Method for synthesizing momordica grosvenori glycoside V by utilizing multi-enzyme cascade catalysis of mogrol

Abstract

The invention discloses a method for synthesizing momordica grosvenori glucoside V. According to the method, a multi-enzyme cascade system of sucrose synthase and glycosyltransferase is constructed to catalyze the momordica grosvenori alcohol to synthesize the momordica grosvenori glucoside V. The sucrose synthase AtSUS1 is introduced to convert sucrose into fructose and UDPG. And then the glycosyl transferase starts a glycosylation reaction to catalytically synthesize mogroside, so that a UDPG cyclic regeneration system is formed, efficient recovery of the UDPG is realized, the cost of taking the UDPG as an auxiliary substrate is reduced, and meanwhile, the yield of the momordica grosvenori glycoside V is increased.

Description

(I) Technical field

[0001] The invention belongs to the technical field of biocatalysis and enzyme engineering, and relates to a method for synthesizing mogroside V by utilizing multi-enzyme cascade catalysis of mogroside alcohol. (II) Background technology

[0002] Mogroside V is a natural sweet compound extracted from monk fruit (Siraitia grosvenorii). It is a disodium glycoside compound and is the component with the highest content and sweetness among monk fruit glycosides. It has many pharmacological activities such as cough and expectorant, anti-cancer, antioxidant, and blood sugar regulation. It has become a natural non-sugar sweetener with therapeutic functions and has broad market prospects. Studies have also found that mogroside V inhibits angiogenesis through a VEGF-dependent mechanism, resulting in a decrease in MVD and reduced tumor growth, thereby preventing pancreatic tumor growth.

[0003] Mogrol is an important precursor for the synthesis of mogroside V. Mogrol (also known as mogroside alcohol or mono mogroside alcohol) is a natural organic compound belonging to the class of polyols. It is obtained by chemical separation and purification of monk fruit extract. Mogrol is relatively safe and has no toxic side effects on the human body. In the case of moderate intake, it generally does not cause indigestion or obvious adverse reactions. Excessive intake may cause mild gastrointestinal discomfort, such as bloating or diarrhea. However, during use, it is still necessary to follow the principle of moderate intake and pay attention to safe use according to personal circumstances.

[0004] The development of plant cell culture, metabolic engineering and synthetic biology has provided a sustainable production strategy for obtaining plant natural products. However, due to the high cost, long time and low yield of plant cell culture, it is difficult to produce specialized natural products; in addition, the complexity of plant cells and the lack of genetic tools and appropriate methods make the metabolic engineering of plant cells challenging for the production of complex natural products such as mogroside V, which requires a multi-step biosynthetic pathway. With the continuous development of enzyme engineering research and the in-depth understanding of the biosynthetic mechanism of glycoside molecules in monk fruit, the synthesis of mogroside V using enzyme engineering technology has become a new way for its large-scale production, which is of great significance and broad prospects in meeting consumers' demand for natural sweeteners.

[0005] Glycosylation reactions catalyzed by glycosyltransferases (UGTs) usually require the use of a large amount of UDP-sugar donors, such as UDP-glucose (UDP-Glc or UDPG). At present, the synthesis process of glycosyl donors is complex, costly, and unstable, and is limited to laboratory and small-scale industrial production. In order to solve this problem, the establishment of an in vitro regeneration system and the reconstruction of endogenous metabolic pathways have become the hot topics of research for scholars at home and abroad. The multi-enzyme cascade catalytic system is to match and couple multiple enzyme-catalyzed reactions to achieve efficient biosynthesis of the target product in the same reaction system. This system can be carried out in vivo or in vitro, and there is no need to separate and purify intermediates, which significantly improves production efficiency and reduces costs. The most widely studied UDPG recycling regeneration system in recent years is the use of UGTs and sucrose synthase coupling reaction for the recycling supply of UDPG. Under weak acid conditions, sucrose synthase can catalyze UDP and sucrose to form active glycosyl donors UDPG and fructose. In addition to reducing the use of UDPG, this method can also avoid the product inhibition effect of UGTs caused by excessive UDP produced by glycosylation. However, at present, the types of recyclable UDP-glycosyl donors are limited, mainly UDP-glucose and UDP-galactose; there are problems in matching and optimizing the reaction kinetics between different enzymes in the cascade catalytic system; the use of UDP-glycosyl donors or cofactors such as UDP cannot be completely avoided; and the efficiency of glycosylation modification based on the UDP-sugar regeneration system is low.

[0006] Therefore, it is necessary to find a method to promote the recycling of UDPG and achieve efficient synthesis of mogroside V by constructing an in vitro multi-enzyme cascade catalytic reaction strategy. (III) Summary of the invention

[0007] The invention aims to provide a method for synthesizing mogroside V from mogroside alcohol by utilizing a multi-enzyme cascade catalysis, construct a multi-enzyme cascade system of sucrose synthase and glycosyltransferase to catalyze the synthesis of mogroside V from mogroside alcohol, convert sucrose into fructose and UDPG by introducing sucrose synthase AtSUS1, and then glycosyltransferase (UGT) initiates a glycosylation reaction to catalyze the synthesis of Mogrosides, thereby forming a UDPG recycling system, realizing efficient recovery of UDPG, not only reducing the cost of UDPG as an auxiliary substrate, but also increasing the yield of mogroside V, thereby solving the problem of expensive UDPG raw materials in the prior art.

