Rhamnosyltransferase mutant as well as coding gene, vector, recombinant bacterium and application thereof

Abstract

The invention discloses a rhamnosyl transferase mutant as well as a coding gene, a carrier, a recombinant bacterium and application thereof, which are characterized in that rhamnosyl transferase from Fagopyrumesculentum is mutated, 17-site Tyr is mutated into Asp, 35-site Ser is mutated into Phe, 74-site Gly is mutated into His and 78-site Gly is mutated into Ala so as to construct a quadruple mutant M4, so that rutin can be catalytically synthesized by glycosylation by taking isoquercetin as a substrate, and the coding gene, the carrier, the recombinant bacterium and the application of the rhamnosyl transferase mutant are obtained. The rutin is expected to be synthesized by using E.coli, and theoretical and technical bases are provided for establishing an industrial rutin production process which is low in production cost, high in production intensity and small in environmental pollution.

Description

Technical Field

[0001] The present invention belongs to the technical field of enzyme engineering, and specifically relates to a rhamnosyltransferase mutant, its coding gene, vector, recombinant bacterium and application. Background Art

[0002] Rutin, with the molecular formula C 14 H 18 N2O2, is a flavonoid compound widely distributed in plants. Rutin has various pharmacological activities: antioxidant, anti-inflammatory, anti-microbial, antibacterial, antiviral, anti-diabetic and anti-cancer effects. Rutin is classified as vitamin P, has multiple cellular targets, can increase the toughness of capillaries, regulate their permeability, and has lipid-lowering, cytoprotective, anti-spasmodic effects and the effects of preventing and treating cardiovascular diseases. Rutin is used in food in various forms, such as colorants, antioxidants, preservatives, stabilizers and ultraviolet absorbers. It is also used as an active ingredient in pharmaceuticals, multivitamin preparations, cosmetics and the chemical industry, as well as animal feed. Because rutin has wide biological activities, a high safety factor and low cost, it is applied in the healthcare system. Currently, rutin tablets are often used in China: mainly for capillary bleeding with increased fragility, and also for the adjuvant treatment of hypertensive encephalopathy, cerebral hemorrhage, retinal hemorrhage, hemorrhagic purpura, acute hemorrhagic nephritis, recurrent epistaxis, traumatic pulmonary hemorrhage and postpartum hemorrhage, etc.; compound rutin tablets (the main components are rutin and vitamin C): mainly for the adjuvant treatment of capillary bleeding, cerebral hemorrhage, purpura, hemorrhagic nephritis, retinal hemorrhage, traumatic pulmonary hemorrhage and postpartum hemorrhage, etc. Therefore, rutin has great market potential.

[0003] The process of synthesizing rutin from isoquercetin requires the participation of rhamnosyltransferase using UDP-rhamnose (Uridine diphosphaterhamnose, UDP-Rha) as a cofactor in the catalytic reaction. Among them, UDP-rhamnose is the glycosyl donor and isoquercetin is the glycosyl acceptor. Therefore, to achieve the synthesis of rutin using isoquercetin as a substrate, it is crucial to construct a rhamnosyltransferase that can efficiently catalyze the synthesis of rutin from isoquercetin. Yin et al. analyzed the gene structures of 106 UGTs based on the tartary buckwheat genome and screened 21 candidate FtUGTs for enzymatic assays. The research results showed that FtUGT73BE5 among them was identified to participate in the synthesis of rutin in vitro and in vivo. Xu et al. studied the GGT genes in the transcriptome-metabolome related network and confirmed that FtUGT79A15 has the rhamnosyltransferase activity to catalyze the formation of rutin from isoquercetin both in vitro and in plants. Therefore, FtUGT79A15 is involved in the biosynthetic pathway of flavonoid diglycosides in tartary buckwheat. Zou et al. identified two highly efficient glycosyltransferases from japonica rice. Sj3GT can regioselectively convert quercetin into isoquercetin, and Sj6”RhaT can catalyze the 6”-O-rhamnosylation of isoquercetin to produce rutin. In recent years, the key enzymes in rutin biosynthesis have been mined, identified, and characterized, but the specific catalytic efficiency is still unknown, and the conversion rate of rhamnosyltransferase for isoquercetin is worthy of study.

