CN113388590B - Mutant of cytochrome P450s - Google Patents

Mutant of cytochrome P450s Download PDF

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CN113388590B
CN113388590B CN202110631786.7A CN202110631786A CN113388590B CN 113388590 B CN113388590 B CN 113388590B CN 202110631786 A CN202110631786 A CN 202110631786A CN 113388590 B CN113388590 B CN 113388590B
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梅超
张晓鹏
宋倩娜
王慧杰
霍利光
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Shanxi Agricultural University
Agricultural Genomics Institute at Shenzhen of CAAS
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Abstract

The invention relates to a mutant of cytochrome P450 s. The mutant not only can be highly expressed in a heterologous host, but also can obviously improve the substrate selection specificity and the yield of a target product 11H-Cuol, and powerfully promote the development of the industrial synthesis technology of mogroside V.

Description

Mutant of cytochrome P450s
Technical Field
The invention relates to a mutant of cytochrome P450s, belonging to the field of bioengineering.
Background
High-sugar diets, whether natural or synthetic, are responsible for a range of modern health problems, such as obesity, diabetes, cardiovascular disease, etc. (sievenpipe, 2014). Therefore, natural non-sugar substances derived from plants are a new generation sweetener meeting the requirement of sweetness due to the advantages of high sweetness, low calorie, high safety and the like. Wherein, the sweetness of the mogroside V which is the main active substance of the grosvenor momordica is 300 times of that of cane sugar, the calorie is low, and the mogroside V has the pharmacological activities of relieving cough, eliminating phlegm, reducing blood sugar, resisting cancer, resisting oxidation and the like (Kasai et al, 1989). Therefore, mogroside V can replace sucrose as a sweetener to be widely applied to various foods as a sugar substitute for patients with obesity or diabetes. However, the growth period of the momordica grosvenori is long, and a plurality of difficulties exist in cultivation; and the content of mogroside V in the fruits is low; traditional acquisition methods have been difficult to meet with the ever-increasing demand (Makapugay et al, 1985). Recently, the rapid development of omics has promoted the analysis of key synthetic pathways and the discovery of key functional genes. The rapid development of metabolic engineering and synthetic biology provides a sustainable production concept for the heterologous biosynthesis of natural products of plants. In 2018, Iktin et al analyzed the mogroside V synthetic pathway according to the Grosvenor momordica transcriptome and genome data, and laid a foundation for constructing cell engineering by synthetic biology and realizing in vitro synthesis (Itkin, 2018).
According to the research of scientists on the heterogenous biosynthesis of mogroside V, the following aspects are mainly shown. Firstly, selection and optimization of the chassis bacteria. Suitable underpan cells are the basis for efficient production of natural products. The MVA pathway of Saccharomyces cerevisiae provides the precursor 2, 3-epoxysqualene for the heterologous synthesis of mogroside, but most of the 2, 3-epoxysqualene in yeast is shunted into the ergosterol pathway by lanosterol synthase (ERG 7). Scientists have increased the metabolic flux of 2, 3-epoxysqualene to mogroside V by treatment with the ERG7 inhibitor R048-8072 or using the strain of saccharomyces cerevisiae lacking ERG7 (GIL77) as a basidiomycete (Dai et al, 2015). Secondly, biosynthesis of cucurbitadienol. Liquassian and the like over-express a triterpene compound synthesis key enzyme and an oxidosqualene cyclase SgCDS in yeast, further improve the expression ratio of different genes, and the yield of cucurbitadienol can reach 1724.10 mg/L. Although cucurbitadienol is not a backbone for mogroside synthesis, mogroside V can be produced using in vitro catalysis by P450 oxidase, a glycosyltransferase (roxburgh et al, 2016). And thirdly, in vitro glycosylation modification. Glycosyl transferase UGT74AC1 was identified by the Miss-Tsunless task group of Tianjin Industrial biology, and UGT74AC1 purified from Escherichia coli can be used for glycosylation of C3-OH in vitro to generate sweet glycoside IE (Dai et al, 2015) by using a sweet glycoside co-precursor substance (mogrol) as a substrate.