[0008] The technical solution adopted by the present invention is:

[0009] The invention provides a method for synthesizing mogroside V from mogroside alcohol by utilizing multi-enzyme cascade catalysis. The method comprises the following steps: using mogroside alcohol and uridine diphosphate (UDP) as substrates, using sucrose synthase AtSUS1, glycosyltransferase UGTM1-3 and glycosyltransferase UGTM2-4 as multi-enzyme cascade catalysts, adding sucrose, MgCl2 and a buffer solution with a pH value of 6-8 to form a reaction system, and reacting at 30-50° C. to generate mogroside V; the nucleotide sequence of a gene encoding the sucrose synthase AtSUS1 is shown in SEQ ID NO.1; the amino acid sequence of the glycosyltransferase UGTM1-3 is shown in SEQ ID NO.2; and the amino acid sequence of the glycosyltransferase UGTM2-4 is shown in SEQ ID NO.4.

[0010] The sucrose synthase AtSUS1 of the present invention is derived from Arabidopsis thaliana (L.) Heynh., GenBank accession number: AT5G20830, and the nucleotide sequence is shown in SEQ ID NO. 1. The nucleotide sequence of the glycosyltransferase UGTM1-3 editing gene is shown in SEQ ID NO. 3; the nucleotide sequence of the glycosyltransferase UGTM2-4 editing gene is shown in SEQ ID NO. 5.

[0011] Furthermore, in the reaction system, the final concentration of mogrosanol added is 0.1-1.0 mM (preferably 0.5 mM); the final concentration of uridine diphosphate added is 2-20 mM (preferably 10 mM); the final concentration of sucrose added is 50-500 mM (preferably 300 mM); and the final concentration of MgCl2 added is 10-50 mM (preferably 15 mM).

[0012] Furthermore, in the reaction system, the final concentrations of sucrose synthase AtSUS1, glycosyltransferase UGTM1-3 and glycosyltransferase UGTM2-4 added were all 10-50 U / L in terms of enzyme activity, wherein the final concentration of sucrose synthase AtSUS1 added was 40 U / L in terms of enzyme activity, the final concentration of glycosyltransferase UGTM1-3 added was 10 U / L in terms of enzyme activity, and the final concentration of glycosyltransferase UGTM2-4 added was 20 U / L in terms of enzyme activity.

[0013] Furthermore, the sucrose synthase AtSUS1, glycosyltransferase UGTM1-3 and glycosyltransferase UGTM2-4 are added in the form of pure enzyme liquid extracted by crushing wet bacteria obtained by fermentation and culture of recombinant Escherichia coli containing their respective encoding genes.

[0014] Further, the pure enzyme solution is prepared according to the following steps:

[0015] (1) Seed culture: The recombinant Escherichia coli containing the encoding gene is activated with LB medium and cultured at 37° C. and 180 rpm for 10-12 hours to obtain seed solution;

[0016] (2) Fermentation culture: The seed solution obtained in step (1) is transferred into LB liquid culture medium at an inoculum concentration of 1-2% by volume, and cultured at 37° C. and 180 r / min for 2-2.5 h until the OD600 reaches 0.6-0.8 to obtain a fermentation liquid;

[0017] (3) Wet cells: add IPTG with a final concentration of 0.1 mmol / L to the fermentation broth obtained in step (2), induce at 20° C. for 12 h, and then centrifuge at 4° C. and 8000 r / min for 10 min to collect the wet cells;

[0018] (4) Crude enzyme solution: The wet cells collected in step (3) were resuspended in 50 mM Tris-HCl (pH 8.0) buffer, placed on ice, and ultrasonically disrupted for 30 min at a power of 130 W for 2 s and a rest period of 4 s. The cell disruption solution was centrifuged at 4° C. and 10,000 rpm for 10 min, and the supernatant was collected to obtain a crude enzyme solution; the volume of the buffer solution used was 10-30 mL / g based on the weight of the wet cells;

[0019] (5) Target protein collection: The crude enzyme solution in step (4) was first filtered through a 0.45 μm filter membrane to remove impurities, and the filtrate was loaded with Ni 2+ Affinity chromatography column, flow rate 2mL / min, first use buffer A to elute impurities until the UV absorption detection value reaches baseline equilibrium; then use buffer B to elute the target protein, collect the eluate when the UV absorption detection value starts to rise and stop collecting when the UV absorption detection value returns to the baseline, which is the target protein;

[0020] Buffer A: 50 mM Tris-HCl solution containing 25 mM imidazole and 300 mM NaCl, pH 8.0;

[0021] Buffer B: 50 mM Tris-HCl solution containing 500 mM imidazole and 300 mM NaCl, pH 8.0;

[0022] (6) Pure enzyme solution: The target protein collected in step (5) was placed in a dialysis bag (3.5 kDa) and dialyzed in 20 mM pH 8.0 phosphate buffer. The solution was changed every 6 hours for 12 hours. The desalting process was ensured to be carried out at low temperature (ice bath 0°C). Finally, the intercepted liquid was collected as the pure enzyme solution, which was divided into 1.5 mL EP tubes, quickly frozen with liquid nitrogen, and stored in a -80°C ultra-low temperature refrigerator to obtain UGTM1-3 pure enzyme solution, UGTM2-4 pure enzyme solution, and AtSUS1 pure enzyme solution, respectively.

