Construction and application of an engineered strain for biosynthesizing mogroside V using mogroside alcohol as substrate

Abstract

The present invention relates to the construction of an engineering strain for biosynthesizing mogroside V using mogroside alcohol as a substrate and its application. The substrate mogroside V is obtained by transforming mogroside alcohol with a recombinant cell containing a glycosyltransferase gene, or a recombinant cell containing a glycosyltransferase gene and a UDP-glucose pyrophosphorylase gene, and the recombinant cell expresses the glucose glycosyltransferase gene and the UDP-glucose pyrophosphorylase gene, and produces UGT enzyme and UDP-glucose pyrophosphorylase. The present invention introduces the UGT enzyme gene and the UDP-glucose pyrophosphorylase gene of an exogenous species by gene recombination technology, transforms them into genetically engineered bacteria for expression, reconstructs a new biosynthetic pathway of mogroside V, and realizes the biosynthesis of mogroside V using mogroside alcohol as a substrate through the overexpression of the UGT enzyme gene and the UDP-glucose pyrophosphorylase gene, with a high synthesis yield.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to the construction and application of an engineering strain for biosynthesizing mogroside V using mogroside alcohol as a substrate. Background Art

[0002] Sweets are a kind of life condiment, and their unique taste gives us endless fun in life. Sweetness mainly comes from traditional sweeteners: sucrose. While satisfying human sweetness needs, sucrose can also provide a certain amount of calories. However, for patients with obesity, hyperlipidemia, hyperglycemia, etc., the intake of sucrose needs to be strictly controlled. The reduction in sugar intake means the reduction of the enjoyment of sweetness. How to reduce sugar intake while satisfying the need for sweetness? Synthetic sweeteners provide people with a possibility, but recent studies have pointed out that synthetic sweeteners will affect the normal intestinal flora, and even worse, will cause metabolic syndrome and glucose intolerance. Natural sugar-free sweeteners that take into account the safety of natural sugars and the calorie-free advantages of synthetic sweeteners have entered people's field of vision. The theme of developing natural sugar-free sweeteners to meet human "needs" for sweetness has attracted great interest from a large number of food practitioners.

[0003] Mogroside is a natural sweetener extracted directly from the Cucurbitaceae plant Siraitiagrosvenorii without chemical synthesis. It contains almost no energy, but its sweetness is about 300 times that of sucrose, making it an ideal sucrose substitute. At the same time, mogroside also has the functions of regulating blood sugar, anti-oxidation, relieving cough and expectoration, protecting the liver, moistening the intestines and promoting bowel movements, and antibacterial and anti-inflammatory. Mogroside is a cucurbitane triterpenoid glycoside compound, including mogroside Ⅰ (Mogroside Ⅰ), mogroside ⅡE (Mogroside ⅡE), mogroside Ⅲ (Mogroside Ⅲ), mogroside ⅢE (Mogroside ⅢE), mogroside Ⅳ (Mogroside Ⅳ), mogroside Ⅴ (Mogroside Ⅴ), 11-oxidized-mogroside Ⅴ (11-Oxo-mogroside V), neomogroside and siamenoside Ⅰ (Siamenoside Ⅰ) and other components. Among them, mogroside V (M5) is the main sweet component with the advantages of high sweetness, low energy, safety and non-toxicity, and has broad application prospects.

[0004] However, the content of mogroside V in monk fruit is only 0.8% to 1.3% (W / W). It is difficult to achieve large-scale production of high-yield and high-purity mogroside V by extracting it from the fruit alone. In addition, due to the harsh planting environment and requirements of monk fruit, the traditional research progress in improving the production of mogroside V through hybrid breeding is limited. Therefore, biosynthesis of mogroside V has become an inevitable trend for large-scale production. However, there are few reports on the use of biosynthesis of mogroside V. Therefore, a method for producing mogroside V by recombining engineered bacteria using key enzymes required for the synthesis of natural mogroside V is provided. Summary of the invention

[0005] In order to solve the above technical problems, the purpose of the present invention is to provide an engineered strain for biosynthesizing mogroside V using mogroside alcohol as a substrate and its application. The engineered strain can convert mogroside alcohol into mogroside V in one step without the use of complicated chemical methods, and has high yield, high purity, and is friendly to the human body and the environment, providing a basis for the large-scale industrial production of mogroside V.