Scientists have explored a heterologous biosynthetic pathway for mogroside (see FIG. 1). We find that obtaining the mogroside V precursor at present mainly depends on a metabolic engineering method, heterogeneously assembles key enzymes required in the way, and does not perform adaptive modification on the enzymes in a prokaryotic system, so that the enzyme activity and stability of the key enzymes are influenced to a certain extent and the maximum function cannot be exerted. Therefore, adaptation of key enzymes is crucial for efficient production of natural products. On the other hand, we can find that the existing synthetic method depends on the original pathway, and basically realizes each reaction step by step on the basis of the original pathway, and although the heterologous biosynthesis is possible, the synthetic pathway of the mogroside V is complex, which is a long process. In order to synthesize mogroside V efficiently and rapidly, the route needs to be shortened and the function of key enzymes needs to be improved. Therefore, the project focuses on realizing the functional improvement of cytochrome P450s which is a key enzyme in the synthetic pathway from cucurbitadienol to mogrol. Wherein, 11H-Cuol is an intermediate compound in the synthesis process from cucurbitadienol to mogrol, and is a precursor substance of mogroside V (shown in figure 2).
Cytochrome P450s is one of the largest family of enzyme proteins in plants and is capable of catalyzing the synthesis of a large number of structurally specific metabolites. These metabolites not only play a role in the growth and development of plants, stress response, etc., but also can be used as precious raw materials to synthesize new drugs (Alonso-gurierrez et al, 2013). Therefore, P450s is usually selected as a target for metabolic engineering, and protein engineering is used to change factors such as enzyme activity, protein stability and the like of P450s to improve the synthesis efficiency of related metabolites. In recent years, scientists have successfully identified and elucidated the catalytic mechanism of P450s in different metabolic pathways, using genome mining and modern biological techniques. P450s catalyzes plant specific metabolic reaction, has low catalytic efficiency (kcat <5s-1) and complex and various substrates. P450s is expressed in very low amounts in heterologous hosts and the transfer of electron flow from NAD (P) H to P450s requires the involvement of its redox partner (Renault et al, 2014 a; Urlacher and Girhard, 2019). The above factors limit the wide application of P450s in metabolic engineering.
The improvement of the application of P450s is mainly shown in the following aspects. First, heterologous host adaptation of plant P450s protein is improved. 1) Plant P450s is normally localized on membrane structures, whereas P450s in prokaryotes is cytosolic and P450 is differentially localized in the two hosts. Therefore, the heterologous expression of P450s in prokaryotic cells can increase the solubility of the protein of eukaryotic P450s by deleting or modifying the N-terminal transmembrane region of the protein. 2) The expression level of P450s in yeast or bacteria is very low, and the production of a desired component in a metabolic process is usually increased by optimizing promoters, codons or copy number (Mcintosh et al, 2014). 3) In addition, the catalytic reaction of P450 can be promoted by adjusting the reaction conditions. Scientists convert norbornene to epoxynorbornane by expressing a cytochrome P450 epoxidase, CytP, in e. The two-phase catalytic system of the CytP enzyme is optimized from the pH and temperature of the reaction, the catalytic activity and specificity of the CytP enzyme are greatly improved, and a foundation is laid for industrial production and green production of epoxy norbornane. Second, the electron transport chain in the redox reaction is optimized. 1) P450s is normally localized on the ER membrane in eukaryotes, so the composition and content of lipids in the ER membrane structure affects the expression of P450s on the ER membrane. Based on this property, oleaginous yeasts are commonly used to express synthetic flavonoids from P450s of plant origin due to their relatively abundant membrane structure (Yongkun et al, 2019). 2) Another approach is to increase the physical distance between P450s and its redox reaction partner on the ER membrane to create better electron transfer. The practical operation is to express P450s and its mate in fusion and to increase the enzyme activity and solubility of P450 s. However, this method may cause cytotoxicity, and therefore balancing the expression ratio of P450s and its chaperone protein is also important for the transfer of electron chains (Renault et al, 2014 b). It can be found that the current P450s function improvement mainly depends on the traditional metabolic engineering method, and the function improvement of mutating P450s by molecular biology or bioinformatics method is still needed.
Disclosure of Invention
In order to solve the above problems, the present invention provides a mutant of cytochrome P450 s.
In one embodiment, the mutant of cytochrome P450s has the amino acid sequence shown in SEQ ID No. 6.
Secondly, the invention also provides a method for efficiently and heterogeneously synthesizing the mogroside V precursor, which comprises the steps of culturing a host containing the nucleic acid under the condition that the mogroside V precursor can be produced, and separating the mogroside V precursor from the metabolite.
The invention also provides a method for preparing mogroside V, which is characterized in that a host containing the nucleic acid is cultured under the condition of producing a mogroside V precursor, and then the mogroside V is synthesized by taking the precursor as a substrate.
The host is selected from prokaryotes, fungi, plant cells or animal cells, and preferably, the host is yeast.
The host for expression in the heterologous synthesis is a prokaryote, e.g., a bacterium, in particular, e.g., e.
