 mM. beta. -mercaptoethanol), 25. mu.L of the crude enzyme solution, 8mM UDP-glucose and 2mM mogrol was prepared in a volume of 100. mu.L for the assay of UGT enzyme activity. After incubating the reaction solution at 37 ℃ for 24h, the reaction was stopped by adding 300. mu.L of methanol solution, followed by brief vortexing. Subsequently, the reaction solution was centrifuged at 20,000 × g for 10min, and the supernatant was collected, and the contents of subsequent products were analyzed as follows:

(1) adding 1mL of chromatographic grade methanol solution into 1mL of the reaction solution to obtain a product dissolved substance;

(2) centrifuging the product dissolved substance at 5000rpm for 10min, and collecting supernatant;

(3) filtering the supernatant with a 0.22 μm filter membrane in a brown liquid bottle to obtain a sample to be detected;

(4) the new product was analyzed by liquid mass nuclear magnetic coupling technique IC-MS-NMR: LC-MS analysis used an Agilent260HPLC system and a Bruker-microtof-II electrospray mass spectrometer. A reversed phase C18 column (4.6X 250mm, 5um, Welch, Shanghai) was used in the HPLC, the amount of sample was 10. mu.L, the flow rate was 0.5mL/min, and the UV detection wavelength was 210 nm. Mobile phase a was water (0.1% formic acid) and mobile phase B was acetonitrile (0.1% formic acid), using gradient elution: 0.00-15.00min, 20% -26% B; 15.00-15.01min, 26% -100% B; 15.01-25.00min, 100% B;

(5) finally, the yield of the mogroside V of 5 engineering bacteria is calculated according to the peak area of the standard product of the mogroside V, and the specific yield is shown in table 3. According to calculation, the maximum mogroside V yield of the bacterial strain 1, namely pET28a-LsUGT engineering bacteria can reach 1.175 g/L.

Table 35 engineering bacteria momordica grosvenori glucoside V yield

Example 4 construction of pET28a-UGT-UDPase plasmid and recombinant Strain of E.coli

Based on the LsUGT and AtUGT transformed engineering bacteria with higher momordica grosvenori glucoside V yield, UGT and UDPase are expressed in series.

The pET28a-UGT-UDPGase plasmid construction method:

(1) PCR amplification was performed using a specific primer using pET28a-UGT vector as a template. The pET28a vector is a commercial vector purchased from Novagen; the primer sequences are shown in Table 4; recovering the PCR product to obtain a linear vector pET28a-UGT reverse amplification fragment;

(2) according to the UDPase protein sequences of Arabidopsis thaliana (Arabidopsis thaliana) and Drosophila melanogaster (Drosophila melanogaster) published by NCBI (https:// www.ncbi.nlm.nih.gov /) database, as shown in SEQ ID NO.12, SEQ ID NO.14, codon optimization was performed according to the codon preference of Escherichia coli, nucleic acid sequences as shown in SEQ ID NO.11, SEQ ID NO.13 were artificially synthesized, and 2 pairs of specific primers were designed to amplify them. The primer sequences are shown in Table 4.

TABLE 4 UDPase primer sequences

(4) Combining and connecting the AtUDPase positive amplified fragment, the DmUDPase positive amplified fragment, the pET28a-LsUGT reverse amplified fragment and the pET28a-RsUGT reverse amplified fragment in pairs to obtain a recombinant plasmid, naming the recombinant plasmid as pET28a-UGT-UDPase, and obtaining 4 successfully constructed pET28a-UGT-UDPase plasmids through sequencing verification.

4pET28a-UGT-UDPase plasmids are transformed into an escherichia coli cloning host by a heat shock transformation method to obtain positive transformants, then transformants with correct sequencing are transformed into an escherichia coli expression host bacterium BL21(DE3) and coated on an LB solid culture medium containing 50 mu g/mL kanamycin, an LB plate is cultured at 37 ℃ to grow out the transformants, and the positive transformants are picked to obtain expected engineering bacteria.

TABLE 5pET28a-UGT-UDPase engineering bacteria types

Example 5 production of mogroside V from pET28 a-UGT-UDPase-engineered bacteria

The induced expression of the target protein of the engineered bacteria and the mogroside V production were determined according to the methods of examples 2-3 above.

According to the peak area of the momordica grosvenori glycoside V standard product, the yield of the momordica grosvenori glycoside V of 4 kinds of pET28a-UGT-UDPase engineering bacteria is finally calculated, and the specific yield is shown in Table 6. Through calculation, the maximum yield of the momordica grosvenori V of the strain 6, namely pET28a-LsUGT-AtUDPase engineering bacteria can reach 1.583 g/L.

