A momordicin glycosyltransferase mutant and application thereof in production of siamenoside i

Abstract

This invention discloses a mogroside glycosyltransferase mutant and its application in the production of sympathomimetic I, belonging to the fields of genetic engineering and enzyme engineering. The amino acid sequence of the mogroside glycosyltransferase mutant is shown in SEQ ID NO:2. Compared with the glycosyltransferase SgUGT94-289-3, the mogroside glycosyltransferase mutant of this invention (M92T / H96M / V148W protein) can significantly increase the production of sympathomimetic I. Specifically, it can increase the yield of sympathomimetic I to 81% and the specificity to 100% in enzyme engineering and biosynthesis, meeting the requirements for industrial production and possessing significant application value.

Description

Technical Field

[0001] This invention relates to the fields of genetic engineering and enzyme engineering, and more specifically to a mogroside glycosyltransferase mutant and its application in the production of symmenidine I. Background Technology

[0002] Mogrosides are the main active ingredient of the traditional Chinese medicine monk fruit (Luo Han Guo), and are also a non-sugar natural sweetener, with a sweetness 300 times that of sucrose. Pharmacological studies have shown that mogrosides have antitussive, expectorant, lung-moistening, liver-protecting, anti-inflammatory, anti-cancer, and hypoglycemic effects. The sweetness of mogrosides is related to the number and linkage of sugar residues. When the total number of glucose residues linked at positions 3 and 24 of the aglycone mogroside is 4-6 glucose residues, it forms mogrosides IV, IVA, V, and VI, as well as symmenidine I, which have a high-intensity sweetness.

[0003] Cimanin I is one of the main components of mogrosides and the sweetest of all known mogrosides, approximately 563 times sweeter than sucrose. Bioactivity studies have shown that cimanin I inhibits maltase and can be used to control postprandial blood glucose in diabetic patients. Currently, cimanin I is mainly extracted from the mature fruit of mogroside. However, because the content of cimanin I in mogroside is only about one ten-thousandth, it is extremely difficult to extract and concentrate from the fruit, limiting its commercial application. Therefore, synthetic biology methods, specifically enzyme engineering to modify the enzymes responsible for producing cimanin I, provide a solution for the industrial production of high-purity cimanin I.

[0004] Currently, the biosynthetic pathway of mogrosides has been fully elucidated. The final steps in the biosynthesis of both symmancoside I and mogroside V are completed by modification of the glycosyltransferase SgUGT94-289-3, and symmancoside I is also an intermediate product in the synthesis of mogroside V. Studies have found that the key terminal glycosyltransferase SgUGT94-289-3 in symmancoside I synthesis suffers from substrate heterogeneity and low in vitro catalytic activity, hindering its application in industrial production.

[0005] Therefore, how to develop a glycosyltransferase with high enzyme activity, strong product specificity, and the ability to meet the requirements of industrial production of symmenidine I is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0006] In view of this, the purpose of the present invention is to provide a mogroside glycosyltransferase mutant and its application in the production of sympathoside I, so as to overcome the shortcomings of the prior art.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] A mutant of mogroside glycosyltransferase, the amino acid sequence of which is shown in SEQ ID NO:2.

[0009] Furthermore, the above-mentioned monk fruit glycosyltransferase mutant is obtained by replacing the methionine residue at position 92, the histidine residue at position 96, and the valine residue at position 148 in the amino acid sequence of glycosyltransferase SgUGT94-289-3, thereby obtaining a protein with monk fruit glycosyltransferase activity; wherein, the amino acid sequence of glycosyltransferase SgUGT94-289-3 is shown in SEQ ID NO:1.

[0010] Furthermore, the above-mentioned mogroside glycosyltransferase mutant is obtained by replacing the methionine residue at position 92, the histidine residue at position 96, and the valine residue at position 148 in the amino acid sequence of glycosyltransferase SgUGT94-289-3, to obtain a protein with mogroside glycosyltransferase activity, and then attaching a tag to the N-terminus and / or C-terminus of the protein to obtain a fusion protein; wherein, the amino acid sequence of glycosyltransferase SgUGT94-289-3 is shown in SEQ ID NO:1.