[0004] Currently, the conventional production method of rutin is plant extraction. However, the plant extraction method has problems such as cumbersome extraction steps, low production efficiency, and large environmental pollution. Given the advantages of less microbial contamination, low cost, fast reproduction, etc. and the ability to improve its catalytic efficiency by site-directed mutagenesis of key enzymes, the purpose of this study is to transform and construct the best rhamnosyltransferase FeF3G6”RhaT mutant to improve the ability of this mutant to catalyze the synthesis of rutin from isoquercetin, so as to achieve the goal of synthesizing rutin in vitro using microorganisms. Summary of the Invention

[0005] The purpose of this part is to outline some aspects of the embodiments of the present invention and briefly introduce some preferred embodiments. Simplifications or omissions may be made in this part, as well as in the abstract and title of the specification of this application, to avoid obscuring the purpose of this part, the abstract, and the title of the invention, and such simplifications or omissions shall not be used to limit the scope of the present invention.

[0006] In view of the above and / or problems existing in the prior art, the present invention is proposed.

[0007] To solve the above technical problems, the present invention provides the following technical solution: A rhamnosyltransferase mutant, characterized in that: the rhamnosyltransferase mutant is FeF3G6”RhaT 17D / 35F / G74H / G78A, it is obtained by mutating Tyr at position 17, Ser at position 35, Gly at position 74, and Gly at position 78 of the parental sequence with the amino acid sequence shown in SEQ ID NO.1 to Asp, Phe, His, and Ala, respectively.

[0008] As a preferred embodiment of the rhamnosyltransferase mutant of the present invention, wherein: the amino acid sequence of the rhamnosyltransferase mutant is shown in SEQ ID NO.3, and the nucleotide sequence is shown in SEQ ID NO.4.

[0009] Another object of the present invention is to overcome the deficiencies in the prior art and provide a coding gene containing the rhamnosyltransferase mutant.

[0010] Another object of the present invention is to overcome the deficiencies in the prior art and provide an expression vector carrying the coding gene.

[0011] As a preferred embodiment of the expression vector of the present invention, wherein: the expression vector is a bacterial plasmid, phage, yeast plasmid, plant cell virus, or mammalian cell virus.

[0012] Another object of the present invention is to overcome the deficiencies in the prior art and provide a recombinant bacterium expressing the rhamnosyltransferase mutant.

[0013] As a preferred embodiment of the recombinant bacterium of the present invention, wherein: the recombinant bacterium uses bacteria, fungi, plants, insects, or animal cells as host cells.

[0014] Another object of the present invention is to overcome the deficiencies in the prior art and provide an application of the rhamnosyltransferase mutant in catalyzing the synthesis of rutin using isoquercetin as a substrate.

[0015] Another object of the present invention is to overcome the deficiencies in the prior art and provide an enzyme preparation of the rhamnosyltransferase mutant.

[0016] As a preferred embodiment of the enzyme preparation of the present invention, wherein: the enzyme preparation is a solid enzyme preparation or a liquid enzyme preparation.

[0017] Advantages of the present invention:

[0018] The present invention mutates the rhamnosyltransferase FeF3G6”RhaT from Fagopyrum esculentum, mutating Tyr at position 17 to Asp, Ser at position 35 to Phe, Gly at position 74 to His, and Gly at position 78 to Ala, enabling it to use isoquercetin as a substrate and significantly improving the catalytic efficiency to efficiently synthesize rutin. This may be promising for the in vitro synthesis of L-erythraline using E. coli, providing a theoretical and technical basis for establishing an industrial process for rutin production with low production costs, high production intensity, and low environmental pollution. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings. Among them:

[0020] Figure 1 For the construction and expression of the FeF3G6”RhaT plasmid in the embodiments of the present invention, in which the M lane is the protein molecular weight standard Marker; the 1st lane is BL21(DE3)pET-28a; the 2nd lane is BL21(DE3)pET-FeRhaT.

[0021] Figure 2 For the enzyme activity and isoquercetin conversion rate of FeF3G6”RhaT and its mutants in the embodiments of the present invention.