Table 62 Momordica grosvenori glycoside V yields of pET28a-UGT-UDPGase engineering bacteria

Nucleotide and amino acid sequence listing of the specification

< 110 > Hebei Weidakang Biotech Ltd

Less than 120 construction and application of engineering strain for synthesizing mogroside V by using mogrol as substrate microorganism

＜160＞ 14

＜210＞ 1

＜211＞1542bp

＜212＞ DNA

< 213 > is artificially synthesized

＜400＞ 1

atgattctga aagaatttgg cgaatatctg agcagccgcg gccatagcgt gacccagatt 60

cgctttaaag atcgcaaccc gagccagccg ccgcagatta aaaacagcaa cattagcgtg 120

attgatctgc cgattagccg ctttaccgat aacgaatgca ttgattatat taacgatgat 180

ggcgaaattg atattaacat tagcgcgacc aaactgctgt ggaaagatgg cgaaagcgtg 240

tataaagtgc cgagcgatat tttttgcgtg acccgcgcgc attgccatac cctgtttacc 300

gtgcatagcg cgaaaatgaa aaaagtgctg gatagcggca actttgaact ggcgattgtg 360

gatattattg cgaacgaatg cggcctggcg atggcgaaaa gcctgggcat tccggtggtg 420

ggcttttgga tgtttagctt tcagggcggc gaaaccctgc gcagcccggt gtttaacccg 480

ccgagcattg tgccgagctt tctgagcagc ctgccgccga aaaaatggaa ctttctggaa 540

cgcgtgtata actttattgt gcatctgacc catcgcgcgc tgctggcgat tcaggaaagc 600

attgcgaacg attttattaa agaacgctat ccgaacctgc cgccggtgcg cgatctgttt 660

tttgatctgg attttacctt tattcatacc aactttctgg tggatagccc gaaactgatt 720

gcgccgaaca gcaaatatgt gggcggcctg catattcgca gcccgcgcaa actgcgcaaa 780

gaactgaaag aatttgtgga aggcgcgaaa aaaggcgtga ttatttttag cctgggcttt 840

accggctata gcagcgataa cgtgccgctg agctttattc gcgatctgct gcatattatt 900

gaaaaactgg aaggctatcg cctgattatg cgctttgata aagatgtgat tagcagcccg 960

ctgccggaaa acgtgctggt gctgaaatgg ctgccgcagc aggatctgct ggcgcatagc 1020

gcggtgaaac tgtttattaa ccatggcggc atgggcggca ttcaggaaag cgtgtattat 1080

ggcgtgccga tggtgattat tccgattttt ggcgatcaga acgataacgc ggcgcgcatt 1140

cagcattatg aactgggcct gtatgtggtg aaaagccgcc tgaactatga taacctgcgc 1200

tttgcgattc atgaagtgct gtttaacgat aaatatagcg aaaaaagcaa atttcatcag 1260

agcctgtgga aagatgaatt ttgcagcagc caggaagaaa ccgcgtattg gattgaactg 1320

ctgattaaat atggccatct ggatcatctg aaaattaaag gcgtgaacat tagcctgttt 1380

cagtatttta gcctggatgt gattctgttt tttctgagca ttctgtttat tagcaccacc 1440

ctggtgctgg gctttattat ttataacatt tttggcaaat gcttttttgt gcatgatacc 1500

tataacaacg gcaccaaacg caaaattatt aaagtgaact aa 1542

＜210＞ 2

＜211＞　513

＜212＞ PRT

< 213 > is artificially synthesized

＜400＞ 1

MILKEFGEYL SSRGHSVTQI RFKDRNPSQP PQIKNSNISV IDLPISRFTD NECIDYINDD 60

GEIDINISAT KLLWKDGESV YKVPSDIFCV TRAHCHTLFT VHSAKMKKVL DSGNFELAIV 120

DIIANECGLA MAKSLGIPVV GFWMFSFQGG ETLRSPVFNP PSIVPSFLSS LPPKKWNFLE 180

RVYNFIVHLT HRALLAIQES IANDFIKERY PNLPPVRDLF FDLDFTFIHT NFLVDSPKLI 240

APNSKYVGGL HIRSPRKLRK ELKEFVEGAK KGVIIFSLGF TGYSSDNVPL SFIRDLLHII 300

EKLEGYRLIM RFDKDVISSP LPENVLVLKW LPQQDLLAHS AVKLFINHGG MGGIQESVYY 360

GVPMVIIPIF GDQNDNAARI QHYELGLYVV KSRLNYDNLR FAIHEVLFND KYSEKSKFHQ 420

SLWKDEFCSS QEETAYWIEL LIKYGHLDHL KIKGVNISLF QYFSLDVILF FLSILFISTT 480

LVLGFIIYNI FGKCFFVHDT YNNGTKRKII KVN 513

＜210＞ 3

＜211＞　1410bp

＜212＞ DNA

< 213 > is artificially synthesized

＜400＞ 1

atggcgccgc cgcattttct gctggtgacc tttccggcgc agggccatgt gaacccgagc 60

ctgcgctttg cgcgccgcct gattaaacgc accggcgcgc gcgtgacctt tgtgacctgc 120