[0011] Furthermore, the methionine residue at position 92 is replaced with a threonine residue.

[0012] Furthermore, the histidine residue at position 96 is replaced with a methionine residue.

[0013] Furthermore, the valine residue at position 148 is replaced with a tryptophan residue.

[0014] The specific mutant of mogroside glycosyltransferase in this invention is the M92T / H96M / V148W protein, with the amino acid sequence shown in SEQ ID NO: 2. It is a mutant obtained by mutating the amino acid combination at positions 92, 96, and 148 of the glycosyltransferase SgUGT94-289-3. The M92T / H96M / V148W protein is obtained by replacing the methionine residue at position 92 (from the N-terminus) shown in SEQ ID NO: 1 with a threonine residue, replacing the histidine residue at position 96 with a methionine residue, and replacing the valine residue at position 148 with a tryptophan residue.

[0015] The present invention also seeks protection for the nucleic acid molecule encoding the above-mentioned Luo Han Guo glycosyltransferase mutant, which is obtained by replacing the three nucleotides ATG shown at positions 274-276 from the 5' end of the gene encoding glycosyltransferase SgUGT94-289-3 with ACA, the three nucleotides CAC shown at positions 286-288 with ATG, and the three nucleotides GTC shown at positions 442-444 with TGG; wherein the nucleotide sequence of the gene encoding glycosyltransferase SgUGT94-289-3 is shown in SEQ ID NO:3.

[0016] The present invention also seeks protection for expression cassettes, recombinant vectors or recombinant microorganisms containing the above-mentioned nucleic acid molecules.

[0017] Furthermore, the aforementioned recombinant vector is a recombinant plasmid obtained by inserting the above nucleic acid molecules into an expression vector (such as the pET28a vector) or a cloning vector, such as the recombinant plasmid M92T / H96M / V148W.

[0018] Furthermore, the recombinant microorganisms mentioned above are recombinant bacteria obtained by introducing a recombinant vector into a starting microorganism. Even further, the starting microorganisms mentioned above are any one of Escherichia coli, yeast, Bacillus subtilis, Corynebacterium glutamicum, or lactic acid bacteria, preferably Escherichia coli, and more preferably Escherichia coli Rosetta (DE3).

[0019] The present invention also seeks protection for the use of the above-mentioned mogroside glycosyltransferase mutant in the production of symbioside I (SIA) and its upstream and downstream products, wherein the upstream and downstream products of symbioside I are mogrosides such as MV, MIIIE and MIII.

[0020] The present invention also seeks protection for the use of the above-mentioned nucleic acid molecules in the production of symbioside I (SIA) and its upstream and downstream products, wherein the upstream and downstream products of symbioside I are mogrosides such as MV, MIIIE and MIII.

[0021] The present invention also claims protection for the use of the above-mentioned expression cassette, recombinant vector or recombinant microorganism in the production of symbioside I (SIA) and its upstream and downstream products, wherein the upstream and downstream products of symbioside I are mogrosides such as MV, MIIIE and MIII.

[0022] As can be seen from the above technical solution, compared with the prior art, the beneficial effects of the present invention are as follows: Compared with the glycosyltransferase SgUGT94-289-3, the mogroside glycosyltransferase mutant of the present invention (M92T / H96M / V148W protein) can significantly increase the production of sympathoside I. Specifically, it can increase the yield of sympathoside I to 81% and the specificity to 100% in enzyme engineering and biosynthesis, which meets the requirements for industrial production and has important application value. Attached Figure Description