[0022] Figure 3 For the structural comparison of the rhamnosyltransferase FeF3G6”RhaT and its mutants in the embodiments of the present invention. (Among them, WT is the wild type of FeF3G6”RhaT, M2 is FeF3G6”RhaT G74H / G78A , M3 is FeF3G6”RhaT 17D / G74H / G78A , M4 is FeF3G6”RhaT 17D / 35F / G74H / G78A .) DETAILED DESCRIPTION OF THE EMBODIMENTS

[0023] To make the above objects, features, and advantages of the present invention more obvious and understandable, the following will provide a detailed description of the specific embodiments of the present invention in combination with the embodiments of the specification.

[0024] In the following description, many specific details are set forth to fully understand the present invention. However, the present invention can also be implemented in other ways different from those described herein. Those skilled in the art can make similar extensions without departing from the connotation of the present invention. Therefore, the present invention is not limited by the specific embodiments disclosed below.

[0025] Secondly, as used herein, "one embodiment" or "an embodiment" refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The appearances of "in one embodiment" in different places in this specification do not all refer to the same embodiment, nor are they separate or alternative embodiments that are mutually exclusive of other embodiments.

[0026] Unless otherwise specified, the raw materials used in the embodiments of the present invention are all commercially available. The starting bacterium Escherichia coli BL21(DE3) belongs to wild-type Escherichia coli.

[0027] Qualitative and quantitative analysis of substrates and products and monitoring of the growth of bacteria: Determination of bacterial liquid concentration: Absorb the sample bacterial liquid, dilute it by a certain multiple with distilled water, use distilled water as a blank control, and measure the OD at a wavelength of 600 nm and a light path of 1 cm using a spectrophotometer. 600 . The contents of isoquercetin and rutin were determined by high performance liquid chromatography (HPLC), and their contents were determined with reference to the standard curve. The crude enzyme solution was purified by an affinity chromatography column according to the His tag on pET-28a.

[0028] Example 1

[0029] Construction of the expression plasmid pET-FeRhaT:

[0030] The rhamnosyltransferase FeF3G6”RhaT encoding gene FeRhaT derived from Fagopyrum esculentum was synthesized by Suzhou Genewiz Biotechnology Co., Ltd., and its sequence was codon-optimized. It was ligated onto the pET-28a plasmid through the restriction enzyme sites BamHⅠ and HindⅢ.

[0031] Example 2

[0032] Transformation of the plasmid into Escherichia coli BL21(DE3) for expression:

[0033] The recombinant expression plasmid pET-FeRhaT was transformed into Escherichia coli BL21(DE3), and recombinant expression strains were screened by culturing on LB + Kan solid medium under the culture condition of 37 °C.

[0034] The starting strain and the recombinant strain were inoculated into liquid TB medium. After induction with IPTG, the bacteria were collected and disrupted by ultrasonic waves. Then, the supernatant and the precipitate were subjected to SDS-PAGE electrophoresis, and a specific band with a molecular weight of about 52.9 kDa ( Figure 1 ) was detected, which was consistent with the reported size of the target protein, indicating that FeRhaT could be correctly expressed in Escherichia coli BL21(DE3).

[0035] Example 3

[0036] Site-directed mutagenesis of the FeRhaT-encoded protein FeF3G6”RhaT:

[0037] Using the expression plasmid pET-FeRhaT as a template, PCR amplifications were performed successively with G74H-F / G74H-R, G78A-F / G78A-R, Y17NNK-F / Y17NNK-R, and S35NNK-F / S35NNK-R (primer sequences are shown in Table 1) as primers, and the plasmid template was removed with DpnI digestion enzyme. Subsequently, the PCR products were purified and transformed into competent Escherichia coli BL21(DE3) cells for spreading culture. The target recombinant strains were screened using the test primers (i.e., FeRhaT-F / FeRhaT-R; primer sequences are shown in Table 1) and sequenced to obtain the target recombinant plasmid pET-FeRhaT Y17D / S35F / G74H / G78A and the target recombinant strain Escherichia coli BL21(DE3) / pET-FeRhaT Y17D / S35F / G74H / G78A .