The host for expression in the heterologous synthesis is a eukaryote, for example a fungus, a plant cell or an animal cell, in particular a yeast.
The mogroside V precursor is 11H-Cuol.
A protein has a sequence shown in SEQ ID No. 6.
A nucleic acid encoding a protein shown as SEQ ID No. 6.
The biological material containing the nucleic acid is any one of the following A1) to A4):
A1) an expression cassette comprising the nucleic acid;
A2) a recombinant vector containing the nucleic acid;
A3) a recombinant microorganism containing said nucleic acid;
A4) a transgenic plant cell line comprising said nucleic acid.
The protein, the nucleic acid or the biological material is applied to the preparation of a mogroside V precursor or a mogroside V.
In one embodiment, the mogroside V precursor is 11H-Cuol.
The structural formula of the 11H-Cuol is as follows:
Figure BDA0003103993670000051
the P450s enzyme of the present invention is superior to the prior art in that:
firstly, the mogroside V precursor can be efficiently synthesized by heterogeneously synthesizing.
Secondly, the momordica glycoside V can be efficiently synthesized in a heterogenous way.
Thirdly, the high-efficiency heterologous expression can be realized.
Therefore, the invention has the advantages of combining the heterogenous high expression of the enzyme of P450s and the specific and high-efficiency catalytic activity to the target product, obviously exceeding the level of the prior art and having very wide industrial practical prospect.
Drawings
FIG. 1 shows the heterologous biosynthesis pathway of mogroside V.
FIG. 2 is a synthetic route from cucurbitadienol to mogrol.
FIG. 3 demonstrates the V2-based mutant catalytic activity and protein expression. Wherein:
a: detecting the yield of the related metabolites in the yeast system by using HPLC-qTOF;
b: the expression of the V2 mutant protein was detected by western blotting.
FIG. 4 demonstrates the V3-based mutant catalytic activity and protein expression. Wherein:
a: detecting the yield of the related metabolites in the yeast system by using HPLC-qTOF;
b: the expression of the V3 mutant protein was detected by western blotting.
FIG. 5 verifies the catalytic activity and protein expression of the mixed mutants. Wherein:
a: detecting the yield of the related metabolites in the yeast system by using HPLC-qTOF;
b: and (3) detecting the expression of the mixed mutant protein by using western blotting.
Note: in the metabolite detection map, the metabolites are sequentially 11H-Cuol (blue in color), 11C-Cuol (red in color), and 11C-20H-Cuol (green in color) from left to right.
Detailed Description
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1 protein expression and detection
The P450 is mutated to obtain a plurality of mutants, such as V1(L48F-S49A-I61F-L120T-T352K-L356P), V2(L48F-S49A-I61F-L120T-K352I-L356P) and the like, and further obtain a mutant V2-W119I-L125D capable of efficiently producing the mogroside precursor 11H-Cuol on the basis.
The mutants are transformed into a yeast expression strain INVSC1, induced expression is carried out for 12h, total yeast protein is extracted by a trichloroacetic acid-acetone method, and the expression of the mutant protein is detected by western blotting. The trichloroacetic acid-acetone method detection method comprises the following steps: 5OD yeast is taken. Resuspend with 1ml of alkaline lysis solution (0.25M NaOH + 1% beta-mercaptoethanol), and stand on ice for 10 min; adding 160ul 50% TCA, mixing, standing on ice for 10min, and centrifuging at 14000g for 10 min; fully discarding the supernatant, adding 1ml of precooled acetone for heavy suspension and precipitation, standing on ice for 10min, and centrifuging at 14000g for 10 min; discard the supernatant, evaporate acetone sufficiently, add 100ul 1 Xprotein loading buffer, boil for 10min, and test the loading (EV is control).
Example 2 metabolite detection
First, yeast strain INVSC1 was modified to ensure sufficient substrate cucurbitadienol. The four key enzymes HMGR, ERG20, ERG9 and ERG1 are important in the biosynthesis pathway of 2, 3-oxidosqualene, and the four genes are integrated on the chromosome of a yeast strain to be overexpressed (the content of cucurbitadienol is increased). On this basis, cyclooxisqualene synthase and the P450 redox partner CPR (Csa1G423150) were transferred.