[0023] Figure 1 The structural formulas of mogrosides MV, SIA, MIVA, MIIIE, MIII and MIIE are given, where Glu is glucose; Figure 2 To determine the retention times of mogroside MV, SIA, MIVA, MIIIE, MIII and MIIE standards using the UPLC-MS system; Figure 3 The results of the detection of glycosyltransferase SgUGT94-289-3 (WT protein) in Example 3; Figure 4 The results of the detection of M92T / H96M / V148W protein in Example 3; Figure 5 The statistical results of the conversion yield of sermonidine I by M92T / H96M / V148W protein and glycosyltransferase SgUGT94-289-3 (WT protein) are presented. Figure 6 Statistical results on the specificity of M92T / H96M / V148W protein and glycosyltransferase SgUGT94-289-3 (WT protein) to sympathoside I product. Detailed Implementation

[0024] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0025] Example 1 Construction of recombinant plasmid expressing mogroside glycosyltransferase mutant (1) The gene encoding the artificially synthesized glycosyltransferase SgUGT94-289-3 The amino acid sequence of the glycosyltransferase SgUGT94-289-3 is shown in SEQ ID NO:1, specifically: MDAAQQGDTTTILLMLPWLGYGHLSAFLELAKSLSRRNFHIYFCSTSVNLDAIKPKLPSSFSDSIQFVELHLPSSPEFPPHLHTTNGLPPTLMPALHQAFSMAAQHFESILQTL APHLLIYDSLQPWAPRVASSLKIPAINFNTTGVFVISQGLHPIHYPHSKFPFSEFVLHNHWKAMYSTADGASTERTRKRGEAFLYCLHASCSVILINSFRELEGKYMDYLSVL LNKKVVPVGPLVYEPNQDGEDEGYSSIKNWLDKKEPSSTVFVSFGSEYFPSKEEMEEIAHGLEASEVNFIWVVRFPQGDNTSGIEDALPKGFLERAGERGMVVKGWAPQAKIL KHWSTGGFVSHCGWNSVMESMMFGVPIIGVPMHVDQPFNAGLVEEAGVGVEAKRDPDGKIQRDEVAKLIKEVVVEKTREDVRKKAREMSEILRSKGEEKFDEMVAEISLLLLKI.

[0026] The nucleotide sequence of the gene encoding the glycosyltransferase SgUGT94-289-3 (named "SgUGT94-289-3 gene") is shown in SEQ ID NO:3, specifically:

[0027] (2) After a large number of experiments, the inventor modified the SgUGT94-289-3 gene, mainly by modifying the nucleotides shown at positions 274-276, 286-288 and 442-444 from the 5' end of the SgUGT94-289-3 gene, and obtained the M92T / H96M / V148W gene.

[0028] Compared with the SgUGT94-289-3 gene, the nucleotide sequence of the M92T / H96M / V148W gene differs in that: the three nucleotides ATG shown at positions 274-276 from the 5' end of the SgUGT94-289-3 gene are replaced with ACA, the three nucleotides CAC shown at positions 286-288 are replaced with ATG, and the three nucleotides GTC shown at positions 442-444 are replaced with TGG, while the other nucleotide sequences remain unchanged.

[0029] The amino acid sequence of the M92T / H96M / V148W protein (hereinafter referred to as "M92T / H96M / V148W protein") encoded by the M92T / H96M / V148W gene is shown in SEQ ID NO:2, and is as follows: MDAAQQGDTTTILMLPWLGYGHLSAFLELAKSLSRRNFHIYFCSTSVNLDAIKPKLPSSFSDSIQFVELHLPSSPEFPPHLHTTNGLPPTLTPALMQAFSMAAQHFESILQTL APHLLIYDSLQPWAPRVASSLKIPAINFNTTGVFWISQGLHPIHYPHSKFPFSEFVLHNHWKAMYSTADGASTERTRKRGEAFLYCLHASCSVILINSFRELEGKYMDYLSVL LNKKVVPVGPLVYEPNQDGEDEGYSSIKNWLDKKEPSSTVFVSFGSEYFPSKEEMEEIAHGLEASEVNFIWVVRFPQGDNTSGIEDALPKGFLERAGERGMVVKGWAPQAKIL KHWSTGGFVSHCGWNSVMESMMFGVPIIGVPMHVDQPFNAGLVEEAGVGVEAKRDPDGKIQRDEVAKLIKEVVVEKTREDVRKKAREMSEILRSKGEEKFDEMVAEISLLLLKI.