[0006] A method for synthesizing mogroside V comprises the following steps: using a recombinant cell containing a glycosyltransferase (UDP-glucosyltransferase, UGT) gene, or a recombinant cell containing a glycosyltransferase (UDP-glucosyltransferase, UGT) gene and a UDP-glucose pyrophosphorylase gene to transform a substrate mogroside alcohol to obtain mogroside V; the recombinant cell expresses the glucose glycosyltransferase (UDP-glucosyltransferase, UGT) gene and the UDP-glucose pyrophosphorylase gene, and produces the UGT enzyme and the UDP-glucose pyrophosphorylase.

[0007] According to the above 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 UDP-glucose pyrophosphorylase gene sequence is shown as SEQ ID NO.11 or SEQ ID NO.13.

[0008] According to the above scheme, the specific steps of the above method are: step (1): using recombinant cells containing glycosyltransferase (UDP-glucosyltransferase, UGT) genes and UDP-glucose pyrophosphorylase genes to obtain crude enzyme solution; step (2): using mogroside alcohol as a substrate, adding the crude enzyme solution, and converting and synthesizing mogroside V in the presence of UDP glucose.

[0009] According to the above scheme, the preparation method of the crude enzyme solution is: inoculating recombinant cells containing glycosyltransferase (UDP-glucosyltransferase, UGT) genes and UDP-glucose pyrophosphorylase genes into LB liquid culture medium to obtain seed liquid, inoculating the seed liquid into LB liquid culture medium, and the culture conditions can be: 37°C, 200rpm, culturing until the bacterial OD600 = 0.4-1.0, and then adding IPTG induction culture to induce the expression of the target protein; after the induction is completed, the bacterial cells are collected by centrifugation, and the bacterial cells are resuspended, and the cells are broken by ultrasound / freezing cycle in liquid nitrogen, and then the cell broken liquid is centrifuged, and the supernatant is collected to obtain the crude enzyme solution.

[0010] According to the above scheme, the reaction conditions in step (2) of step (2) are 25-45°C and the reaction time is 1-48h.

[0011] A recombinant vector comprises a glycosyltransferase (UDP-glucosyltransferase, UGT) gene, or comprises a glycosyltransferase (UDP-glucosyltransferase, UGT) gene and a UDP-glucose pyrophosphorylase gene.

[0012] A method for preparing a recombinant vector comprises the following steps: (1) using a vector as a template and performing PCR amplification using specific primers to obtain a linearized vector reverse amplification fragment; (2) using a glycosyltransferase gene as a template and performing PCR amplification to obtain a UGT forward amplification fragment, which is recovered and connected with the vector reverse amplification to obtain a first recombinant vector; and further, using the first recombinant vector as a template and performing PCR amplification to obtain a linearized vector reverse amplification fragment; using a UDP-glucose pyrophosphorylase gene as a template and performing PCR amplification to obtain a UDPase amplification fragment, which is recovered and connected with the aforementioned vector reverse amplification fragment to obtain a second recombinant vector.

[0013] A genetically engineered bacterium for biosynthesizing mogroside V using mogroside alcohol as a substrate. The genetically engineered bacterium expresses a glycosyltransferase (UDP-glucosyltransferase, UGT) gene and a UDP-glucose pyrophosphorylase gene to produce UGT enzyme and UDP-glucose pyrophosphorylase.

[0014] The method for constructing the genetically engineered bacteria comprises transforming the recombinant vector into a host cell, culturing the transformant, and obtaining an engineered strain that biosynthesizes mogroside V using mogroside alcohol as a substrate.

[0015] According to the above scheme, the host cell is Escherichia coli.

[0016] Provided is the use of the recombinant vector or genetically engineered bacteria in the biosynthesis of mogroside V.