Then, the enzyme mutant is transformed into EY10-Bi chassis yeast containing a redox partner CPR, the induction is carried out for 48H, the yeast with the same mass is taken, the total metabolites of the yeast are extracted by an alkaline lysis method, and the yield of the compound (the target product 11H-Cuol and the byproducts 11C-Cuol and 11C-20H-Cuol) in the synthetic pathway is detected by HPLC-qTOF. The important detection parameters of metabolites are as follows: the Q-TOF system and the APCI ion source detect positive ions; HHS T33.5um, 4.6 multiplied by 150mm column; the column temperature is 35 ℃; the mobile phase is A phase: 0.1% (v/v) aqueous formic acid, phase B: 0.1% (v/v) formic acid-methanol solution; the flow rate is 1 mL/min; gradient as follows B (tmin, B%): (1,90), (3,100), (10,100), (12,90), and (15, 90); the MS parameters were as follows: corona current is 4 uA; the capillary voltage is 5.0 kV; the skimmer voltage is 65V; segment fragment 135V; the gas temperature is 350 ℃; the vapourizer temperature is 400 ℃; drying gas flow rate is 8L/min; nebulizer pressure 60 psi; dynamic range 150-.
The detection result of the metabolite shows that:
compared with the mutant V2, the mutant V2-W119I-L125D has a larger increase in the amount of 11H-Cuol produced, and the protein expression level is equivalent to that of the mutant V2 and is much higher than that of the wild type (FIGS. 3-5).
Example 3 Effect of other mutants
The P450s mutant is based on the amino acid sequence of CPY87D20 of cucumber (cucumber sativus), and is shown as SEQ ID No.1, and a plurality of mutants are obtained in sequence, such as: the amino acid sequence of the mutant V1 is shown as SEQ ID No. 2; the amino acid sequence of the mutant V2 is shown as SEQ ID No. 3; the amino acid sequence of the mutant V3 is shown in SEQ ID No. 4.
For another example, a mutant further mutated on the basis of the above mutants: V2-I46L-A49L, V2-W119I-L125D, V2-R385Y, V2-W399K, V2-I439H, V2-E463P, V2-I46L-A49L-C343Y, V Y-C343Y-S49Y, V Y-C343Y-I46Y, V Y-K73Y, V Y-F89Y, V Y-Y432Y, V Y-L125Y, V Y-R383Y, V Y-W399Y and the like, which are tested in batches under the same conditions as in examples 1-2, and whether the yield of the target product 11H-Cuol and the selectivity of the substrate for the enzyme and the expression level of the protein are significantly higher than those of the wild type (see FIG. 3-W Y) as shown in SEQ ID No. Y-Y (see SEQ ID Y-Y).
In conclusion, the invention provides a mutant of P450 enzyme, which not only optimizes the performance of the mutant in expression in yeast, but also obviously improves the yield of the target product 11H-Cuol and the substrate selection specificity of the enzyme.
While the invention has been described in detail with respect to specific embodiments thereof, the foregoing description is not intended to limit the scope of the invention.
SEQUENCE LISTING
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Val Lys Arg Tyr Gly Pro Ile Phe Lys Thr Cys Leu Ala Gly Arg Pro