[0030] The M92T / H96M / V148W protein is a mutant of the glycosyltransferase SgUGT94-289-3 (i.e., the Luo Han Guo glycosyltransferase mutant), specifically a mutant obtained by mutating the 92nd, 96th, and 148th amino acids of the glycosyltransferase SgUGT94-289-3.

[0031] (3) Replace the small DNA fragment between the restriction endonucleases BamHI and XhoI in the pET28a vector (Merck Millipore product, catalog number 69864) with the SgUGT94-289-3 gene to obtain the recombinant plasmid pET28a-WT. Following the above steps, replace the SgUGT94-289-3 gene with the M92T / H96M / V148W gene to obtain the recombinant plasmid M92T / H96M / V148W.

[0032] Example 2 Expression and purification of mogroside glycosyltransferase mutant (1) Transform the recombinant plasmid pET28a-WT into Escherichia coli Rosetta (DE3) (a product of Beijing TransGen Biotech Co., Ltd., product catalog number CD801-02) to obtain transgenic Escherichia coli containing the recombinant plasmid pET28a-WT, named recombinant bacteria WT; following the above steps, replace the recombinant plasmid pET28a-WT with the recombinant plasmid M92T / H96M / V148W respectively to obtain recombinant bacteria M92T / H96M / V148W; (2) Inoculate single clones of recombinant bacteria (recombinant bacteria WT, recombinant bacteria M92T / H96M / V148W) into 10 ml LB liquid medium and incubate overnight at 37°C and 220 rpm to obtain culture solution 1; (3) Inoculate culture solution 1 into 1L LB liquid medium and culture at 37℃ and 220 rpm with shaking until the OD600 nm of the system is 0.6-0.8; then add IPTG and make its concentration in the system 0.5 mM, and induce culture at 18℃ and 180 rpm for 20 h to obtain culture solution 2; (4) Take 2 liters of the culture solution, centrifuge at 8000 rpm for 10 min, and collect the bacterial cells; (5) Add lysis buffer (solute and concentration of 500mM NaCl, 5% glycerol and 10mM imidazole, solvent of pH 8.0, 50mM Tris-HCl buffer) to the bacterial cells collected in step (4) to resuspend the bacterial cells and obtain bacterial cell lysis buffer; the bacterial cell lysis buffer should be placed on ice; (6) The bacterial lysate obtained in step (5) is ultrasonically broken (ultrasonic parameters are 20Hz, 20min), then centrifuged at 4℃ and 18000g for 30min, and the supernatant is collected; the supernatant is the crude enzyme solution of mutant M92T / H96M / V148W or glycosyltransferase WT. (7) Take the supernatant collected in step (6) and purify it by nickel column affinity chromatography to obtain the purified mutant M92T / H96M / V148W protein or WT protein; the specific steps are as follows: (71) Add nickel packing material (GE Life Sciences product, catalog number 17-0575-02) to the chromatography column, first add 5 column volumes of deionized water, then add 10 column volumes of lysis buffer to equilibrate the column; (72) Using a peristaltic pump, the supernatant collected in step (6) is loaded into the column that completed step (71). Elution is performed with 50 column volumes of elution buffer containing 100 mM imidazole (solute and concentration of 500 mM NaCl, solvent of pH 8.0, 50 mM Tris-HCl buffer) (the purpose is to remove contaminating proteins); then, the column pass solution is collected with elution buffer containing 300 mM imidazole; the entire elution process is kept on ice. (73) Replace the column-passed solution collected in step (72) with an ultrafiltration tube (Merck Millipore, C7719) to a buffer A (solute and concentration of 0.1 mM MEDTA and 5 mM β-ME, solvent of pH 8.0, 20 mM Tris-HCl buffer) (to fully reduce the concentration of imidazole and NaCl), and collect the solution; (74) The solution collected in step (73) was added to a RESOURCEQ anion exchange chromatography column (GE LifeSciences, 17-1177-01). A linear gradient elution was performed using a buffer system consisting of buffer A and buffer B (the solute and concentration of buffer B were 500 mM NaCl, 0.1 mM EDTA and 5 mM β-ME, and the solvent was pH 8.0, 20 mM Tris-HCl buffer) to obtain the purified mogroside glycosyltransferase mutant or glycosyltransferase SgUGT94-289-3. Specifically, gradient elution was performed using a gradient elution condition of increasing buffer B (B) and decreasing buffer A (A), as detailed below (% are all volume percentages): 0-5 min, 100% A; 5-20 min, 0-70% B; 20-25 min, 100% B.