gtgagcgtgt ttcataacag catgattgcg aaccataaca aagtggaaaa cctgagcttt 180

ctgaccttta gcgatggctt tgatgatggc ggcattagca cctatgaaga tcgccagaaa 240

cgcagcgtga acctgaaagt gaacggcgat aaagcgctga gcgattttat tgaagcgacc 300

aaaaacggcg atagcccggt gacctgcctg atttatacca ttctgctgaa ctgggcgccg 360

aaagtggcgc gccgctttca gctgccgagc gcgctgctgt ggattcagcc ggcgctggtg 420

tttaacattt attataccca ttttatgggc aacaaaagcg tgtttgaact gccgaacctg 480

agcagcctgg aaattcgcga tctgccgagc tttctgaccc cgagcaacac caacaaaggc 540

gcgtatgatg cgtttcagga aatgatggaa tttctgatta aagaaaccaa accgaaaatt 600

ctgattaaca cctttgatag cctggaaccg gaagcgctga ccgcgtttcc gaacattgat 660

atggtggcgg tgggcccgct gctgccgacc gaaattttta gcggcagcac caacaaaagc 720

gtgaaagatc agagcagcag ctataccctg tggctggata gcaaaaccga aagcagcgtg 780

atttatgtga gctttggcac catggtggaa ctgagcaaaa aacagattga agaactggcg 840

cgcgcgctga ttgaaggcaa acgcccgttt ctgtgggtga ttaccgataa aagcaaccgc 900

gaaaccaaaa ccgaaggcga agaagaaacc gaaattgaaa aaattgcggg ctttcgccat 960

gaactggaag aagtgggcat gattgtgagc tggtgcagcc agattgaagt gctgagccat 1020

cgcgcggtgg gctgctttgt gacccattgc ggctggagca gcaccctgga aagcctggtg 1080

ctgggcgtgc cggtggtggc gtttccgatg tggagcgatc agccgaccaa cgcgaaactg 1140

ctggaagaaa gctggaaaac cggcgtgcgc gtgcgcgaaa acaaagatgg cctggtggaa 1200

cgcggcgaaa ttcgccgctg cctggaagcg gtgatggaag aaaaaagcgt ggaactgcgc 1260

gaaaacgcga aaaaatggaa acgcctggcg atggaagcgg gccgcgaagg cggcagcagc 1320

gataaaaaca tggaagcgtt tgtggaagat atttgcggcg aaagcctgat tcagaacctg 1380

tgcgaagcgg aagaagtgaa agtgaaataa 1410

＜210＞ 4

＜211＞　469

＜212＞ PRT

< 213 > is artificially synthesized

＜400＞ 1

MAPPHFLLVT FPAQGHVNPS LRFARRLIKR TGARVTFVTC VSVFHNSMIA NHNKVENLSF 60

LTFSDGFDDG GISTYEDRQK RSVNLKVNGD KALSDFIEAT KNGDSPVTCL IYTILLNWAP 120

KVARRFQLPS ALLWIQPALV FNIYYTHFMG NKSVFELPNL SSLEIRDLPS FLTPSNTNKG 180

AYDAFQEMME FLIKETKPKI LINTFDSLEP EALTAFPNID MVAVGPLLPT EIFSGSTNKS 240

VKDQSSSYTL WLDSKTESSV IYVSFGTMVE LSKKQIEELA RALIEGKRPF LWVITDKSNR 300

ETKTEGEEET EIEKIAGFRH ELEEVGMIVS WCSQIEVLSH RAVGCFVTHC GWSSTLESLV 360

LGVPVVAFPM WSDQPTNAKL LEESWKTGVR VRENKDGLVE RGEIRRCLEA VMEEKSVELR 420

ENAKKWKRLA MEAGREGGSS DKNMEAFVED ICGESLIQNL CEAEEVKVK 469

＜210＞ 5

＜211＞　1410bp

＜212＞ DNA

< 213 > is artificially synthesized

＜400＞ 1

atgagccagc cgcattttct gctggtgacc tttccggcgc agggccatat taacccgacc 60

ctgcagtttg cgaaacatct gattcgcatt ggcgtgcgcg tgacctttat tacctgcatt 120

agcgcgcgcc gccgcatgac caaagtgccg accgcgcagg gcctgacctt tctgccgttt 180

agcgatggct atgatgatgg ctttcagccg ggcgatgata ttgaacatta tctgagcgaa 240

ctgcgccgcc gcggcaaaga agcgattagc gaatttatta ccagcagcga atatgaaggc 300

aaaccggtga cctgcattgt gtataccctg tttattcatt gggcgagcga agtggcgcgc 360

aaacatcata ttccggcggc gctgctgtgg attcagccgg cgaccgtgtt tgatatttat 420

tatttttatt ttaacggcta tgaaagcacc attaaagtgc cggtggatga aaccaacccg 480

aaacgcagca ttaaactgcc gggcctgccg ctgctggcga cccgcgatct gccgagcttt 540

gtgaccgcga gcaacgtgta tcgctgggcg ctgagcctgt ttcaggaaca gatggatatt 600

ctggcggatg aaagcaaccc gaaaattctg gtgaacacct ttgatgcgct ggaacaggaa 660

gcgctgaacg cgattgaaaa ctttaacatg gtgggcattg gcccgctgat tccgagcagc 720