[0038] Table 1 Primer sequences required for PCR amplification

[0039]

[0040]

[0041]

[0042]

[0043]

[0044]

[0045] Example 4

[0046] Determination of the enzyme activities of wild-type FeF3G6”RhaT and mutants:

[0047] Cultivate the target recombinant strain E.coli / pET-FeRhaT and mutants in 10 mL LB liquid medium for 12 h, then take 1 mL and transfer it into 50 mL TB liquid medium, and culture at 37 °C until OD 600 is between 0.5 and 0.6, add IPTG with a final concentration of 0.1 mM, and induce expression at 25 °C for 20 h. After expression, collect the bacteria, wash them twice with PBS buffer, suspend the bacteria and control them at the same OD 600 , and then disrupt the bacteria with an ultrasonic disruptor, and centrifuge to take the supernatant to obtain the crude enzyme solution.

[0048] Enzyme activity assay reaction system: The 200 μL reaction solution contains 300 μmol·L -1Isoquercetin, 400 μmol·L -1 UDP-Rha, 50 mmol·L -1 Tris-HCl (pH 8.0) solution and 80 μg of pure enzyme were reacted at 30 °C for 2 h and monitored for 10 min under the condition of 350 nm. The enzyme activity unit (U) was defined as the amount of enzyme required to consume 1 μmol L -1 rutin per minute under the above reaction conditions. The enzyme activities of wild-type FeF3G6”RhaT and different FeF3G6”RhaT mutants are as Figure 2 shown. The amino acid sequence of FeF3G6”RhaT is shown in SEQ ID NO.1, and the DAN sequence is shown in SEQ ID NO.2. It was found from Figure 2 that wild-type FeF3G6”RhaT had a certain ability to catalyze the synthesis of rutin from isoquercetin. The quadruple mutant FeF3G6”RhaT constructed by mutating Tyr at position 17 to Asp, Ser at position 35 to Phe, Gly at position 74 to His, and Gly at position 78 to Ala 17D / 35F / G74H / G78A significantly improved the catalytic ability towards isoquercetin.

[0049] Example 5

[0050] Effect of different FeF3G6”RhaT on rutin synthesis:

[0051] Enzyme conversion system: 200 μL of the reaction solution contained 300 μmol·L -1 isoquercetin, 400 μmol·L -1 UDP-Rha, 50 mmol·L -1 Tris-HCl (pH 8.0) solution and 100 μg of pure enzyme, and reacted at 30 °C for 2 h. After the reaction, the rutin content was determined by high performance liquid chromatography (HPLC), and the results are as Figure 2 shown.

[0052] It can be seen from Figure 2 that the rutin yield was consistent with the enzyme activity level, that is, the FeF3G6”RhaT mutants with high enzyme activity also showed high rutin yields. In summary, mutant M4 (FeF3G6”RhaT 17D / 35F / G74H / G78A ) showed the highest enzyme activity (466.8 ± 4.2 U / mg) and isoquercetin conversion rate (91.2 ± 3.4%). Its amino acid sequence is shown in SEQ ID NO.3, and the DNA sequence is shown in SEQ ID NO.4.

[0053] Example 6

[0054] Purification of wild-type FeF3G6”RhaT and mutants and analysis of their kinetic parameters:

[0055] The crude enzyme solution obtained by expressing the above mutant enzyme was purified using an affinity chromatography column. Isoquercetin solutions with mass concentrations of 0.5 mg / mL, 1.0 mg / mL, 1.5 mg / mL, 2.0 mg / mL, 2.5 mg / mL, and 3.0 mg / mL and 100 mmol / L UDP-rha were respectively prepared, and 1 mg of the purified enzyme was added. According to the Lineweaver-Burk double reciprocal method for plotting, the catalytic constant (kcat), Michaelis constant (Km), and kcat / Km of FeF3G6”RhaT and its mutants with isoquercetin as the substrate could be calculated. It was found that mutating Tyr at position 17 to Asp, Ser at position 35 to Phe, Gly at position 74 to His, and Gly at position 78 to Ala could effectively improve the ability of rhamnosyltransferase to catalyze the synthesis of rutin from isoquercetin. The results are shown in Table 2. Figure 3 This is the structural comparison of the rhamnosyltransferase FeF3G6”RhaT and its mutants in the examples of the present invention. According to the above results, positions 17, 35, 74, and 78 are important substrate recognition sites of FeF3G6”RhaT and are highly related to the glycosylation of the substrate. Therefore, mutating these sites to other simple amino acids may change the enzyme cavity structure and have a great impact on improving the enzyme catalytic efficiency with isoquercetin as the substrate, which has a great impact on enzyme research and industrial production.