[0033] Example 3 In vitro catalysis and product analysis of mogroside glycosyltransferase mutant In this embodiment, the structural formulas of mogrosides MV, SIA, MIV, MIVA, MIIIE, MIII, and MIIE are shown below. Figure 1 The UPLC-MS system was used to detect mogrosides MIV, MIVA, MV, SIA, MIIIE, MIIE, and MIII standards (CAS accession numbers 88901-41-1, 88901-36-4, 126105-12-2, 88901-37-5, 88901-38-6, and 130567-83-8, respectively). Specific retention times are detailed in [link to relevant documentation]. Figure 2 .

[0034] (1) Preparation of the reaction system The reaction system consisted of 100 μL of UDP-glucose, 10 µg of mogroside mutant M92T / H96M / V148W or WT protein, mogroside MIIE, MIIIE or MIII, pH 6.5, 50 mM, and PBS buffer. The concentration of UDP-glucose in the reaction system was 8 mM, and the concentration of mogroside substrate was 0.1 mM. (2) Take the reaction system prepared in step (1) and incubate it at 45°C for 40 min; (3) After completing step (2), add 50 μL of methanol to terminate the reaction, then vortex to mix, centrifuge at 13000g for 10 min, and collect the supernatant. (4) The supernatant collected in step (3) was detected by UPLC-MS system. The peak area was used to calculate the yield of mogroside produced by the mogroside mutant and WT. The conversion yield of sympathoside I was further calculated (conversion yield = sympathoside I peak integral area / total integral area). The UPLC-MS reaction parameters are as follows: the mobile phase consists of 0.1% formic acid aqueous solution (solution A) and acetonitrile (solution B); the flow rate is 0.2 ml / min, and gradient elution is performed using gradient elution conditions with increasing solution B and decreasing solution A, specifically as follows: 0~10 min, 23% solution B; 10~20 min, 23~100% solution B; 20~30 min, 100% solution B. Mass spectrometry conditions: negative ion scanning; ionization voltage 4500 V; ion source temperature 550℃; curtain gas: nitrogen (purity ≥ 97%), pressure 20 psi; nebulizer gas: nitrogen (purity ≥ 97%), pressure 55 psi; auxiliary gas: nitrogen (purity ≥ 97%), pressure 45 psi; detection method: multiple reaction monitoring (MRM).

[0035] The retention times and monitoring ions of mogroside compounds are shown in Table 1.

[0036] Table 1. Retention time and monitoring ion pair information of mogrosides from Luo Han Guo.

[0037] The results are as follows Figure 3-6 As shown: (1) Regarding the conversion rate and specificity of siamanside I product Using MIIE, MIIIE, or MIII as substrates, the yield of SIA of M92T / H96M / V148W proteins was significantly increased compared with WT protein after 40 min of reaction, with yields of 14.96%, 81.57%, and 77.68%, respectively.