tttctgaaca gcaacgatag cctggataac agcctgcgca ccgatctgtt tcagagcgat 780

agcaaagatt atattcagtg gctggatagc aaaccgaaaa gcgcggtggt gtatgtgagc 840

tttggcagca ttgcggtgct gaccaaacag caggtggaag aaattgcgcg cgcgctgatt 900

agcagccgcc gcccgtttct gtgggtggtg cgcaactgga aagatggcgt ggaagaagaa 960

aaagaagaag ataaactgac ctggcgcgaa gaactggaac gctttggcat ggtggtgccg 1020

tggtgcagcc aggtggaagt gctgagccat ccgagcctgg gctgctttgt gacccattgc 1080

ggctggaaca gcaccctgga aagcatggtg gcgggcgtgc cggtggtggc gtttccgcag 1140

tggaccgatc agggcaccaa cgcgaaactg attgaagatg tgtggggcaa cggcgtgcgc 1200

gtgagcgcga acgaagaagg catggtggaa cgcgatgaaa ttgtgcgctg cctggatctg 1260

gtgatgggcg atgatgaaaa aggcatggaa gtgaaaaaaa acgtggaaaa atgggaaggc 1320

ctggcgcgcg aagcgagcat ggaaggcggc agcatggata tgaacctgaa agcgtttgtg 1380

gatgatgtgg cgcagggctg ctggaaataa 1410

＜210＞ 6

＜211＞　469

＜212＞ PRT

< 213 > is artificially synthesized

＜400＞ 1

MSQPHFLLVT FPAQGHINPT LQFAKHLIRI GVRVTFITCI SARRRMTKVP TAQGLTFLPF 60

SDGYDDGFQP GDDIEHYLSE LRRRGKEAIS EFITSSEYEG KPVTCIVYTL FIHWASEVAR 120

KHHIPAALLW IQPATVFDIY YFYFNGYEST IKVPVDETNP KRSIKLPGLP LLATRDLPSF 180

VTASNVYRWA LSLFQEQMDI LADESNPKIL VNTFDALEQE ALNAIENFNM VGIGPLIPSS 240

FLNSNDSLDN SLRTDLFQSD SKDYIQWLDS KPKSAVVYVS FGSIAVLTKQ QVEEIARALI 300

SSRRPFLWVV RNWKDGVEEE KEEDKLTWRE ELERFGMVVP WCSQVEVLSH PSLGCFVTHC 360

GWNSTLESMV AGVPVVAFPQ WTDQGTNAKL IEDVWGNGVR VSANEEGMVE RDEIVRCLDL 420

VMGDDEKGME VKKNVEKWEG LAREASMEGG SMDMNLKAFV DDVAQGCWK 469

＜210＞ 7

＜211＞　1395bp

＜212＞ DNA

< 213 > is artificially synthesized

＜400＞ 1

atggcgccgc cgccgccgca ttttctgctg gtgacctttc cggcgcaggg ccatgtgaac 60

ccgagcctgc gctttgcgta tcgcctgatt cgcaccaccg gcgcgcgcgt gacctttgtg 120

acctgcgcga gcgtgtttca tcgcagcatg attagcaacc atagcgatct ggataacctg 180

agctttctga cctttagcga tggctttgat cagggcggcc tgaccaccgc ggaagatcat 240

aaaaaacgca gcgcgaacct ggaaattaac ggcgataaag cgctgagcga atttattaaa 300

gcgaacgaaa acggcgatag cccggtgacc tgcctggtgt ataccatttt tctgaactgg 360

gcgccgaaag tggcgagccg ctttcagctg ccgagcgcgc tgctgtggat tcagccggcg 420

ctggtgtttg atatttatta taaccatttt aacggcgata acaacaacag ctatctggaa 480

tttaaaaacc tgccgagcct ggcgattcgc gatctgccga gctttctgac cccgaccaac 540

accaaccagg cggcgtatgc gagctttcag gaactgatgg aactgctgaa aaaagaaacc 600

aacccgaaaa ttctggtgaa cacctttgat agcctggaac aggaagcgct gaaagcgatt 660

ccgagcgtgg gcatggtggc ggtgggcccg ctgctgccga gcgatatgtt taccggcagc 720

gaaccggtga aagatctgag caaagaacag accggcagca gctatagccg ctggctggaa 780

agcaaaaccg aaagcagcgt gatttatgtg agctttggca ccatggtgga actgagcaaa 840

aaacagattg aagaactggc gcgcgcgctg attgaaggca aacgcccgtt tctgtgggtg 900

attaccgata aaagcaaccg cgaagcgaaa accgaaggcg aagatgaaac cgaaattgaa 960

aaaattgcgg gctttcgcca tgaactggaa gaagtgggca tgattgtgag ctggtgcagc 1020

caggtggaag tgctgaaaca tcgcgcggtg ggctgctttg tgacccattg cggctggagc 1080

agcaccctgg aaagcctggt gctgggcgtg ccggtggtgg cgtttccgat gtggagcgat 1140

cagccgacca gcgcgaaact gctggaagaa ctgtggcgca ccggcgtgcg cgtgcgcgaa 1200

aacgaagaag gcctggtgga acgcgaagaa attcgccgct gcctggaagc ggtgatggat 1260

gaacgcctgg tggaactgcg cgaaaacgcg gtgaaatgga aacgcctggc ggtggaagcg 1320