[0017] Mogroside V is a triterpenoid saponin compound, and its synthetic precursors are isopentenylpyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP). IPP and DMAPP are generated from acetyl-CoA via the mevalonic acid pathway (MVA pathway), and then catalyzed by geranyl pyrophosphate synthase (PS), farnesylpyrophosphate synthase (FPPS), squalene synthase (SQS), cyclase and cucurbitadienol synthase (CDS) to synthesize 24,25-epoxycucurbitadienol. Subsequently, 24,25-epoxycucurbitadienol is converted into mogroside by the action of epoxide hydrolase (EPH) and cytochrome P450 (CYP450). Finally, mogroside V is synthesized after gradual glycosylation modification by UDP-glucosyltransferase (UGT) using mogroside as a substrate. UGT is the decisive enzyme in the conversion of mogroside V from mogroside alcohol. It uses mogroside alcohol as a substrate and UDP-glucose as a glycosyl donor to catalyze the glycosylation reaction of the glucose branch chains at C24, C3, and C3, C24 of mogroside alcohol, and synthesizes mogroside V through tetraglycosylation intermediates. At the same time, as a glycosyl donor, the supply of UDP-glucose in the catalytic process also affects the glycosylation efficiency of mogroside alcohol. When the glycosyl supply is insufficient, the glycosylation reaction of mogroside alcohol will be restricted. Therefore, increasing the UDP-glucose content may significantly promote the glycosylation efficiency of mogroside alcohol.

[0018] To this end, the present invention selects UGT and UDPase enzyme genes, creates a genetic engineering strain that co-expresses UGT and UDPase, and uses high expression in the host strain and the addition of mogroside alcohol as a substrate to synthesize mogroside V. The method is simple to operate, does not pollute the environment, has high yield, high purity, and low cost, and provides a reference for the industrial production of mogroside V.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] The invention introduces UGT enzyme genes and UDP-glucose pyrophosphorylase genes of exogenous species through gene recombination technology, transforms them into genetically engineered bacteria for expression, reconstructs a new biosynthetic pathway of mogroside V, and realizes the biosynthesis of mogroside V using mogroside alcohol as a substrate through overexpression of the UGT enzyme gene and the UDP-glucose pyrophosphorylase gene, with high synthesis yield.

[0021] The method is simple to operate, does not pollute the environment, and has high yield, high purity and low cost, thus providing a basis for the industrial production of mogroside V. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Schematic diagram of the construction of engineered strains expressing UGT and UGT-UDPase.

[0023] Figure 2 The yield of mogroside V in the engineered bacteria. DETAILED DESCRIPTION

[0024] Example 1 Construction of pET28a-UGT plasmid and recombinant Escherichia coli strain

[0025] Construction method of pET28a-UGT plasmid:

[0026] (1) Using pET28a vector as template, PCR amplification was performed using specific primers. The pET28a vector is a commercial vector purchased from Novagen; the primer sequences are shown in Table 1; after the PCR product was recovered, a linearized vector pET28a-reverse amplification was obtained, and the linearized vector fragment size was 5369 bp;

[0027] (2) Based on the UGT protein sequences of five organisms, namely, Lepeophtheirussalmonis, Arabidopsis thaliana, Gossypiumhirsutum, Raphanussativus, and Drosophila simulans, published in the NCBI (https: / / www.ncbi.nlm.nih.gov / ) database, the protein sequences are shown in SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.6, SEQ ID NO.8, or SEQ ID NO.10, and codon optimization is performed according to the codon preference of Escherichia coli. The nucleic acid sequences of LsUGT, AtUGT, GhUGT, RsUGT, and DsUGT are shown in SEQ ID NO.1, SEQ ID NO.3, SEQ ID NO.5, SEQ ID NO.7, or SEQ ID NO.9, respectively. Six pairs of specific primers are designed to amplify them. The amino acid sequence and nucleic acid sequence of the UGT are shown in SEQ Listing, and the primer sequences are shown in Table 1.

[0028] Table 1 Primer sequences

[0029]

[0030]

[0031] (3) The PCR amplification program was as follows: 95°C pre-denaturation for 5 min, 94°C denaturation for 45 s, 60°C annealing for 45 s, 72°C extension for 1.5 min, 35 cycles, and 72°C extension for 10 min.

[0032] (4) After the UGT forward amplification fragments were recovered, they were connected with the recovered pET28a reverse amplification fragments to obtain a recombinant plasmid, which was named pET28a-UGT. The recombinant plasmid was successfully constructed after sequencing verification.

[0033] Method for constructing recombinant E. coli strains:

[0034] The pET28a-UGT plasmid was transformed into the Escherichia coli cloning host using the heat shock transformation method to obtain positive transformants. The correctly sequenced transformant plasmid was then transferred to the Escherichia coli expression host BL21 (DE3) and spread on LB solid culture medium containing 50 μg / mL kanamycin. The LB plate was cultured at 37°C until transformants grew, and the positive transformants were picked to obtain the expected engineered bacteria.