[0056] Table 2 Kinetic parameters of rhamnosyltransferase and its mutants in the glycosylation reaction

[0057]

[0058] Note: WT is the wild type of FeF3G6”RhaT, M2 is FeF3G6”RhaT G74H / G78A ,M3 is FeF3G6”RhaT 17D / G74H / G78A ,M4 is FeF3G6”RhaT 17D / 35F / G74H / G78A

[0059] It should be noted that the above examples are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the spirit and scope of the technical solutions of the present invention, and they should all be covered within the scope of the present invention.

[0060] SEQ ID NO.1

[0061] Amino acid sequence of the parent (FeF3G6”RhaT):

[0062] MGTQANTTDLHIAVFPYFAFGHINPFVHISNKLASHGIKISFFSAPGNIPRIKSSLSTSPLISIVPLTFPHVDGLPAGFESTADITPAIAELLKVALDKMQPQIRSLLTQLKPDVVFFDFAQNWIPSLASELGIKTVMFSVFSLISNSYLMTPARLSSDEIPTIEELKKPPQGYPNPDLSLKTFQAKDLLYPFRRFNGGPSALERNYAGIQGCDAIAYKSCHEMEGPYWSYFKKVIGKPIIMAGIPIPETSSSGDLDSNWATWLAKFPPKSVTLCSFGSETFLTDVQVQELALGLELTELPFLMVLSSNGFDQERLNKILPEGFLERVKDRGLIHIGWVPQQKIMAHENVGCYVNHAGFGSVIEAIVTDCQLVLLPFKGDQFLNSKLLSLDMKVGVEVNRRDEDGHFGKEDIFEAVRIVTVDGDKEPGKKIRGNLVKWKELLMNKEFEEKYVLELVKEVKALVGN

[0063] SEQ ID NO.2

[0064] DNA sequence of the parent (FeF3G6”RhaT):

[0065]

[0066] SEQ ID NO.3

[0067] Amino acid sequence of rhamnosyltransferase mutant M4:

[0068] MGTQANTTDLHIAVFPDFAFGHINPFVHISNKLAFHGIKISFFSAPGNIPRIKSSLSTSPLISIVPLTFPHVDHLPAAFESTADITPAIAELLKVALDKMQPQIRSLLTQLKPDVVFFDFAQNWIPSLASELGIKTVMFSVFSLISNSYLMTPARLSSDEIPTIEELKKPPQGYPNPDLSLKTFQAKDLLYPFRRFNGGPSALERNYAGIQGCDAIAYKSCHEMEGPYWSYFKKVIGKPIIMAGIPIPETSSSGDLDSNWATWLAKFPPKSVTLCSFGSETFLTDVQVQELALGLELTELPFLMVLSSNGFDQERLNKILPEGFLERVKDRGLIHIGWVPQQKIMAHENVGCYVNHAGFGSVIEAIVTDCQLVLLPFKGDQFLNSKLLSLDMKVGVEVNRRDEDGHFGKEDIFEAVRIVTVDGDKEPGKKIRGNLVKWKELLMNKEFEEKYVLELVKEVKALVGN

[0069] SEQ ID NO.4

[0070] DNA sequence of rhamnosyltransferase mutant M4:

[0071] ATGGGCACCCAAGCGAACACCACCGATCTGCATATTGCGGTGTTTCCGGATTTTGCGTTTGGCCATATTAACCCGTTTGTGCATATTAGCAACAAACTGGCGTTTCATGGCATTAAAATTAGCTTTTTTAGCGCGCCGGGCAACATTCCGCGCATTAAAAGCAGCCTGAGCACGAGCCCGCTGATTAGCATTGTGCCGCTGACCTTTCCGCATGTGGATcatCTGCCGGCGGCGTTTGAAAGCACCGCGGATATTACCCCGGCGATTGCGGAACTGCTGAAAGTGGCGCTGGATAAAATGCAGCCGCAGATTCGCAGCCTGCTGACGCAGCTGAAACCGGATGTGGTGTTTTTTGATTTTGCGCAGAACTGGATTCCGAGCCTGGCGAGCGAACTGGGCATTAAAACCGTGATGTTTAGCGTGTTTAGCCTGATTAGCAACAGCTATCTGATGACCCCGGCGCGCCTGAGCAGCGATGAAATTCCGACCATTGAAGAACTGAAAAAACCGCCGCAAGGCTATCCGAACCCGGATCTGAGCCTGAAAACCTTTCAAGCGAAAGATCTGCTGTATCCGTTTCGCCGCTTTAACGGCGGCCCGAGCGCGCTGGAACGCAACTATGCGGGCATTCAAGGCTGCGATGCGATTGCGTATAAAAGCTGCCATGAAATGGAAGGCCCGTATTGGAGCTATTTTAAAAAAGTGATTGGCAAACCGATTATTATGGCGGGCATTCCGATTCCGGAAACGAGCAGTAGCGGCGATCTGGATAGCAACTGGGCGACCTGGCTGGCGAAATTTCCGCCGAAAAGCGTGACCCTGTGCAGCTTTGGTAGTGAAACCTTTCTGACCGATGTGCAAGTGCAAGAACTGGCGCTGGGCCTGGAACTGACCGAACTGCCGTTTCTGATGGTGCTGAGCAGCAACGGCTTTGATCAAGAACGCCTGAACAAAATTCTGCCGGAAGGCTTTCTGGAACGCGTGAAAGATCGCGGCCTGATTCATATTGGCTGGGTGCCGCAGCAGAAAATTATGGCGCATGAAAACGTGGGCTGCTATGTGAACCATGCGGGCTTTGGCAGCGTGATTGAAGCGATTGTGACCGATTGTCAGCTGGTGCTGCTGCCGTTTAAAGGCGATCAGTTTCTGAACAGCAAACTGCTGAGCCTGGATATGAAAGTGGGCGTGGAAGTGAACCGCCGCGATGAAGATGGCCATTTTGGCAAAGAAGATATTTTTGAAGCGGTGCGCATTGTGACCGTGGATGGCGATAAAGAACCGGGCAAAAAAATTCGCGGCAACCTGGTGAAATGGAAAGAACTGCTGATGAACAAAGAATTTGAAGAAAAATATGTGCTGGAACTGGTGAAAGAAGTGAAAGCGCTGGTGGGCAACTAA

Claims

1. A rhamnosyltransferase mutant, characterized in that: The rhamnosyltransferase mutant is FeF3G6”RhaT 17D / 35F / G74H / G78A , which is obtained by mutating Tyr at position 17, Ser at position 35, Gly at position 74, and Gly at position 78 of the parental sequence with the amino acid sequence shown in SEQ ID NO.1 to Asp, Phe, His, and Ala, respectively.

2. The rhamnosyltransferase mutant according to claim 1, characterized in that: The amino acid sequence of the rhamnosyltransferase mutant is shown in SEQ ID NO.3, and the DNA sequence is shown in SEQ ID NO.

4.

3. The coding gene of the rhamnosyltransferase mutant according to claim 1.

4. An expression vector carrying the coding gene according to claim 3.

5. The expression vector according to claim 4, characterized in that, The vector is a bacterial plasmid, phage, yeast plasmid, plant cell virus or mammalian cell virus.

6. A recombinant bacterium expressing the rhamnosyltransferase mutant according to claim 1.

7. The recombinant bacterium according to claim 6, characterized in that, The recombinant bacterium uses a bacterium, fungus, plant, insect or animal cell as the host cell.

8. Use of the rhamnosyltransferase mutant according to claim 1 in the catalytic synthesis of rutin using isoquercetin as a substrate.

9. An enzyme preparation comprising the rhamnosyltransferase mutant according to claim 1.

10. The enzyme preparation according to claim 9, characterized in that, The enzyme preparation is a solid enzyme preparation or a liquid enzyme preparation.

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