[0023] The recombinant Escherichia coli containing the glycosyltransferase UGTM1-3 encoding gene of the present invention is constructed according to the following steps: taking the recombinant plasmid pET28a-UGTM1-3 containing the glycosyltransferase UGTM1-3 encoding gene and the molecular chaperone plasmid pGro7 and adding them to the E. coli BL21 (DE3) competent cells at the same time, ice bathing for 30 minutes and then accurately heat shocking at 42°C for 90 seconds, and then placing on ice for 3 minutes; adding antibiotic-free LB liquid culture medium in a clean bench, incubating at 37°C and 180rpm for 1 hour; centrifuging at 5000rpm for 3 minutes, discarding 4 / 5 volume of the supernatant; suspending the bacteria in the remaining supernatant, and evenly coating it on a plate containing 50μg·mL -1 Kanamycin and 20 μg mL -1 On the LB solid medium plate with double antibiotics of chloramphenicol, culture at 37℃ for 12h. After the transformants grow out, the monoclonal colonies on the plate are screened as templates, and the positive transformants are identified by colony PCR; the amplification conditions of the colony PCR system are: 98℃ pre-denaturation for 5min, 1 cycle; 98℃ denaturation for 1min, 58℃ annealing for 1min, 72℃ extension for 1min30s, 30 cycles; 72℃ final extension for 10min. After the amplification is completed, take an appropriate amount of colony PCR product and spot it in the agarose gel well. 160V, electrophoresis for 15min. Then observe the gel electrophoresis results under the imager. To verify the correct clone, use the gun tip to gently pick up the single colony from the LB plate and inoculate it in the LB liquid medium containing the corresponding antibiotics for cultivation. After the transformants grow out, the plasmid is extracted for verification. If the verification is correct, the recombinant bacteria containing the recombinant plasmid pET28a-UGTM1-3 and the molecular chaperone plasmid pGro7 can be obtained, which is recorded as the recombinant bacteria M1-3; the preparation method of the recombinant Escherichia coli containing the glycosyltransferase UGTM2-4 encoding gene is the same as the recombinant Escherichia coli containing the glycosyltransferase UGTM1-3 encoding gene. The recombinant Escherichia coli containing the sucrose synthase AtSUS1 encoding gene is obtained by transforming the recombinant plasmid pET28a-AtSUS1 containing the sucrose synthase AtSUS1 encoding gene into E. coli BL21 (DE3) competent cells and verifying that it is correct. The chaperone plasmid pGro7 is used to improve the soluble expression of the glycosyltransferase UGTM1-3 and UGTM2-4 proteins.

[0024] The present invention also provides a glycosyltransferase UGTM1-3 for synthesizing mogroside V, wherein the glycosyltransferase UGTM1-3 is synthesized by converting the mogroside-derived gene UGT74AC1 M7(T79Y / L48M / R28H / L109I / S15A / M76L / H47R) The valine at position 58 of the encoded protein (named UGTM1) is mutated into leucine, the serine at position 181 is mutated into phenylalanine, and the alanine at position 371 is mutated into serine; the amino acid sequence is shown in SEQ ID NO.2, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.3.

[0025] The present invention also provides a glycosyltransferase UGTM2-4 for synthesizing mogroside V. The glycosyltransferase UGTM2-4 is synthesized by converting the gene UGT94-298-3 derived from mogroside M7(S34A / F77L / V146A / T344V / A313V / M360L / A391V) The proline at position 54 of the encoded protein (named UGTM2) was mutated to alanine, the glycine at position 152 was mutated to threonine, the proline at position 241 was mutated to glutamic acid, and the serine at position 272 was mutated to histidine.

[0026] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0027] (1) The present invention utilizes a multi-enzyme cascade method of glycosyltransferase UGTM1-3, glycosyltransferase UGTM2-4 and sucrose synthase AtSUS1 to catalyze the conversion of mogroside alcohol to mogroside V under mild conditions, thereby reducing the cost of directly using UDPG as a co-substrate.

[0028] (2) The reaction time for preparing mogroside V by the method of the present invention is shorter than that by the plant extraction method, and there are fewer intermediate operations. At the same time, compared with the catalysis by chemical synthesis, the reaction cost is greatly reduced, and environmental pollution is reduced, which has important value and significance. (IV) Description of the drawings

[0029] Figure 1 , Schematic diagram of recombinant plasmid pET28a-UGTM1-3.

[0030] Figure 2 , Schematic diagram of recombinant plasmid pET28a-UGTM2-4.

[0031] Figure 3 , Schematic diagram of recombinant plasmid pET28a-AtSUS1.

[0032] Figure 4 , A curve chart showing the effect of the enzyme ratio of UGTM1-3 and UGTM2-4 in the in vitro dual enzyme cascade system on the yield of mogroside V.

[0033] Figure 5, Bar graph showing the effect of the enzyme ratio of UGTM1-3, UGTM2-4 and AtSUS1 in the in vitro three-enzyme cascade system on the production of mogroside V.

[0034] Figure 6 , bar chart showing the effect of different pH on the yield of mogroside V.

[0035] Figure 7 , bar chart showing the effect of different sucrose concentrations on the yield of mogroside V.

[0036] Figure 8 , bar chart showing the effect of different UDP addition amounts on the yield of mogroside V. (V) Specific implementation methods

[0037] The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto:

[0038] The culture medium involved in the following examples is as follows:

[0039] LB solid medium: 10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, 0.2 g / L agar powder, solvent is deionized water, pH value is natural.