[0035] Table 2 Five types of pET28a-UGT engineered bacteria

[0036]

[0037] Example 2 Induced expression of target protein

[0038] Five engineered bacteria were inoculated into 5 mL LB liquid medium and cultured overnight at 37°C and 200 rpm. Then, 0.8 mL of the overnight culture was inoculated into 20 mL LB liquid medium and cultured at 37°C and 200 rpm until the bacterial OD600 = 0.6. IPTG was then added to a final concentration of 1 mM and cultured at room temperature for 16 h to induce the expression of the target protein. After induction, the bacterial cells were collected by centrifugation at 10,000 × g for 5 min, and the bacterial cells were resuspended in 1.5 mL of a mixed solution (50 mMTris·HCl (pH 7.0), 15% (vol / vol) glycerol, 0.1 mM EDTA and 5 mM β-mercaptoethanol), and the cells were broken by ultrasound for 3 min. Subsequently, the E. coli cell lysate was centrifuged at 20,000 × g for 10 min, and the supernatant was collected to obtain a crude enzyme solution for enzyme characterization. The crude enzyme solution was stored at -20°C until further analysis.

[0039] Example 3 Determination of Mogroside V Content

[0040] The mogrosanol was dissolved to 20 mM with 50% DMSO. 50 mMTris·HCl (pH 7.0, containing 5 mM β-mercaptoethanol), 25 μL crude enzyme solution, 8 mM UDP-glucose and 2 mM mogrosanol were added to prepare a reaction system with a volume of 100 μL for the determination of UGT enzyme activity. After the reaction solution was incubated at 37°C for 24 hours, 300 μL of methanol solution was added to terminate the reaction, and then vortexed briefly. Subsequently, the reaction solution was centrifuged at 20,000 × g for 10 minutes, and the supernatant was collected. The subsequent product content analysis was as follows:

[0041] (1) adding 1 mL of chromatography grade methanol solution to 1 mL of the above reaction solution to obtain a product solution;

[0042] (2) After the product solution was centrifuged at 5000 rpm for 10 min, the supernatant was collected;

[0043] (3) The supernatant is filtered through a 0.22 μm filter membrane into a brown liquid phase bottle to obtain the sample to be tested;

[0044] (4) The new product was analyzed by liquid chromatography-mass spectrometry and nuclear magnetic resonance technology IC-MS-NMR: LC-MS analysis was performed using an Agilent 260 HPLC system and a Bruker-microtof-II electrospray mass spectrometer. A reversed phase C18 column (4.6×250 mm, 5 um, Welch, Shanghai) was used in the high performance liquid phase, the injection volume was 10 μL, the flow rate was 0.5 mL / 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). A gradient elution method was used: 0.00-15.00 min, 20%-26% B; 15.00-15.01 min, 26%-100% B; 15.01-25.00 min, 100% B;

[0045] (5) According to the peak area of ​​the mogroside V standard, the yields of mogroside V of the five engineered bacteria were finally calculated, and the specific yields are shown in Table 3. It was calculated that the highest mogroside V yield of strain 1, i.e., pET28a-LsUGT engineered bacteria, could reach 1.175 g / L.

[0046] Table 3 Production of mogroside V from five engineered bacteria

[0047]

[0048]

[0049] Example 4 Construction of pET28a-UGT-UDPase plasmid and recombinant E. coli strain

[0050] Based on the engineered bacteria with high production of mogroside V transformed with LsUGT and AtUGT, UGT and UDPase were expressed in tandem.

[0051] Construction method of pET28a-UGT-UDPGase plasmid:

[0052] (1) Using pET28a-UGT vector as template, PCR amplification was performed using specific primers. The pET28a vector is a commercial vector purchased from Novagen; the primer sequences are shown in Table 4; after the PCR product was recovered, the linearized vector pET28a-UGT reverse amplification fragment was obtained;

[0053] (2) According to the UDPase protein sequences of Arabidopsis thaliana and Drosophila melanogaster published in the NCBI (https: / / www.ncbi.nlm.nih.gov / ) database, as shown in SEQ ID NO.12 and SEQ ID NO.14, codon optimization was performed according to the codon preference of Escherichia coli, and the nucleic acid sequence was artificially synthesized. The nucleic acid sequence is shown in SEQ ID NO.11 and SEQ ID NO.13, and two pairs of specific primers were designed to amplify it. The primer sequences are shown in Table 4.