ggccgcgaag gcggcctgag cgataaaaac atggaagcgt ttgtggaaga aatttgcgaa 1380

gatagcgtgc tgtaa 1395

＜210＞ 8

＜211＞　464

＜212＞ PRT

< 213 > is artificially synthesized

＜400＞ 1

MAPPPPHFLLVTFPAQGHVNPSLRFAYRLIRTTGARVTFVTCASVFHRSMISNHSDLDNL 60

SFLTFSDGFD QGGLTTAEDH KKRSANLEIN GDKALSEFIK ANENGDSPVT CLVYTIFLNW 120

APKVASRFQL PSALLWIQPA LVFDIYYNHF NGDNNNSYLE FKNLPSLAIR DLPSFLTPTN 180

TNQAAYASFQ ELMELLKKET NPKILVNTFD SLEQEALKAI PSVGMVAVGP LLPSDMFTGS 240

EPVKDLSKEQ TGSSYSRWLE SKTESSVIYV SFGTMVELSK KQIEELARAL IEGKRPFLWV 300

ITDKSNREAK TEGEDETEIE KIAGFRHELE EVGMIVSWCS QVEVLKHRAV GCFVTHCGWS 360

STLESLVLGV PVVAFPMWSD QPTSAKLLEE LWRTGVRVRE NEEGLVEREE IRRCLEAVMD 420

ERLVELRENA VKWKRLAVEA GREGGLSDKN MEAFVEEICE DSVL 464

＜210＞ 9

＜211＞　1647bp

＜212＞ DNA

< 213 > is artificially synthesized

＜400＞ 1

atgcgcagct tttatggcta tcgcggcatt ccgattcgct ttgtgcagct gtggagcgaa 60

ctgcagcatg aacgcaccga aagcgtgaaa atgtgcctgg gctattgctg gctgaccgtg 120

tttctgtgcc tgctgctgca gcaggatctg catcaggatc aggcggaagc ggcgaacatt 180

ctgggcattt ttccgtatcg ccatattagc ccgttttttg tgatgcagcc gctggtgcgc 240

accctggcgg aacgcggcca taacgtgacc ctgattaccc cgagcggcct gccgaacgat 300

attgaaggcg tgcgccatat tcgcgtggcg cagctgaacg aacgcattaa agatcaggtg 360

ctggattttc tgattaacaa atggaccgaa agcgcgctga ccgcgaaagc gctgtataac 420

gcgagcaacg atattctgag cgatccgggc gtgcagcgca tgctgcatga taaaagcgaa 480

cgctttgatc tgattattat ggaaccgagc agcctggatg cgctgtatgg cctggtggaa 540

ttttataacg cgaccctgat tggcctgagc agcattcgca ttaactggca gaccgatgaa 600

ctggcgggca acccggcgcc gagcatttat gaaccgatta gcccggtggg ctttagcctg 660

gaaaccagcc tgtttagccg cgtgtataac tggattcata ttatggaaga aaaactggtg 720

gattatctga ttctgcgccc ggcgcagctg catctgtttc agaaattttt tggctatagc 780

gcgcagaaaa tgaacgaact gcgcaaccgc tttagcctga tgctgattaa cagccattat 840

agcatgggca aagtgcgcgc gaacgcgccg aacattattg aagtgggcgg cctgcatctg 900

agcgaaccgc cggaaccgag cgatgaagaa ctgcagaaat ttctggataa agcgaaccat 960

ggcgtgattt attttagcat gggcaacgat gtgctgatta aatttctgcc ggcgaacatt 1020

caggaactgc tgctgcagac ctttgcgaaa ctgaaagaaa gcattatttg gaaaagcgaa 1080

ctgctgtgca tgccgaacaa aagcgataac gtgtatgtga ttgaacaggc gccgcagcgc 1140

catattctga accatccgaa cgtgcgcctg tttattacca acggcggcct gctgagcgtg 1200

attgaagcgg tggatagcgg cgtgccgatg ctgggcctgc cgatgttttt tgatcagttt 1260

gcgaacatgc gctgggtgca gctgagcggc atggcggaag tgatggatat taacattctg 1320

aacaaagata ccctgaccga aaccattaaa catatgctgg cgagcgatag ctattatctg 1380

aaagcgaaag aaatgagcca gttttttaaa gatcgcccga tgagcccgct ggataccgcg 1440

gtgtggtgga ccgaatatgc gctgcgcaac cgcaacatta cccgcatgcg cctgaacctg 1500

gaagaaattc cgctgattga atattatcgc attgatagca ttctggcgtt tagcctgcgc 1560

tttggcctgg tggcggcgag cctgattttt ctggtgtata ccctgtttct gaaatatcgc 1620

attcgcctgc gccgcctgca tccgtaa 1647

＜210＞ 10

＜211＞　548

＜212＞ PRT

< 213 > is artificially synthesized

＜400＞ 1

MRSFYGYRGI PIRFVQLWSE LQHERTESVK MCLGYCWLTV FLCLLLQQDL HQDQAEAANI 60

LGIFPYRHIS PFFVMQPLVR TLAERGHNVT LITPSGLPND IEGVRHIRVA QLNERIKDQV 120

LDFLINKWTE SALTAKALYN ASNDILSDPG VQRMLHDKSE RFDLIIMEPS SLDALYGLVE 180

FYNATLIGLS SIRINWQTDE LAGNPAPSIY EPISPVGFSL ETSLFSRVYN WIHIMEEKLV 240

DYLILRPAQL HLFQKFFGYS AQKMNELRNR FSLMLINSHY SMGKVRANAP NIIEVGGLHL 300