[0040] LB liquid culture medium: 10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, solvent is deionized water, pH value is natural.

[0041] 1M MgSO4 solution: Weigh 24.074 g of MgSO4 solid and dilute to 200 mL with sterile water.

[0042] CCB1 solution: Weigh 2.06 g of CaCl2 solid and dilute to 80 mL with sterile water. Sterilize at 121°C for 20 min. After cooling to room temperature, add 1 mL of 1M MgSO4 solution and shake to mix.

[0043] CCB2 solution: weigh 1.03 g CaCl2 and dissolve it in 64 mL sterile water, add 36 mL glycerol, and sterilize at 121°C for 20 min.

[0044] 50 mM Tris-HCl buffer: weigh 6.05 g of tris(hydroxymethyl)aminomethane powder and dissolve it in 1 L of ultrapure water, and adjust the pH to different values ​​(7.0-9.0) with hydrochloric acid.

[0045] Binding buffer: Prepare 50 mM Tris-HCl solution, then add 300 mM NaCl, adjust the pH to 8.0, and sterilize with a 0.22 μm filter and degas by ultrasonication.

[0046] Buffer A: Prepare Binding buffer solution, then add 25 mM imidazole, adjust the pH to 8.0, sterilize with a 0.22 μm filter, and degas by ultrasonication.

[0047] Buffer B: Prepare Binding buffer solution, then add 500 mM imidazole, adjust the pH to 8.0, sterilize with a 0.22 μm filter membrane and degas by ultrasonication. Prepare the reagents before use.

[0048] Example 1: Construction of recombinant plasmid

[0049] 1. Recombinant plasmid pET28a-UGTM1-3

[0050] The Luo Han Guo gene UGT74AC1 reported by Li J team (Li J, Yang JG, Mou SC, et al. Efficient O-glycosylation of triterpenes enabled by protein engineering of plant glycosyltransferase UGT74AC1 [J]. Chemicals & Chemistry, 2020.) M7(T79Y / L48M / R28H / L109I / S15A / M76L / H47R) The sequence was used as a template and the whole gene was synthesized by GenScript (Nanjing) Co., Ltd. The nucleotide sequence is shown in SEQ ID NO.6 and named UGTM1. The gene was inserted into the pET28a vector, and a 6×His-tag was introduced at the N-terminus and the C-terminus to obtain the recombinant expression plasmid pET28a-UGTM1 containing the glycosyltransferase gene.

[0051] Using pET28a-UGTM1 plasmid as template, primers F-58 and R-58, F-181 and R-181, F-371 and R-371 in Table 1 were used to perform site-directed mutagenesis PCR of the whole plasmid.

[0052] Table 1 Primers related to site-directed mutagenesis

[0053]

[0054] PCR amplification system: 1 μL of 1 ng / μL pET28a-UGTM1 plasmid as template, 1 μL of 10 μM primer F and primer R, 25 μL of 2× PrimeSTAR HSDNA Polymerase high-fidelity DNA polymerase, and 22 μL of ultrapure water.

[0055] PCR reaction program: pre-denaturation at 95°C for 5 min, then enter temperature cycle at 95°C for 10 sec; 58°C for 15 sec; 72°C for 1 min; a total of 30 cycles, with a termination temperature of 4°C.

[0056] The whole plasmid site-directed mutagenesis PCR product was digested with endonuclease Dpn I at 37°C for 2h, and the purified product obtained by purification kit was transferred into E. coli DH5α competent cells. The bacterial solution was evenly spread on LB solid medium containing 100μg / mL ampicillin (Amp) and cultured at 37°C overnight to obtain a site-directed mutagenesis library. Single transformants were picked and positive transformants were identified by colony PCR. After three rounds of whole plasmid site-directed mutagenesis, the recombinant plasmid pET28a-UGTM1-3 was obtained, as shown in Figure 1 As shown, glycosyltransferase UGTM1-3, the amino acid sequence is shown in SEQ ID NO.2, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.3.

[0057] 2. Recombinant plasmid pET28a-UGTM2-4

[0058] The gene UGT94-298-3 from Monk Fruit reported by Li J team (Li J, Mou SC, Yang JG, et al. Glycosyltransferase engineering and multi-glycosylation routes development facilitating synthesis of high-intensity sweetener mogrosides[J]. iScience, 2022, 25(10): 105222-105222.) M7(S34A / F77L / V146A / T344V / A313V / M360L / A391V) The whole gene was synthesized by GenScript (Nanjing) Co., Ltd. with the nucleotide sequence shown in SEQ ID NO.7, named UGTM2. The gene was inserted into the pET28a vector, and a 6×His-tag was introduced at the N-terminus and the C-terminus to obtain the recombinant expression plasmid pET28a-UGTM2 containing the glycosyltransferase gene.

[0059] Using the pET28a-UGTM2 plasmid as a template, the primers in Table 2 were used to perform site-directed mutagenesis PCR according to the method in step 1 to obtain the recombinant plasmid pET28a-UGTM2-4. Figure 2 As shown, glycosyltransferase UGTM2-4, the amino acid sequence is shown in SEQ ID NO.4, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.5.