[0054] Table 4 UDPase primer sequences

[0055]

[0056] (3) The PCR amplification program was as follows: 95°C pre-denaturation for 5 min, 94°C denaturation for 45 s, 60°C annealing for 45 s, 72°C extension for 1.5 min, 35 cycles, and 72°C extension for 10 min.

[0057] (4) The AtUDPase forward amplification fragment, the DmUDPase forward amplification fragment, the pET28a-LsUGT reverse amplification fragment, and the pET28a-RsUGT reverse amplification fragment were connected in pairs to obtain a recombinant plasmid, which was named pET28a-UGT-UDPase. The plasmid was verified to be correct by sequencing, and four pET28a-UGT-UDPase plasmids were successfully constructed.

[0058] Method for constructing recombinant E. coli strains:

[0059] The four pET28a-UGT-UDPase plasmids were transformed into the Escherichia coli cloning host by heat shock transformation method to obtain positive transformants. Then, the correctly sequenced transformant plasmids were transferred into the Escherichia coli expression host BL21 (DE3) and spread on LB solid culture medium containing 50 μg / mL kanamycin. The LB plates were cultured at 37°C until transformants grew, and the positive transformants were picked to obtain the expected engineered bacteria.

[0060] Table 5 pET28a-UGT-UDPase engineering bacteria types

[0061]

[0062] Example 5: Production of Mogroside V by pET28a-UGT-UDPase Engineering Bacteria

[0063] According to the method of Examples 2-3 above, the induced expression of the target protein of the engineered bacteria and the determination of the yield of mogroside V were carried out.

[0064] According to the peak area of ​​the mogroside V standard, the yields of mogroside V of the four pET28a-UGT-UDPase engineered bacteria were finally calculated, and the specific yields are shown in Table 6. It was calculated that strain 6, i.e., pET28a-LsUGT-AtUDPase engineered bacteria, had the highest yield of mogroside V, which could reach 1.583 g / L.

[0065] Table 6 The yields of mogroside V from two pET28a-UGT-UDPGase engineered bacteria

[0066]