SEPPEPSDEE LQKFLDKANH GVIYFSMGND VLIKFLPANI QELLLQTFAK LKESIIWKSE 360

LLCMPNKSDN VYVIEQAPQR HILNHPNVRL FITNGGLLSV IEAVDSGVPM LGLPMFFDQF 420

ANMRWVQLSG MAEVMDINIL NKDTLTETIK HMLASDSYYL KAKEMSQFFK DRPMSPLDTA 480

VWWTEYALRN RNITRMRLNL EEIPLIEYYR IDSILAFSLR FGLVAASLIF LVYTLFLKYR 540

IRLRRLHP 548

＜210＞ 11

＜211＞　1437bp

＜212＞ DNA

< 213 > is artificially synthesized

＜400＞ 1

atggcggcga ccaccgaaaa cctgccgcag ctgaaaagcg cggtggatgg cctgaccgaa 60

atgagcgaaa gcgaaaaaag cggctttatt agcctggtga gccgctatct gagcggcgaa 120

gcgcagcata ttgaatggag caaaattcag accccgaccg atgaaattgt ggtgccgtat 180

gaaaaaatga ccccggtgag ccaggatgtg gcggaaacca aaaacctgct ggataaactg 240

gtggtgctga aactgaacgg cggcctgggc accaccatgg gctgcaccgg cccgaaaagc 300

gtgattgaag tgcgcgatgg cctgaccttt ctggatctga ttgtgattca gattgaaaac 360

ctgaacaaca aatatggctg caaagtgccg ctggtgctga tgaacagctt taacacccat 420

gatgataccc ataaaattgt ggaaaaatat accaacagca acgtggatat tcataccttt 480

aaccagagca aatatccgcg cgtggtggcg gatgaatttg tgccgtggcc gagcaaaggc 540

aaaaccgata aagaaggctg gtatccgccg ggccatggcg atgtgtttcc ggcgctgatg 600

aacagcggca aactggatac ctttctgagc cagggcaaag aatatgtgtt tgtggcgaac 660

agcgataacc tgggcgcgat tgtggatctg accattctga aacatctgat tcagaacaaa 720

aacgaatatt gcatggaagt gaccccgaaa accctggcgg atgtgaaagg cggcaccctg 780

attagctatg aaggcaaagt gcagctgctg gaaattgcgc aggtgccgga tgaacatgtg 840

aacgaattta aaagcattga aaaatttaaa atttttaaca ccaacaacct gtgggtgaac 900

ctgaaagcga ttaaaaaact ggtggaagcg gatgcgctga aaatggaaat tattccgaac 960

ccgaaagaag tggatggcgt gaaagtgctg cagctggaaa ccgcggcggg cgcggcgatt 1020

cgcttttttg ataacgcgat tggcgtgaac gtgccgcgca gccgctttct gccggtgaaa 1080

gcgagcagcg atctgctgct ggtgcagagc gatctgtata ccctggtgga tggctttgtg 1140

acccgcaaca aagcgcgcac caacccgagc aacccgagca ttgaactggg cccggaattt 1200

aaaaaagtgg cgacctttct gagccgcttt aaaagcattc cgagcattgt ggaactggat 1260

agcctgaaag tgagcggcga tgtgtggttt ggcagcagca ttgtgctgaa aggcaaagtg 1320

accgtggcgg cgaaaagcgg cgtgaaactg gaaattccgg atcgcgcggt ggtggaaaac 1380

aaagcggtga tgattctggt gcaggatttt cagagcaacc atcagaacga aaactaa 1437

＜210＞ 12

＜211＞　478

＜212＞ PRT

< 213 > is artificially synthesized

＜400＞ 1

MAATTENLPQ LKSAVDGLTE MSESEKSGFI SLVSRYLSGE AQHIEWSKIQ TPTDEIVVPY 60

EKMTPVSQDV AETKNLLDKL VVLKLNGGLG TTMGCTGPKS VIEVRDGLTF LDLIVIQIEN 120

LNNKYGCKVP LVLMNSFNTH DDTHKIVEKY TNSNVDIHTF NQSKYPRVVA DEFVPWPSKG 180

KTDKEGWYPP GHGDVFPALM NSGKLDTFLS QGKEYVFVAN SDNLGAIVDL TILKHLIQNK 240

NEYCMEVTPK TLADVKGGTL ISYEGKVQLL EIAQVPDEHV NEFKSIEKFK IFNTNNLWVN 300

LKAIKKLVEA DALKMEIIPN PKEVDGVKVL QLETAAGAAI RFFDNAIGVN VPRSRFLPVK 360

ASSDLLLVQS DLYTLVDGFV TRNKARTNPS NPSIELGPEF KKVATFLSRF KSIPSIVELD 420

SLKVSGDVWF GSSIVLKGKV TVAAKSGVKL EIPDRAVVEN KAVMILVQDF QSNHQNEN 478

＜210＞ 13

＜211＞1542bp

＜212＞ DNA

< 213 > is artificially synthesized

＜400＞ 1

atgctggatg tgccgcatga agcgaaagtg cgcggccatc agcgcgcgcc gagcgatagc 60

aaagaatttc atgaagtgac caaacgcgat gcgctgcgcc tgctggaaca tgatgtggat 120

cgcctgctgg aaaccaccga aaaagcgcgc cagccggcgc tgaaagcgga aatgggccgc 180

tttgcggatc tgtttggccg ctttattcag gaagaaggcc cggcgctgga ttggaacaaa 240

