A BS-YjiC mutant for transforming and generating ginsenosides and its application

Abstract

The present invention relates to the field of genetic engineering technology, and specifically to a BS-YjiC mutant for converting and generating ginsenosides and its application. The BS-YjiC mutant is obtained by mutation of one or more sites of wild-type glycosyltransferase BS-YjiC, including K125I / A322C, P313W / A322C, K125I / D142I / N178I / and K125I / N178I / Q189N. The glycosyltransferase BS-YjiC mutant of the present invention is more suitable for catalyzing protopanaxadiol (PPD) to generate rare ginsenosides F12 and Rh2 than the wild-type, and is more conducive to the flexibility of the production process.

Description

Technical Field

[0001] The invention relates to the technical field of genetic engineering, and in particular to a BS-YjiC mutant for transforming and generating ginsenosides and an application thereof. Background Art

[0002] Ginsenosides are an important class of active ingredients in precious Chinese medicinal materials such as ginseng, Panax notoginseng, and American ginseng. At least 289 ginsenosides have been identified. Ginsenosides have diverse structures and generally have activities such as improving the cardiovascular and cerebrovascular, immune system, nervous system, and anti-tumor effects of the human body. According to the different skeletons of ginsenoside aglycones, ginsenosides can be divided into two major categories: oleanane type and dammarane type. Among them, dammarane type ginsenosides are mainly composed of protopanaxadiol (PPD), protopanaxatriol (PPT) and pseudo-ginsenosides as aglycones.

[0003] Glycosylation is an essential modification reaction in the biosynthesis of natural products. It can enhance substrate solubility, bioavailability, stability and biological activity by forming a variety of natural glucosides. The physiological functions of ginsenosides are closely related to their glycosylation sites. Natural ginsenosides are not easy to absorb and need to be degraded and deglycosylated before they can be absorbed and utilized by the human body; while rare ginsenosides with less glycosides have more prominent pharmacological activity and bioavailability. Bs-YjiC from Bacillus subtilis 168 is a promiscuous and powerful UGT that can not only efficiently catalyze the C3-OH and C12-OH of PPD to synthesize rare ginsenoside Rh2 and unnatural ginsenoside F12 (published in: Longhai D, Can L, Jiao L, et al. One-Pot Synthesis of Ginsenoside Rh2 and Bioactive Unnatural Ginsenoside by Coupling Promiscuous Glycosyltransferase from Bacillus subtilis 168 to Sucrose Synthase. [J]. Journal of agricultural and food chemistry, 2018, 66(11): 2830-2837.), but can also glycosylate at the C3-OH, C6-OH and C12-OH sites of PPT to produce ginsenoside Rh1 and four unnatural ginsenosides. The traditional extraction method of ginsenosides cannot meet the needs of new drug development because ginseng plants have a long growth cycle and are easily affected by environmental factors such as climate, soil and rainfall. In addition, the saponin content in ginseng plants is low and the extraction method is complicated.

[0004] Therefore, the glycosyltransferase BS-YjiC conversion method is an effective method for industrial production of rare ginsenosides and has good application prospects. However, Bs-YjiC has certain defects, such as low stability, low thermal stability, and low relative enzyme activity. Summary of the invention

[0005] In order to improve the stability, thermal stability and relative enzyme activity of glycosyltransferase, the present invention provides a BS-YjiC mutant for converting and generating ginsenosides and its application. The mutant provided by the present invention is more suitable for catalyzing protopanaxadiol PPD to generate rare natural ginsenoside Rh2 and non-natural ginsenosides F12 and Rh2 than the wild type, and is more conducive to the flexibility of the production process.

[0006] To achieve the above object, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides a glycosyltransferase BS-YjiC mutant, wherein the glycosyltransferase BS-YjiC mutant is any one of the following methods:

[0008] A) The proline at position 313 of the glycosyltransferase BS-YjiC was mutated to tryptophan, and the alanine at position 322 was mutated to cysteine;

[0009] B) mutating the lysine at position 125 of the glycosyltransferase BS-YjiC to isoleucine to obtain a K125I mutant, and further performing amino acid mutations at one or more sites on the K125I mutant, including:

[0010] B1) mutating the alanine at position 322 of the K125I mutant to cysteine;

[0011] B2) the aspartic acid at position 142 of the K125I mutant is mutated to isoleucine, and the asparagine at position 178 is mutated to isoleucine;

[0012] B3) the asparagine at position 178 of the K125I mutant was mutated to isoleucine, and the glutamine at position 189 was mutated to asparagine;

[0013] The amino acid sequence of the glycosyltransferase BS-YjiC is shown in SEQ ID NO.1.

[0014] In a second aspect, the present invention provides the use of the glycosyltransferase BS-YjiC mutant in the preparation of ginsenosides.

[0015] Furthermore, the application is to use the glycosyltransferase BS-YjiC mutant to catalyze protopanaxadiol to produce non-natural ginsenoside F12 and natural ginsenoside Rh2.

[0016] In a third aspect, the present invention provides a gene encoding the glycosyltransferase BS-YjiC mutant.