[0067] Nucleotide and amino acid sequence listing <110> Hebei Vidakang Biotechnology Co., Ltd. <120> Construction and application of an engineered strain for synthesizing mogroside V using mogroside alcohol as substrate <160> 14 <210> 1 <211>1542bp <212> DNA <213> Artificial synthesis <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> Synthetic <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> Synthetic <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 acctgaagt gaacggcgat aaagcgctga gcgatttat tgaagcgacc 300 aaaaacggcg atagcccggt gacctgcctg attatta ttctgctgaa ctggcgccg 360 aaagtggcgc gccgctttca gctgccgagc gcgctgctgt ggttcagcc ggggctgtg 420 tttaacattt attataccca tttatgggc aaaaaagcg tgttgaact gccgaacctg 480 agcagcctgg aaattcgcga tctgccgagc ttctgaccc cgagcacac caacaaggc 540 gcgtatgatg cgtttcagga aatgatggaa tttctgatta aagaaaccaa accgaaaatt 600 ctgattaca cctttgatag cctggaccg gaagcgctga ccgcgttttcc gaacattgat 660 atggtggcgg tggcccgct gctgccgacc gaattttta gcggcagcac caaaaagc 720 gtgaaagatc agagcagcag ctataccctg tggctggata gcaaaccga aagcagcgtg 780 atttatgtga gctttggcac catggtggaa ctgagcaaaaacattga agaactggcg 840 cgcgcgctga tgaggcaa acgcccgttt ctgtgggtga ttaccgataa aagcaaccgc 900 gaaaccaaaa ccgaaggcga agaagaacc gaaattgaa aaattgcggg ctttcgccat 960 gaactggaag aagtgggcat gattgtgagc tggtgcagcc agattgaagt gctgagccat 1020 gaactggaag aagtgggcat gattgtgagc tggtgcagcc agattgaagt gctgagccat 1020 cgcgcggtgg gctgctttgt gacccattgc ggctggagca gcaccctgga aagcctggtg 1080 cgcgcggtgg gctgctttgt gacccattgc ggctggagca gcaccctgga aagcctggtg 1080 ctgggcgtgc cggtggtggc gtttccgatg tggagcgatc agccgaccaa cgcgaaactg 1140 ctgggcgtgc cggtggtggc gtttccgatg tggagcgatc agccgaccaa cgcgaaactg 1140 ctggaagaaa gctggaaaac cggcgtgcgc gtgcgcgaaa acaaagatgg cctggtggaa 1200 ctggaagaaa gctggaaaac cggcgtgcgc gtgcgcgaaa acaaagatgg cctggtggaa 1200 cgcggcgaaa ttcgccgctg cctggaagcg gtgatggaag aaaaaagcgt ggaactgcgc 1260 cgcggcgaaa ttcgccgctg cctggaagcg gtgatggaag aaaaaagcgt ggaactgcgc 1260 gaaaacgcga aaaaatggaa acgcctggcg atggaagcgg gccgcgaagg cggcagcagc 1320 gaaaacgcga aaaaatggaa acgcctggcg atggaagcgg gccgcgaagg cggcagcagc 1320 gataaaaaca tggaagcgtt tgtggaagat atttgcggcg aaagcctgat tcagaacctg 1380 gataaaaaca tggaagcgtt tgtggaagat atttgcggcg aaagcctgat tcagaacctg 1380 tgcgaagcgg aagaagtgaa agtgaaataa 1410 tgcgaagcgg aagaagtgaa agtgaaataa 1410 <210> 4<210> 4 <211> 469<211> 469 <212> PRT <212> PRT <213> 人工合成 <213> Synthetic <400> 1 <400> 1 MAPPHFLLVT FPAQGHVNPS LRFARRLIKR TGARVTFVTC VSVFHNSMIA NHNKVENLSF 60 MAPPHFLLVT FPAQGHVNPS LRFARRLIKR TGARVTFVTC VSVFHNSMIA NHNKVENLSF 60 LTFSDGFDDG GISTYEDRQK RSVNLKVNGD KALSDFIEAT KNGDSPVTCL IYTILLNWAP 120 LTFSDGFDDG GISTYEDRQK RSVNLKVNGD KALSDFIEAT KNGDSPVTCL IYTILLNWAP 120 KVARRFQLPS ALLWIQPALV FNIYYTHFMG NKSVFELPNL SSLEIRDLPS FLTPSNTNKG 180 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> Synthetic <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 gcggggcgtgc cggtggtggc gtttccgcag 1140 tggaccgatc agggcaccaa cgcgaaactg attgaagatg tgtggggcaa cggcgtgcgc gtgagcgcga acgaagaagg catggtggaa cgcgatgaaa ttgtgcgctg cctggatctg gtgatgggcg atgatgaaaa aggcatgga gtgaaaaaa acgtggaaaa atgggaaggc ctggcgcgcg aagcgagcat ggaaggcggc agcatggata tgaacctgaa agcgtttgtg gatgatgtgg cgcagggctg ctggaata 9210 / 6 1211 / 469 12120 PRT 12130 PERFORMANCE 4000> 60. MSQPHFLLVT FPAQGHINPT LQFAKHLIRI GVRVTFITCI SARRRMTKVP TAQGLTFLPF SDGYDDGFQP GDDIEHYLSE LRRRGKEAIS EFITSSEYEG KPVTCIVYTL FIHWASEVAR 120 KHHIPAALLW IQPATVFDIY YFYFNGYEST IKVPVDETNP KRSIKLPGLP LLATRDLPSF VTASNVYRWA LSLFQEQMDI CHARGER SNPKIL 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> Synthetic <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 attached 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> Synthetic <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> Synthetic <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> Synthetic <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> Synthetic <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 gatgataccc ataaaattgt ggaaaaatat accaacagca acgtggatat tcataccttt aaccagagca aatatccgcg cgtggtggcg gatgaatttg tgccgtggcc gagcaaaggc aaaaccgata aagaaggctg gtatccgccg ggccatggcg atgtgtttcc ggcgctgatg 600 aacagcggca aactggatac ctttctgagc cagggcaaag aatatgtgtt tgtggcgaac agcgataacc tgggcgcgat tgtggatctg accattctga aacatctgat tcagaacaaa aacgaatatt gcatggaagt gaccccgaaa accctggcgg atgtgaaagg cggcaccctg attagctatg aaggcaaagt gcagctgctg gaaattgcgc aggtgccgga tgaacatgtg aacgaattta aaagcattga aaaatttaaa atttttaaca ccaacaacct gtgggtgaac ctgaaagcga ttaaaaaact ggtggaagcg gatgcgctga aaatggaat tattccgaac ccgaaagaag tggatggcgt gaaagtgctg cagctggaaa ccgcggcggg cgcggcgatt cgcttttttg father tggcgtgaac gtgccgcgca gccgctttct gccggtgaaa 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> Synthetic <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> Synthetic <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> Synthetic <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, characterized in that: The substrate mogroside V is obtained by transforming mogroside alcohol using a recombinant cell comprising a glycosyltransferase gene and a UDP-glucose pyrophosphorylase gene. The recombinant cell expresses the glycosyltransferase gene and the UDP-glucose pyrophosphorylase gene, produces UGT enzyme and UDP-glucose pyrophosphorylase, the glycosyltransferase gene sequence is shown in SEQ ID NO.1, and the UDP-glucose pyrophosphorylase gene sequence is shown in SEQ ID NO.