[0060] Table 2 Primers related to site-directed mutagenesis

[0061]

[0062] 3. Recombinant plasmid pET28a-AtSUS1

[0063] Using pTrc99a-AtSUS1 plasmid (AtSUS1 target gene was synthesized after codon optimization by Beijing Qingke Biotechnology Co., Ltd. and constructed between EcoRⅠ and Eco53kⅠ restriction nuclease sites of pTrc99a vector) as template, using primers in Table 3, PCR amplification was performed using the PCR reaction system and procedure in step 1 to obtain the AtSUS1 fragment and the pET28a linearized fragment. The gene AtSUS1 was inserted into the pET28a vector through one-step cloning and connection, and transformed into E. coli DH5α competent cells, and cultured using the method in step 1. Colony PCR verification was performed using primers F-28a-P and RP-AtSUS1, and the plasmid was extracted and sent for testing to obtain the recombinant plasmid pET28a-AtSUS1, as shown in FIG. Figure 3 As shown, the nucleotide sequence of the gene encoding sucrose synthase AtSUS1 is shown as SEQ ID NO.1.

[0064] Table 3 Primers related to plasmid construction

[0065]

[0066] Colony PCR amplification system: Pick several single clones on the transformation plate as templates, 1 μL each of 10 μM primer F and primer R, 10 μL of 2× Rapid Taq Master Mix enzyme, and 8 μL of ultrapure water.

[0067] PCR reaction program: pre-denaturation at 98°C for 5 min, then enter temperature cycle at 98°C for 10 sec; 58°C for 15 sec; 72°C for 30 sec; a total of 30 cycles, with a termination temperature of 4°C.

[0068] Example 2: Preparation of competent E. coli BL21 (DE3)

[0069] Take out the preserved wild-type E.coli BL21 (DE3) bacterial solution from the -80℃ ultra-low temperature refrigerator in the laboratory, draw the bacterial solution under a sterile environment, and divide and streak on the non-resistant LB solid culture medium plate. The plate is placed in a 37℃ constant temperature incubator for overnight culture. Then pick a single colony with a pipette tip and inoculate it in the non-resistant LB liquid culture medium, and culture it overnight to form a seed solution. Transfer it to a new LB culture medium according to the volume concentration of 2% inoculation, culture for 2h until the bacterial solution OD600 = about 0.6, and stop the culture. After the bacterial solution is pre-cooled, transfer it to a sterile 50mL centrifuge tube and centrifuge it at 4℃, 4500rpm for 5min. Discard the supernatant, add about 40mL of CCB1 solution that has been pre-cooled, grind it gently, ice bath for 30min, centrifuge it at 4℃, 4500rpm for 5min, discard the supernatant, draw 1mL of pre-cooled CCB1 solution and CCB2 solution respectively, and gently blow the suspended cells. Finally, pipette 100 μL into sterilized centrifuge tubes, mark them, and store them in a -80°C refrigerator.

[0070] Example 3: Construction of recombinant bacteria

[0071] 1. Construction of the M1-3 strain co-expressing molecular chaperone

[0072] Take 200 μg of the recombinant plasmid pET28a-UGTM1-3 prepared in Example 1 and 200 μg of the molecular chaperone plasmid pGro7 (purchased from GenScript (Nanjing) Co., Ltd.) and add them to 100 μL of E. coli BL21 (DE3) competent cells prepared in Example 2. After ice bathing for 30 minutes, heat shock at 42°C for 90 seconds, and then place on ice for 3 minutes. Add 1 mL of antibiotic-free LB liquid culture medium to the clean bench and incubate at 37°C and 180 rpm for 1 hour. Centrifuge at 5000 rpm for 3 minutes and discard 800 μL of the supernatant. The remaining 200 μL of culture medium is used to suspend the bacteria and is evenly applied to a plate containing 50 μg·mL -1 Kanamycin and 20 μg mL -1On the LB solid medium plate with double antibiotics of chloramphenicol, culture at 37℃ for 12h. After the transformants grow out, the monoclonal colonies on the plate are screened as templates, and the positive transformants are identified by colony PCR. The amplification conditions of the colony PCR system are: 98℃ pre-denaturation for 5min, 1 cycle; 98℃ denaturation, 1min, 58℃ annealing, 1min, 72℃ extension for 1min30s, 30 cycles; 72℃ final extension for 10min. After the amplification is completed, take an appropriate amount of colony PCR products and spot them in the agarose gel well. 160V, electrophoresis for 15min. Then observe the gel electrophoresis results under the imager. Verify the correct clones and use the gun tip to gently pick up the single colony from the LB plate and inoculate it in the LB liquid medium containing the corresponding antibiotics for culture. After the transformants grow out, extract the plasmid for verification. If the verification is correct, the recombinant bacteria containing the recombinant plasmid pET28a-UGTM1-3 and the molecular chaperone plasmid pGro7 can be obtained, which is recorded as the recombinant bacteria M1-3.

[0073] 2. Construction of the M2-4 recombinant bacterial strain co-expressing molecular chaperones

[0074] The recombinant plasmid pET28a-UGTM1-3 in step 1 was replaced with pET28a-UGTM2-4, and other operations were the same to obtain the recombinant bacteria M2-4.

[0075] 3. Construction of recombinant bacteria AtSUS1

[0076] Using the method in step 1, the recombinant plasmid pET28a-AtSUS1 was transformed into E. coli BL21 (DE3) competent cells and verified to be correct, thereby obtaining the recombinant bacteria AtSUS1.