65 70 75 80
Val Val Val Ser Thr Asp Ala Glu Phe Asn His Tyr Ile Met Leu Gln
85 90 95
Glu Gly Arg Ala Val Glu Met Trp Tyr Leu Asp Thr Leu Ser Lys Phe
100 105 110
Phe Gly Leu Asp Thr Glu Trp Thr Lys Ala Leu Gly Leu Ile His Lys
115 120 125
Tyr Ile Arg Ser Ile Thr Leu Asn His Phe Gly Ala Glu Ser Leu Arg
130 135 140
Glu Arg Phe Leu Pro Arg Ile Glu Glu Ser Ala Arg Glu Thr Leu His
145 150 155 160
Tyr Trp Ser Thr Gln Thr Ser Val Glu Val Lys Glu Ser Ala Ala Ala
165 170 175
Met Val Phe Arg Thr Ser Ile Val Lys Met Phe Ser Glu Asp Ser Ser
180 185 190
Lys Leu Leu Thr Glu Gly Leu Thr Lys Lys Phe Thr Gly Leu Leu Gly
195 200 205
Gly Phe Leu Thr Leu Pro Leu Asn Leu Pro Gly Thr Thr Tyr His Lys
210 215 220
Cys Ile Lys Asp Met Lys Gln Ile Gln Lys Lys Leu Lys Asp Ile Leu
225 230 235 240
Glu Glu Arg Leu Ala Lys Gly Val Lys Ile Asp Glu Asp Phe Leu Gly
245 250 255
Gln Ala Ile Lys Asp Lys Glu Ser Gln Gln Phe Ile Ser Glu Glu Phe
260 265 270
Ile Ile Gln Leu Leu Phe Ser Ile Ser Phe Ala Ser Phe Glu Ser Ile
275 280 285
Ser Thr Thr Leu Thr Leu Ile Leu Asn Phe Leu Ala Asp His Pro Asp
290 295 300
Val Val Lys Glu Leu Glu Ala Glu His Glu Ala Ile Arg Lys Ala Arg
305 310 315 320
Ala Asp Pro Asp Gly Pro Ile Thr Trp Glu Glu Tyr Lys Ser Met Asn
325 330 335
Phe Thr Leu Asn Val Ile Cys Glu Thr Leu Arg Leu Gly Ser Val Ile
340 345 350
Pro Ala Leu Pro Arg Lys Thr Thr Lys Glu Ile Gln Ile Lys Gly Tyr
355 360 365
Thr Ile Pro Glu Gly Trp Thr Val Met Leu Val Thr Ala Ser Arg His
370 375 380
Arg Asp Pro Glu Val Tyr Lys Asp Pro Asp Thr Phe Asn Pro Trp Arg
385 390 395 400
Trp Lys Glu Leu Asp Ser Ile Thr Ile Gln Lys Asn Phe Met Pro Phe
405 410 415
Gly Gly Gly Leu Arg His Cys Ala Gly Ala Glu Tyr Ser Lys Val Tyr
420 425 430
Leu Cys Thr Phe Leu His Ile Leu Phe Thr Lys Tyr Arg Trp Arg Lys
435 440 445
Leu Lys Gly Gly Lys Ile Ala Arg Ala His Ile Leu Arg Phe Glu Asp
450 455 460
Gly Leu Tyr Val Asn Phe Thr Pro Lys Glu
465 470
<210> 4
<211> 474
<212> PRT
<213> Artificial Synthesis
<400> 4
Met Trp Thr Ile Leu Leu Gly Leu Ala Thr Leu Ala Ile Ala Tyr Tyr
1 5 10 15
Ile His Trp Val Asn Lys Trp Lys Asp Ser Lys Phe Asn Gly Val Leu
20 25 30
Pro Pro Gly Thr Met Gly Leu Pro Leu Ile Gly Glu Thr Ile Gln Leu
35 40 45
Ser Arg Pro Ser Asp Ser Leu Asp Val His Pro Phe Ile Gln Arg Lys
50 55 60
Val Lys Arg Tyr Gly Pro Ile Phe Lys Thr Cys Leu Ala Gly Arg Pro
65 70 75 80
Val Val Val Ser Thr Asp Ala Glu Phe Asn His Tyr Ile Met Leu Gln
85 90 95
Glu Gly Arg Ala Val Glu Met Trp Tyr Leu Asp Thr Phe Ser Lys Phe
100 105 110
Leu Gly Leu Asp Thr Glu Trp Leu Lys Ala Leu Gly Leu Ile His Lys
115 120 125
Tyr Ile Arg Ser Ile Thr Leu Asn His Phe Gly Ala Glu Ser Leu Arg
130 135 140
Glu Arg Phe Leu Pro Arg Ile Glu Glu Ser Ala Arg Glu Thr Leu His
145 150 155 160
Tyr Trp Ser Thr Gln Thr Ser Val Glu Val Lys Glu Ser Ala Ala Ala
165 170 175
Met Val Phe Arg Thr Ser Ile Val Lys Met Phe Ser Glu Asp Ser Ser
180 185 190
Lys Leu Leu Thr Glu Gly Leu Thr Lys Lys Phe Thr Gly Leu Leu Gly
195 200 205
Gly Phe Leu Thr Leu Pro Leu Asn Leu Pro Gly Thr Thr Tyr His Lys
210 215 220