[0038] Mogroside IVA, mogroside IV, and symbioside I are competitive substrates in the reaction. This invention determines the specificity of mutants in producing symbioside I based on the percentage of symbioside I, mogroside IV, and mogroside IVA (specificity = peak integral area of ​​symbioside I / total integral area of ​​symbioside I, mogroside IV, and mogroside IVA). Using MIIE, MIIIE, or MIII as substrates, when the reaction reaches 40 min, the specificity of SIA products of M92T / H96M / V148W proteins all reach 100% compared with WT proteins.

[0039] (2) Regarding upstream and downstream products In this invention, upstream and downstream products refer to other mogrosides (such as MIII, MIIIE, and MV) besides symmenidine I in the reaction system. Due to the regioselectivity and substrate heterogeneity of the glycosyltransferase SgUGT94-289-3, upstream and downstream products such as MIII, MIIIE, and MV are generated in the reaction products.

[0040] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A mogrosider glycosyltransferase mutant, characterized in that, Its amino acid sequence is shown in SEQ ID NO:

2.

2. The mogroside glycosyltransferase mutant according to claim 1, characterized in that, By replacing the methionine residue at position 92, the histidine residue at position 96, and the valine residue at position 148 in the amino acid sequence of the glycosyltransferase SgUGT94-289-3, a protein with mogroside glycosyltransferase activity was obtained. The amino acid sequence of the glycosyltransferase SgUGT94-289-3 is shown in SEQ ID NO:

1.

3. The mogroside glycosyltransferase mutant according to claim 1, characterized in that, By replacing the methionine residue at position 92, the histidine residue at position 96, and the valine residue at position 148 in the amino acid sequence of the glycosyltransferase SgUGT94-289-3, a protein with mogroside glycosyltransferase activity was obtained, and a fusion protein was obtained by attaching a tag to the N-terminus and / or C-terminus of the protein. The amino acid sequence of the glycosyltransferase SgUGT94-289-3 is shown in SEQ ID NO:

1.

4. A mogrosiderin glycosyltransferase mutant according to claim 2 or 3, characterized in that, The methionine residue at position 92 is replaced with a threonine residue; the histidine residue at position 96 is replaced with a methionine residue; and the valine residue at position 148 is replaced with a tryptophan residue.

5. A nucleic acid molecule encoding a *Mogroscus grosvenorii* glycosyltransferase mutant as described in any one of claims 1-4, characterized in that, The nucleic acid molecule was obtained by replacing the three nucleotides ATG at positions 274-276 from the 5' end of the gene encoding glycosyltransferase SgUGT94-289-3 with ACA, the three nucleotides CAC at positions 286-288 with ATG, and the three nucleotides GTC at positions 442-444 with TGG. The nucleotide sequence of the gene encoding the glycosyltransferase SgUGT94-289-3 is shown in SEQ ID NO:

3.

6. An expression cassette, recombinant vector, or recombinant microorganism containing the nucleic acid molecule as described in claim 5.

7. The expression cassette, recombinant vector, or recombinant microorganism according to claim 6, characterized in that, The recombinant vector is a recombinant plasmid obtained by inserting the nucleic acid molecule as described in claim 5 into an expression vector or cloning vector; The recombinant microorganism is a recombinant bacterium obtained by introducing a recombinant vector into the starting microorganism.

8. The application of the mogroside glycosyltransferase mutant according to any one of claims 1-4 in the production of symbioside I and its upstream and downstream products, characterized in that, The upstream and downstream products of the symbioside I are MV, MIIIE and MIII.

9. The application of the nucleic acid molecule as described in claim 5 in the production of sympathoside I and its upstream and downstream products, characterized in that, The upstream and downstream products of the symbioside I are MV, MIIIE and MIII.

10. The application of the expression cassette, recombinant vector, or recombinant microorganism as described in claim 6 or 7 in the production of symbioside I and its upstream and downstream products, characterized in that, The upstream and downstream products of the symbioside I are MV, MIIIE and MIII.

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