[0077] Example 4: Preparation of pure enzyme solution and detection of enzyme activity

[0078] 1. Preparation of enzyme source

[0079] (1) The recombinant bacteria obtained in Example 3 were activated with LB medium and cultured at 37° C. and 180 rpm for 12 h to obtain seed solution;

[0080] (2) the seed solution obtained in step (1) was transferred into 50 mL of LB liquid culture medium at a volume concentration of 1%, and cultured at 37° C. and 180 r / min for 2-2.5 h until the OD600 reached 0.6-0.8 to obtain a fermentation liquid;

[0081] (3) adding IPTG at a final concentration of 0.1 mmol / L to the fermentation broth obtained in step (2), inducing at 20° C. for 12 h, and centrifuging at 4° C. and 8000 r / min for 10 min to collect wet cells;

[0082] (4) Weigh 1 g of the wet cells collected in step (3), resuspend them in 20 mL of 50 mM Tris-HCl (pH 8.0) buffer, place them on ice, and ultrasonically disrupt them for 30 min at a power of 130 W for 2 s and a rest period of 4 s. Centrifuge the cell disruption solution at 4 ° C and 10,000 r / min for 10 min, collect the supernatant, and obtain a crude enzyme solution;

[0083] (5) In order to remove the impurities in the crude enzyme solution, the crude enzyme solution was first filtered through a 0.45 μm filter membrane, and the filtrate was then filtered through a Ni 2+ Protein was purified by affinity chromatography. The buffer was cooled on ice before purification. 2+ The affinity column was cleaned with ultrapure water and binding buffer, and Ni 2+ The column was flushed to equilibrium again. The collected filtrate was loaded with sample at a flow rate of 2 mL / min. Buffer A was used to elute the impurities first until the UV absorption detection value reached baseline equilibrium; then the target protein was eluted with buffer B. When the UV absorption detection value began to rise, the eluate was collected and the collection was stopped when the UV absorption detection value returned to the baseline. This was the target protein. Finally, the binding buffer was used to flush to baseline equilibrium, and the column was preserved with 20% ethanol.

[0084] (6) The purified target protein was desalted by dialysis. Before dialysis, the dialysis bag (3.5 kDa) was leak tested to ensure good sealing of the dialysis bag. The target protein was placed in a 20 mM pH 8.0 phosphate buffer in a dialysis bag. The solution was changed every 6 hours. The dialysis lasted for 12 hours and the desalting process was ensured to be carried out at low temperature (ice bath 0°C). Finally, the intercepted liquid was collected as the pure enzyme solution, which was divided into 1.5 mL EP tubes, quickly frozen with liquid nitrogen, and stored in a -80°C ultra-low temperature refrigerator to obtain UGTM1-3 pure enzyme solution, UGTM2-4 pure enzyme solution, and AtSUS1 pure enzyme solution, respectively.

[0085] 2. Detection of enzyme activity

[0086] (1) Determination of UGTM1-3 enzyme activity

[0087] Definition of enzyme activity unit (U): The amount of enzyme required to convert 1 micromole of substrate per minute is one enzyme activity unit (U).

[0088] The enzyme activity detection reaction system consists of: 0.5mM Mogrol, 10mM UDPG, 10mM MgCl2, UGTM1-3 pure enzyme solution prepared in step 1 (protein content 50mg) and 50mM Tris-HCl (pH 8.0) to 300μL. After reacting at 40℃ for 10min, the enzyme was immediately heated at 95℃ for 5min to inactivate the enzyme, followed by adding the same volume of methanol for dilution, centrifuging at 12000×g for 5min, and filtering the supernatant with a 0.22μm organic filter membrane. The filtrate was analyzed for the content of mogroside V using a high performance liquid chromatography system (HPLC). Each reaction was repeated three times.

[0089] Determination method of mogroside V:

[0090] HPLC used Aglient 1260 liquid chromatograph: C18 column (250 mm×4.6 mm, 5 μm); mobile phase A (acetonitrile + 0.1% formic acid) and mobile phase B (water + 0.1% formic acid); injection volume 5 μL; elution conditions: 0-20 min, 10%-90% A+90%-10% B gradient elution; 20-22 min, 10% A+90% B elution; 1 mL / min, wavelength 210 nm, column temperature 30°C.

[0091] (2) Determination of UGTM2-4 enzyme activity

[0092] Definition of enzyme activity unit (U): The amount of enzyme required to convert 1 micromole of substrate per minute is one enzyme activity unit (U).

[0093] The enzyme activity detection reaction system consists of: 0.5mM MogrosideⅡE (MogrosideⅡE), 10mM UDPG, 10mM MgCl2, UGTM2-4 (protein content 50mg) pure enzyme solution prepared in step 1 and 50mM Tris-HCl (pH 8.0) to 300μL. After reacting at 40℃ for 10min, the enzyme was immediately heated at 95℃ for 5min to inactivate the enzyme, followed by adding the same volume of methanol for dilution, centrifuging at 12000×g for 5min, and filtering the supernatant with a 0.22μm organic filter membrane. The content of Mogroside V was analyzed by high performance liquid chromatography (HPLC) system in step (1). Each reaction was repeated three times.

[0094] (3) Determination of sucrose synthase AtSUS1 activity

[0095] Enzyme activity unit (U) definition: the amount of enzyme that converts sucrose to produce 1 μmol of fructose in 1 min.

[0096] The enzyme activity assay reaction system consisted of 500 mM sucrose, 10 mM UDP (uridine diphosphate) and the pure AtSUS1 enzyme solution prepared in step 1 with a protein content of 0.5 mg, and 50 mM Tris-HCl buffer (pH 8.0) was added to 500 μL and added to an EP tube. The reaction was carried out at 40°C for 10 min. The yield of reduced fructose was determined by the DNS method.