Cys Ile Lys Asp Met Lys Gln Ile Gln Lys Lys Leu Lys Asp Ile Leu
225 230 235 240
Glu Glu Arg Leu Ala Lys Gly Val Lys Ile Asp Glu Asp Phe Leu Gly
245 250 255
Gln Ala Ile Lys Asp Lys Glu Ser Gln Gln Phe Ile Ser Glu Glu Phe
260 265 270
Ile Ile Gln Leu Leu Phe Ser Ile Ser Phe Ala Ser Phe Ala Ser Ile
275 280 285
Ser Thr Thr Leu Thr Leu Ile Leu Asn Phe Leu Ala Asp His Pro Asp
290 295 300
Val Val Lys Glu Leu Glu Ala Glu His Glu Ala Ile Arg Lys Ala Arg
305 310 315 320
Ala Asp Pro Asp Gly Pro Ile Thr Trp Glu Glu Tyr Lys Ser Met Asn
325 330 335
Phe Thr Leu Asn Val Ile Cys Glu Thr Leu Arg Leu Gly Ser Val Thr
340 345 350
Pro Ala Leu Leu Arg Lys Thr Thr Lys Glu Ile Gln Ile Lys Gly Tyr
355 360 365
Thr Ile Pro Glu Gly Trp Thr Val Met Leu Val Thr Ala Ser Arg His
370 375 380
Arg Asp Pro Glu Val Tyr Lys Asp Pro Asp Thr Phe Asn Pro Trp Arg
385 390 395 400
Trp Lys Glu Leu Asp Ser Ile Thr Ile Gln Lys Asn Phe Met Pro Phe
405 410 415
Gly Gly Gly Leu Arg His Cys Ala Gly Ala Glu Tyr Ser Lys Val Tyr
420 425 430
Leu Cys Thr Phe Leu His Ile Leu Phe Thr Lys Tyr Arg Trp Arg Lys
435 440 445
Leu Lys Gly Gly Lys Ile Ala Arg Ala His Ile Leu Arg Phe Glu Asp
450 455 460
Gly Leu Tyr Val Asn Phe Thr Pro Lys Glu
465 470
<210> 5
<211> 474
<212> PRT
<213> Artificial Synthesis
<400> 5
Met Trp Thr Ile Leu Leu Gly Leu Ala Thr Leu Ala Ile Ala Tyr Tyr
1 5 10 15
Ile His Trp Val Asn Lys Trp Lys Asp Ser Lys Phe Asn Gly Val Leu
20 25 30
Pro Pro Gly Thr Met Gly Leu Pro Leu Ile Gly Glu Thr Leu Gln Phe
35 40 45
Leu Arg Pro Ser Asp Ser Leu Asp Val His Pro Phe Phe Gln Arg Lys
50 55 60
Val Lys Arg Tyr Gly Pro Ile Phe Lys Thr Cys Leu Ala Gly Arg Pro
65 70 75 80
Val Val Val Ser Thr Asp Ala Glu Phe Asn His Tyr Ile Met Leu Gln
85 90 95
Glu Gly Arg Ala Val Glu Met Trp Tyr Leu Asp Thr Leu Ser Lys Phe
100 105 110
Phe Gly Leu Asp Thr Glu Trp Thr Lys Ala Leu Gly Leu Ile His Lys
115 120 125
Tyr Ile Arg Ser Ile Thr Leu Asn His Phe Gly Ala Glu Ser Leu Arg
130 135 140
Glu Arg Phe Leu Pro Arg Ile Glu Glu Ser Ala Arg Glu Thr Leu His
145 150 155 160
Tyr Trp Ser Thr Gln Thr Ser Val Glu Val Lys Glu Ser Ala Ala Ala
165 170 175
Met Val Phe Arg Thr Ser Ile Val Lys Met Phe Ser Glu Asp Ser Ser
180 185 190
Lys Leu Leu Thr Glu Gly Leu Thr Lys Lys Phe Thr Gly Leu Leu Gly
195 200 205
Gly Phe Leu Thr Leu Pro Leu Asn Leu Pro Gly Thr Thr Tyr His Lys
210 215 220
Cys Ile Lys Asp Met Lys Gln Ile Gln Lys Lys Leu Lys Asp Ile Leu
225 230 235 240
Glu Glu Arg Leu Ala Lys Gly Val Lys Ile Asp Glu Asp Phe Leu Gly
245 250 255
Gln Ala Ile Lys Asp Lys Glu Ser Gln Gln Phe Ile Ser Glu Glu Phe
260 265 270
Ile Ile Gln Leu Leu Phe Ser Ile Ser Phe Ala Ser Phe Glu Ser Ile
275 280 285
Ser Thr Thr Leu Thr Leu Ile Leu Asn Phe Leu Ala Asp His Pro Asp
290 295 300
Val Val Lys Glu Leu Glu Ala Glu His Glu Ala Ile Arg Lys Ala Arg
305 310 315 320
Ala Asp Pro Asp Gly Pro Ile Thr Trp Glu Glu Tyr Lys Ser Met Asn
325 330 335
Phe Thr Leu Asn Val Ile Tyr Glu Thr Leu Arg Leu Gly Ser Val Ile
340 345 350
Pro Ala Leu Pro Arg Lys Thr Thr Lys Glu Ile Gln Ile Lys Gly Tyr
355 360 365
Thr Ile Pro Glu Gly Trp Thr Val Met Leu Val Thr Ala Ser Arg His