attcagaaac tgccggaaaa cgcggtgatg aactatagca acctgaaaag cccgaaaaac 300

gaacagaacg aaattcgcaa catgctggat aaactggtgg tgattaaact gaacggcggc 360

ctgggcacca gcatgggctg ccatggcccg aaaagcgtga ttccggtgcg cagcgatctg 420

acctttctgg atctgaccgt gcagcagatt gaacatctga acaaaaccta tgatgcgaac 480

gtgccgctgg tgctgatgaa cagctttaac accgatgaag ataccgaaaa aattgtgcgc 540

aaatataaag gctttcgcgt gcagattcat acctttaacc agagctgctt tccgcgcatt 600

agccgcgaac attatctgcc ggtggcgaaa gattttgatg tggaaaaaga tatggaagcg 660

tggtatccgc cgggccatgg cgatttttat gatacctttc gcaacagcgg cctgctgaaa 720

aaatttattg aagaaggccg cgaatattgc tttctgagca acattgataa cctgggcgcg 780

accgtggatc tgaacattct gaacaaactg gtgggcgaag aacgcgcgac caccccggtg 840

gaatttgtga tggaagtgac cgataaaacc cgcgcggatg tgaaaggcgg caccctgatt 900

cagatggaaa acaaactgcg cctgctggaa attgcgcagg tgccgccgga acatgtggat 960

gattttaaaa gcgtgaaaac ctttaaattt tttaacacca acaacatttg ggcgaacctg 1020

gcggcgattg atcgcgtgct gcgcgaacgc accctgaaca tggaaattat tgtgaacaac 1080

aaaaccctgg aaaacggcac ccgcgtgatt cagctggaaa ccgcggtggg cgcggcgatg 1140

aaatgctttg atggcgcgat tggcattaac gtgccgcgca gccgctttct gccggtgaaa 1200

aaaagcagcg atctgctgct ggtgatgagc aacctgtata ccctgaaaaa cggcagcctg 1260

gtgatgagcc cgcagcgcat gtttccgacc accccgctgg tgaaactggg cgaaaaccat 1320

tttagcaaag tgaaagaatt tctgggccgc tttgcgaaca ttccggatat tattgaactg 1380

gatcatctga ccgtgagcgg cgatgtgacc tttggccgcg gcgtgagcct gcgcggcacc 1440

gtgattatta ttgcgaacca tggcgatcgc attgatattc cggcgggcgc gattctggaa 1500

aacaaaattg tgagcggcaa catgcgcatt ctggatcatt aa 1542

＜210＞ 14

＜211＞　513

＜212＞ PRT

< 213 > is artificially synthesized

＜400＞ 1

MLDVPHEAKV RGHQRAPSDS KEFHEVTKRD ALRLLEHDVD RLLETTEKAR QPALKAEMGR 60

FADLFGRFIQ EEGPALDWNK IQKLPENAVM NYSNLKSPKN EQNEIRNMLD KLVVIKLNGG 120

LGTSMGCHGP KSVIPVRSDL TFLDLTVQQI EHLNKTYDAN VPLVLMNSFN TDEDTEKIVR 180

KYKGFRVQIH TFNQSCFPRI SREHYLPVAK DFDVEKDMEA WYPPGHGDFY DTFRNSGLLK 240

KFIEEGREYC FLSNIDNLGA TVDLNILNKL VGEERATTPV EFVMEVTDKT RADVKGGTLI 300

QMENKLRLLE IAQVPPEHVD DFKSVKTFKF FNTNNIWANL AAIDRVLRER TLNMEIIVNN 360

KTLENGTRVI QLETAVGAAM KCFDGAIGIN VPRSRFLPVK KSSDLLLVMS NLYTLKNGSL 420

VMSPQRMFPT TPLVKLGENH FSKVKEFLGR FANIPDIIEL DHLTVSGDVT FGRGVSLRGT 480

VIIIANHGDR IDIPAGAILE NKIVSGNMRI LDH 513

Claims

1. A method for synthesizing mogroside V, which is characterized by comprising the following steps: converting a substrate mogrol by a recombinant cell containing a glycosyltransferase gene or a recombinant cell containing the glycosyltransferase gene and a UDP-glucose pyrophosphorylase gene to obtain the mogroside V, and expressing the glycosyltransferase gene and the UDP-glucose pyrophosphorylase gene by the recombinant cell to produce UGT enzyme and UDP-glucose pyrophosphorylase.

2. The method of claim 1, wherein: the method comprises the following specific steps: step (1): obtaining crude enzyme solution by using recombinant cells containing glycosyltransferase genes and UDP-glucose pyrophosphorylase genes; step (2): adding crude enzyme solution into mogrol as a substrate, and converting and synthesizing the mogroside V in the presence of UDP glucose.