[0097] Fructose determination method: Place the reaction solution EP tube in a high-speed centrifuge at 12,000 rpm for 1 minute, transfer the supernatant to a new EP tube, add the same volume of DNS reagent to the new EP tube and immediately boil for 10 minutes, cool to room temperature, take out 200 μL of sample and dilute with 800 μL of sterile water, determine its absorbance at a wavelength of 540 nm, and calculate the fructose content using the fructose standard curve.

[0098] Example 5: Optimization of conditions for the method of synthesizing mogroside V from mogroside alcohol catalyzed by multi-enzyme cascade

[0099] 1. Study on the optimal enzyme ratio of in vitro dual enzyme cascade system

[0100] The reaction system was prepared according to Table 4. 100mM mother solutions of mogroside alcohol, MgCl2 and UDPG were prepared with deionized water respectively. UGTM1-3 and UGTM2-4 are pure enzyme solutions prepared in step 1 of Example 4, and the added amount is based on enzyme activity. The addition amount of UGTM1-3 was fixed at 10U / L, and different amounts of UGTM2-4 (3.33, 5, 10, 20, 30 and 40U / L) were added to make the enzyme activity ratio of UGTM1-3:UGTM2-4 3:1, 2:1, 1:1, 1:2, 1:3, 1:4. The reaction was carried out at 40°C for 24h, and samples were taken at 2, 4, 6, 12, and 24h respectively. The yield of mogroside V was detected by the method of Example 4. The results are shown in Table 4. Figure 4 .

[0101] Table 4. Reaction system

[0102]

[0103] Figure 4The results showed that under the condition of sufficient UDPG, the ratio of different enzyme amounts of UGTM1-3 and UGTM2-4 was controlled, and the enzyme amount of UGTM1-3 was fixed at 10U / L. With the increase of the addition amount of UGTM2-4 enzyme, the yield of mogroside V continued to increase. When the addition amount of UGTM2-4 was 20U / L, the yield reached the maximum, the yield was 0.073mM, and the substrate conversion rate was 14.6%. When the addition amount of UGTM2-4 enzyme continued to increase, the reaction rate (curve slope) and yield did not increase greatly, and the accumulated end product no longer increased after 12h. Therefore, in the in vitro dual enzyme reaction system with mogroside alcohol as the substrate, the optimal enzyme addition ratio of UGTM1-3 and UGTM2-4 was 1:2 (10U / L: 20U / L).

[0104] 2. Study on the optimal enzyme ratio of the in vitro three-enzyme cascade system

[0105] The reaction system was prepared according to Table 5. Mogrosanol, UDP and MgCl2 were prepared into 100 mM stock solutions with deionized water, and sucrose was prepared into 2 M stock solution with deionized water. UGTM1-3, UGTM2-4 and AtSUS1 were pure enzyme solutions prepared in step 1 of Example 4, and the added amount was calculated based on enzyme activity.

[0106] The addition amount of UGTM1-3 was fixed at 10U / L, the addition amount of UGTM2-4 was fixed at 20U / L, and different amounts of AtSUS1 pure enzyme solution (10, 20, 30, 40 and 50U / L) were added to make the addition ratio of the three enzymes 1:2:1, 1:2:2, 1:2:3, 1:2:4, 1:2:5. The reaction was carried out at 40°C for 24h, and the samples were taken. The yield of mogroside V was detected by the method of Example 4. The results are shown in FIG. Figure 5 .

[0107] Table 5. Reaction system

[0108]

[0109] Figure 5 It shows that after fixing the enzyme amount of UGTM1-3 and UGTM2-4 at 1:2, the enzyme amount of AtSUS1 is continuously increased, and the yield obtained gradually increases. When the enzyme amount of AtSUS1 is 40U / L, the yield of the reaction system reaches the highest, and the accumulated yield of mogroside V is 0.058mM. After continuously increasing the enzyme addition amount of AtSUS1, the yield decreases. This may be because AtSUS1 sucrose synthase hydrolyzes more UDPG, which makes the glycosyltransferase unable to use the glycosyl donor in time, inhibiting the reaction. Therefore, the enzyme amount ratio of the three-enzyme system was finally determined to be 1:2:4 (10U / L: 20U / L: 40U / L).

[0110] 3. Optimization of pH of in vitro three-enzyme cascade system reaction

[0111] Glycosyltransferase UGT and sucrose synthase AtSUS1 each have different optimal pH values, so pH is an important factor affecting the coupling reaction. The change in pH value is a key factor affecting the direction of the SUS reversible reaction. Under acidic conditions, SUS is more inclined to hydrolyze sucrose to produce fructose and UDP-glucose, which is more conducive to the cascade reaction. The reaction system was prepared according to Table 5. The addition amounts of UGTM1-3, UGTM2-4, and AtSUS1 were 10U / L, 20U / L, and 40U / L, respectively. The pH of the buffer was changed to 6.0, 6.5, 7.0, 7.5, and 8.0, respectively. Other operations were the same. The results are shown in Table 5. Figure 6 shown.