370 375 380
Arg Asp Pro Glu Val Tyr Lys Asp Pro Asp Thr Phe Asn Pro Trp Arg
385 390 395 400
Trp Lys Glu Leu Asp Ser Ile Thr Ile Gln Lys Asn Phe Met Pro Phe
405 410 415
Gly Gly Gly Leu Arg His Cys Ala Gly Ala Glu Tyr Ser Lys Val Tyr
420 425 430
Leu Cys Thr Phe Leu His Ile Leu Phe Thr Lys Tyr Arg Trp Arg Lys
435 440 445
Leu Lys Gly Gly Lys Ile Ala Arg Ala His Ile Leu Arg Phe Glu Asp
450 455 460
Gly Leu Tyr Val Asn Phe Thr Pro Lys Glu
465 470
<210> 6
<211> 474
<212> PRT
<213> Artificial Synthesis
<400> 6
Met Trp Thr Ile Leu Leu Gly Leu Ala Thr Leu Ala Ile Ala Tyr Tyr
1 5 10 15
Ile His Trp Val Asn Lys Trp Lys Asp Ser Lys Phe Asn Gly Val Leu
20 25 30
Pro Pro Gly Thr Met Gly Leu Pro Leu Ile Gly Glu Thr Ile Gln Phe
35 40 45
Ala Arg Pro Ser Asp Ser Leu Asp Val His Pro Phe Phe Gln Arg Lys
50 55 60
Val Lys Arg Tyr Gly Pro Ile Phe Lys Thr Cys Leu Ala Gly Arg Pro
65 70 75 80
Val Val Val Ser Thr Asp Ala Glu Phe Asn His Tyr Ile Met Leu Gln
85 90 95
Glu Gly Arg Ala Val Glu Met Trp Tyr Leu Asp Thr Leu Ser Lys Phe
100 105 110
Phe Gly Leu Asp Thr Glu Ile Thr Lys Ala Leu Gly Asp Ile His Lys
115 120 125
Tyr Ile Arg Ser Ile Thr Leu Asn His Phe Gly Ala Glu Ser Leu Arg
130 135 140
Glu Arg Phe Leu Pro Arg Ile Glu Glu Ser Ala Arg Glu Thr Leu His
145 150 155 160
Tyr Trp Ser Thr Gln Thr Ser Val Glu Val Lys Glu Ser Ala Ala Ala
165 170 175
Met Val Phe Arg Thr Ser Ile Val Lys Met Phe Ser Glu Asp Ser Ser
180 185 190
Lys Leu Leu Thr Glu Gly Leu Thr Lys Lys Phe Thr Gly Leu Leu Gly
195 200 205
Gly Phe Leu Thr Leu Pro Leu Asn Leu Pro Gly Thr Thr Tyr His Lys
210 215 220
Cys Ile Lys Asp Met Lys Gln Ile Gln Lys Lys Leu Lys Asp Ile Leu
225 230 235 240
Glu Glu Arg Leu Ala Lys Gly Val Lys Ile Asp Glu Asp Phe Leu Gly
245 250 255
Gln Ala Ile Lys Asp Lys Glu Ser Gln Gln Phe Ile Ser Glu Glu Phe
260 265 270
Ile Ile Gln Leu Leu Phe Ser Ile Ser Phe Ala Ser Phe Glu Ser Ile
275 280 285
Ser Thr Thr Leu Thr Leu Ile Leu Asn Phe Leu Ala Asp His Pro Asp
290 295 300
Val Val Lys Glu Leu Glu Ala Glu His Glu Ala Ile Arg Lys Ala Arg
305 310 315 320
Ala Asp Pro Asp Gly Pro Ile Thr Trp Glu Glu Tyr Lys Ser Met Asn
325 330 335
Phe Thr Leu Asn Val Ile Cys Glu Thr Leu Arg Leu Gly Ser Val Ile
340 345 350
Pro Ala Leu Pro Arg Lys Thr Thr Lys Glu Ile Gln Ile Lys Gly Tyr
355 360 365
Thr Ile Pro Glu Gly Trp Thr Val Met Leu Val Thr Ala Ser Arg His
370 375 380
Arg Asp Pro Glu Val Tyr Lys Asp Pro Asp Thr Phe Asn Pro Trp Arg
385 390 395 400
Trp Lys Glu Leu Asp Ser Ile Thr Ile Gln Lys Asn Phe Met Pro Phe
405 410 415
Gly Gly Gly Leu Arg His Cys Ala Gly Ala Glu Tyr Ser Lys Val Tyr
420 425 430
Leu Cys Thr Phe Leu His Ile Leu Phe Thr Lys Tyr Arg Trp Arg Lys
435 440 445
Leu Lys Gly Gly Lys Ile Ala Arg Ala His Ile Leu Arg Phe Glu Asp
450 455 460
Gly Leu Tyr Val Asn Phe Thr Pro Lys Glu
465 470
<210> 7
<211> 1425
<212> DNA
<213> Artificial Synthesis
<400> 7
atgtggacga tcttgctcgg tttggcgacg ttggcaattg cctactatat tcattgggtt 60
aacaaatgga aggattctaa attcaacgga gttttgccgc cgggcaccat ggggctgccc 120
ctcatcggag aaacccttca atttcttcgc cctagtgact cccttgatgt tcatcctttc 180