11.

2. The method according to claim 1, characterized in that: The specific steps are as follows: step (1): obtaining a crude enzyme solution using a recombinant cell containing a glycosyltransferase gene and a UDP-glucose pyrophosphorylase gene; step (2): using mogroside alcohol as a substrate, adding the crude enzyme solution, and converting and synthesizing mogroside V in the presence of UDP glucose.

3. The method according to claim 1, characterized in that: The preparation method of the crude enzyme solution comprises the following steps: inoculating recombinant cells containing glycosyltransferase genes and UDP-glucose pyrophosphorylase genes into LB liquid culture medium to obtain seed solution, inoculating the seed solution into LB liquid culture medium, culturing until the bacterial OD600 is 0.4-1.0, then adding IPTG for induction culture to induce the expression of the target protein; after the induction is completed, centrifuging and collecting the bacterial cells, resuspending the bacterial cells, breaking the cells in liquid nitrogen through an ultrasonic / freezing cycle, then centrifuging the cell breaking solution, and collecting the supernatant to obtain the crude enzyme solution.

4. The method according to claim 2, characterized in that: The reaction conditions in step (2) are 25-45° C. and the reaction time is 1-48 h.

5. A recombinant vector comprising a glycosyltransferase gene and a UDP-glucose pyrophosphorylase gene, wherein the glycosyltransferase gene sequence is shown in SEQ ID NO.1, and the UDP-glucose pyrophosphorylase gene sequence is shown in SEQ ID NO.

11.

6. The method for preparing the recombinant vector according to claim 5, characterized in that: The steps are as follows: (1) using a vector as a template and performing PCR amplification using specific primers to obtain a linearized vector reverse amplification fragment; (2) using a glycosyltransferase gene as a template and performing PCR amplification to obtain a UGT forward amplification fragment, which is recovered and connected with the vector reverse amplification to obtain a first recombinant vector containing a glycosyltransferase gene; and further, using the first recombinant vector as a template and performing PCR amplification to obtain a linearized vector reverse amplification fragment; using a UDP-glucose pyrophosphorylase gene as a template and performing PCR amplification to obtain a UDPase amplification fragment, which is recovered and connected with the aforementioned vector reverse amplification fragment to obtain a second recombinant vector containing a glycosyltransferase gene and a UDP-glucose pyrophosphorylase gene.

7. A genetically engineered bacterium for biosynthesizing mogroside V using mogroside alcohol as a substrate, characterized in that: The genetically engineered bacteria express glycosyltransferase genes and UDP-glucose pyrophosphorylase genes, produce UGT enzymes and UDP-glucose pyrophosphorylase, the glycosyltransferase gene sequence is shown in SEQ ID NO.1, and the UDP-glucose pyrophosphorylase gene sequence is shown in SEQ ID NO.

11.

8. The method for constructing a genetically engineered bacterium according to claim 7, characterized in that: The recombinant vector according to claim 5 is transformed into a host cell, and the transformant is cultured to obtain an engineered strain that biosynthesizes mogroside V using mogroside alcohol as a substrate.

9. The method for constructing a genetically engineered bacterium according to claim 7, characterized in that: The host cell is Escherichia coli.

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