3. The method of claim 1, wherein: the preparation method of the crude enzyme solution comprises the following steps: inoculating a recombinant cell containing a glycosyltransferase gene and a UDP-glucose pyrophosphorylase gene into an LB liquid culture medium to obtain a seed solution, inoculating the seed solution into the LB liquid culture medium, culturing until the OD600 of a thallus is 0.4-1.0, adding IPTG (isopropyl-beta-thiogalactoside) to induce and culture, and inducing a target protein to express; after induction, the cells were collected by centrifugation, resuspended, disrupted by ultrasonic/freezing cycles in liquid nitrogen, and then centrifuged, and the supernatant was collected to obtain a crude enzyme solution.

4. The method of claim 2, wherein: the reaction condition in the step (2) is 25-45 ℃, and the reaction time is 1-48 h.

5. A recombinant vector comprising a glycosyltransferase gene, or comprising a glycosyltransferase gene and a UDP-glucose pyrophosphorylase gene.

6. The method for producing a recombinant vector according to claim 5, wherein: the method comprises the following steps: (1) carrying out PCR amplification by using a specific primer by using a carrier as a template to obtain a linear carrier reverse amplification fragment; (2) taking a glycosyltransferase gene as a template, carrying out PCR amplification to obtain a UGT positive amplification fragment, recovering the UGT positive amplification fragment, and carrying out reverse amplification connection on the recovered UGT positive amplification fragment and the vector to obtain a first recombinant vector containing the glycosyltransferase gene; further, performing PCR amplification by using the first recombinant vector as a template to obtain a linear vector reverse amplification fragment; carrying out PCR amplification by taking UDP-glucose pyrophosphorylase gene as a template to obtain an UDPase amplified fragment, recovering the UDPase amplified fragment, and linking the recovered UDPase amplified fragment with the vector reverse-amplified fragment to obtain a second recombinant vector containing the glycosyltransferase gene and the UDP-glucose pyrophosphorylase gene.

7. A genetic engineering bacterium for biosynthesizing mogroside V by taking mogrol as a substrate is characterized in that: the genetic engineering bacteria express glycosyltransferase gene and UDP-glucose pyrophosphorylase gene to produce UGT enzyme and UDP-glucose pyrophosphorylase.

8. The method of claim 1, 5 or 7, wherein: the glycosyltransferase gene sequence is shown in SEQ ID NO.1, SEQ ID NO.3, SEQ ID NO.5, SEQ ID NO.7 or SEQ ID NO. 9; the sequence of the UDP-glucose pyrophosphorylase gene is shown as SEQ ID NO.11 or SEQ ID NO. 13.

9. The method for constructing a genetically engineered bacterium according to claim 7, wherein: transforming a host cell with the recombinant vector of claim 5, and culturing the transformant to obtain an engineering strain for biosynthesizing mogroside V by using mogrol as a substrate.

10. The method for constructing a genetically engineered bacterium according to claim 9, wherein: the host cell is Escherichia coli.