tttcaacgca aagttaaaag atatggaccg atcttcaaga cttgtttggc gggaaggccg 240
gtggtggttt caacggatgc agagtttaac cattacataa tgctccaaga aggaagggcc 300
gtagaaatgt ggtatttgga tacactctct aaattctttg gccttgacac tgaatggacc 360
aaagcccttg gcctcatcca caaatacatt agaagcatta ctttgaacca ctttggtgct 420
gagtcccttc gtgagcgttt ccttcctcgt atcgaagaat ccgctcgaga aacccttcat 480
tattggtcaa ctcaaaccag cgttgaagtc aaggaatcag ccgctgcgat ggttttcaga 540
acttcgattg ttaagatgtt tagtgaagat tctagtaaat tactgacaga aggtctcact 600
aagaagttca caggacttct cggaggtttt ctcaccttgc ctctaaattt gcctggcact 660
acctatcata aatgcataaa ggacatgaag caaatccaaa agaagctaaa agacatttta 720
gaggaaagat tggctaaagg ggttaaaatt gatgaagatt tcttggggca agccattaaa 780
gataaagaat ctcaacaatt catttcagag gaattcatta tccagttgtt gttttccatc 840
agctttgcta gctttgagtc catctctacc actcttactt tgattctcaa cttcctcgcc 900
gatcaccccg acgtagtgaa agaattggag gctgagcatg aggctattag aaaggcaagg 960
gcagatccag atggaccaat cacttgggaa gaatacaaat ccatgaattt cacactcaat 1020
gtcatctatg aaacacttag gttgggaagt gtaatacctg ctttgccgag gaagacaacc 1080
aaggaaattc aaataaaagg atacacaatt ccagaaggat ggacagtaat gcttgtgacc 1140
gcttctcgtc atagagatcc agaagtgtac aaggatcccg ataccttcaa tccatggcgt 1200
tggaaggagt tggactcaat tactattcaa aagaacttca tgccatttgg gggaggctta 1260
aggcattgtg ctggtgctga atactctaaa gtctatttgt gcactttcct tcatatcctt 1320
ttcaccaaat acagatggag aaaactaaag ggaggaaaga ttgcaagggc tcatatattg 1380
aggtttgaag atgggttata tgtgaacttc actcccaagg aatga 1425

Claims (9)

1. A protein has a sequence shown in SEQ.ID.No. 6.
2. A nucleic acid encoding the protein of claim 1.
3. The biomaterial containing the nucleic acid according to claim 2, which is any one of the following A1) to A4):
A1) an expression cassette comprising the nucleic acid of claim 2;
A2) a recombinant vector comprising the nucleic acid of claim 2;
A3) a recombinant microorganism comprising the nucleic acid of claim 2;
A4) a transgenic plant cell line comprising the nucleic acid of claim 2.
4. Use of the protein of claim 1, or the nucleic acid of claim 2, or the biomaterial of claim 3 in the preparation of mogroside V precursor or mogroside V, said mogroside V precursor being 11H-Cuol.
5. A method for producing mogroside V precursor, comprising culturing a host comprising the nucleic acid of claim 2 under conditions such that the mogroside V precursor is produced, and isolating the mogroside V precursor from the metabolites, wherein the mogroside V precursor is 11H-Cuol.
6. A method for producing mogroside V, comprising culturing a host containing the nucleic acid of claim 2 under conditions that produce a mogroside V precursor, which is 11H-Cuol, and then synthesizing mogroside V using the precursor as a substrate.
7. The method of claim 5 or 6, wherein the host is selected from the group consisting of prokaryotes and eukaryotes.
8. The method of claim 5 or 6, wherein the host is a bacterial, fungal or plant cell.
9. The method of claim 8, wherein the fungus is a yeast.
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