[0112] Figure 6 It shows that compared with weakly acidic conditions, neutral and weakly alkaline conditions are more conducive to the coupling reaction, and the optimal pH is 7.0. The optimal reaction pH of the multi-enzyme cascade system is the result of a comprehensive balance of the properties of the two enzymes. Under pH 7.0, the activities of glycosyltransferase and sucrose synthase reach a balance, and the yield accumulation reaches the maximum value. At this time, the UDPG generated by sucrose synthase is consumed by UGT in time and UDP is generated. Driven by excess sucrose, sucrose synthase quickly catalyzes the reverse reaction to generate UDPG, thereby achieving a dynamic balance between UDPG and UDP.

[0113] 4. Study on sucrose concentration in in vitro three-enzyme cascade system

[0114] The reaction system was prepared according to Table 5. The addition amounts of UGTM1-3, UGTM2-4, and AtSUS1 were 10U / L, 20U / L, and 40U / L, respectively. The final concentration of sucrose was changed to 50, 100, 200, 300, and 500mM, respectively. Other operations were the same. The results are shown in Figure 7 shown.

[0115] Figure 7 It shows that low concentration of sucrose leads to slow sucrose reverse reaction, low UDPG production, and limited utilization of glycosyl donor by glycosyltransferase; while high concentration of sucrose can rapidly promote the reverse reaction of sucrose synthase. The maximum yield of the system was observed when the final concentration of sucrose was 300mM, and there was a slight change when the sucrose concentration continued to increase, but it was not obvious.

[0116] 5. Study on the amount of UDP added in the in vitro three-enzyme cascade system

[0117] Both UDP and UDPG can be used as substrates to initiate cascade reactions. UDPG is the direct substrate of UGT, but its price is more expensive than UDP. When UDP is added as a substrate to the reaction system, AtSUS1 is the starting enzyme in the cascade reaction, first converting sucrose into fructose and UDPG, and then UGT initiates the glycosylation reaction to catalyze the synthesis of Mogrosides.

[0118] The reaction system was prepared according to Table 5. The addition amounts of UGTM1-3, UGTM2-4, and AtSUS1 were 10U / L, 20U / L, and 40U / L, respectively. The final concentration of UDP was changed to 2, 5, 10, 15, and 20mM, respectively. Other operations were the same. The results are shown in Table 5. Figure 8 shown.

[0119] Figure 8 It shows that when the UDP concentration is low, the amount of UDPG that drives sucrose synthase to generate is low, and the sugar donor cannot be supplied to the system in time, so the product generated by UGT catalysis is relatively low. At the appropriate UDP concentration, the system undergoes sucrose hydrolysis and glycosylation reactions simultaneously, and UDPG and UDP are circulated. We found that high concentrations of UDP inhibited the formation of products, which may be because the catalytic efficiency of the two glycosyltransferases is not as good as that of sucrose synthase, resulting in the gradual accumulation of UDP concentration, which is not conducive to promoting the cycle of cascade catalytic reactions.

[0120] Based on the above results, the optimal pH value of the reaction system is 7.0, the final concentration of sucrose added is 300mM, the final concentration of UDP added is 10mM, the final concentration of MgCl2 added is 10mM, the final concentration of mogroside alcohol added is 0.5mM, and the conversion is carried out at 40℃ for 24h. The yield of mogroside V obtained is 0.091mM, and the conversion rate is 18.2%.

[0121] The above is a detailed implementation method and a specific operation flow of the present invention, which are implemented on the premise of the technical solution of the present invention, but the protection scope of the present invention is not limited to the above embodiments.

Claims

1. A method for synthesizing mogroside V from mogroside alcohol using multi-enzyme cascade catalysis, characterized in that: The method comprises the following steps: using mogroside alcohol and uridine diphosphate as substrates, using sucrose synthase AtSUS1, glycosyltransferases UGTM1-3 and glycosyltransferases UGTM2-4 as multi-enzyme cascade catalysts, adding sucrose, MgCl2 and a buffer solution with a pH of 6-8 to form a reaction system, and reacting at 30-50° C. to generate mogroside V; the nucleotide sequence of the sucrose synthase AtSUS1 encoding gene is shown in SEQ ID NO.1; the amino acid sequence of the glycosyltransferases UGTM1-3 is shown in SEQ ID NO.2; and the amino acid sequence of the glycosyltransferases UGTM2-4 is shown in SEQ ID NO.

4.

2. The method according to claim 1, characterized in that In the reaction system, the final concentration of mogrosanol added is 0.1-1.0 mM, the final concentration of uridine diphosphate added is 2-20 mM, the final concentration of sucrose added is 50-500 mM, and the final concentration of MgCl2 added is 10-50 mM.

3. The method according to claim 1, characterized in that The sucrose synthase AtSUS1, glycosyltransferase UGTM1-3 and glycosyltransferase UGTM2-4 are added in the form of pure enzyme liquid extracted from wet bacteria obtained by fermentation and culture of recombinant Escherichia coli containing their respective encoding genes and then crushed.

4. The method according to claim 3, characterized in that In the reaction system, sucrose synthase AtSUS1, glycosyltransferase UGTM1-3 and glycosyltransferase UGTM2-4 are added to a final concentration of 10-50 U / L in terms of enzyme activity.

5. The method according to claim 4, characterized in that Sucrose synthase AtSUS1 was added at a final concentration of 40 U / L based on enzyme activity, glycosyltransferase UGTM1-3 was added at a final concentration of 10 U / L based on enzyme activity, and glycosyltransferase UGTM2-4 was added at a final concentration of 20 U / L based on enzyme activity.

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