[0017] In a fourth aspect, the present invention provides a recombinant expression vector carrying the encoding gene.

[0018] In a specific embodiment of the present invention, the recombinant expression vector uses pET-28a(+) vector as the original expression vector.

[0019] In a fifth aspect, the present invention provides a genetically engineered bacterium transformed by the recombinant expression vector.

[0020] Furthermore, the genetically engineered bacteria uses Escherichia coli as a host.

[0021] Furthermore, the Escherichia coli includes BL21 (DE3).

[0022] In a sixth aspect, the present invention provides the use of the encoding gene, the recombinant expression vector or the genetically engineered bacteria in the preparation of ginsenosides.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] 1. Based on the glycosyltransferase BS-YjiC variants K125I / N178I and P313W (disclosed in Chinese patent application No. 202310299778.6), the present invention transforms the molecular structure of glycosyltransferase BS-YjiC through rational design and combined with site-directed mutagenesis biotechnology, analyzes the influence of the mutated residues on the thermal stability of the enzyme, and finally obtains a combined mutant strain with improved stability (mutant strains K125I / A322C, P313W / A322C, K125I / D142I / N178I / and K125I / N178I / Q189N).

[0025] 2. The half-life of the natural glycosyltransferase BS-YjiC is 3.12 h. The half-life of the glycosyltransferase BS-YjiC mutant K125I / D142I / N178I / provided by the present invention reaches 20 h, the half-life of the glycosyltransferase BS-YjiC mutant K125I / N178I / Q189N reaches 10 h, the half-life of the glycosyltransferase BS-YjiC mutant P313W / A322C reaches 4.16 h, and the half-life of the glycosyltransferase BS-YjiC mutant K125I / A322C reaches 10.8 h.

[0026] 3. The glycosyltransferase BS-YjiC mutant provided by the present invention has significantly improved thermal stability while the enzyme activity is not affected. Among them, after heat treatment at 45°C for 90 minutes, the mutants K125I / D142I / N178I, K125I / A322C, P313W / A322C and K125I / N178I / Q189N retained 99.6%, 89.6%, 85.9% and 94.8% of the relative enzyme activity, respectively, while the control group retained 84.47% of the relative enzyme activity.

[0027] 4. The glycosyltransferase BS-YjiC mutant obtained in the present invention is more suitable for catalyzing the synthesis of rare ginsenoside Rh2 from protopanaxadiol PPD than the wild type, and is more conducive to the flexibility of the production process. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The pure enzyme solution was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Bands 1 to 3 were unpurified WT enzyme solution, purified WT enzyme solution, and purified K125I / D142I / N178I enzyme solution, respectively.

[0029] Figure 2 These are the test results of enzyme activity changes of wild-type glycosyltransferase BS-YjiC and glycosyltransferase BS-YjiC mutants K125I / A322C, P313W / A322C, K125I / D142I / N178I / and K125I / N178I / Q189N after incubation at 45°C for different times. DETAILED DESCRIPTION

[0030] The present invention is described in detail below in conjunction with the accompanying drawings and specific examples, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0031] In the following examples, the culture medium and formulation involved are as follows:

[0032] 1. LB liquid medium: 10 g / L peptone, 5 g / L yeast powder, 10 g / L NaCl.

[0033] 2. LB solid medium: Add 2% agar to LB liquid medium.

[0034] The detection methods involved in the following embodiments are as follows:

[0035] 1. Glycosyltransferase BS-YjiC enzyme activity assay method: Take 50 μL of pure enzyme with a concentration of 1.5 mg / mL and add it to a solution containing 1 mM PPD, 5 mM UDPG, 8 mM MgCl 2 The reaction mixture was added to 950 μL of a 50 mM Tris-HCl (pH 8.0) buffer solution; the reaction was carried out in a 35°C water bath for 30 min, and then an equal volume of methanol was added to terminate the enzymatic reaction. The supernatant was collected by centrifugation and filtered with a 0.22 μm filter, and the residual amount of protopanaxadiol PPD in the reaction solution was detected by HPLC.

[0036] 2. Definition of enzyme activity: The amount of enzyme required to convert 1 μmol of ginsenoside PPD per minute under the conditions of 35°C and pH 8.0 is defined as one enzyme activity unit U.

[0037] Example 1: Construction of a recombinant plasmid containing a glycosyltransferase BS-YjiC mutant

[0038] (1) Construction of a recombinant plasmid containing wild-type glycosyltransferase BS-YjiC

[0039] The gene sequence of wild-type glycosyltransferase BS-YjiC (amino acid sequence as shown in SEQ ID NO.1) with a nucleotide sequence as shown in SEQ ID NO.2 was chemically synthesized, and the pET-28a(+) vector was digested with BamH I and Sal I enzymes and ligated to prepare the recombinant vector pET-28a(+)-BS-YjiC.

[0040] (2) Obtaining recombinant vectors containing mutants

[0041] The whole plasmid PCR technique was used to perform site-directed mutagenesis using the recombinant vector pET-28a(+)-BS-YjiC prepared in step (1) as a template to obtain the following recombinant plasmid containing the mutant gene:

[0042] pET-28a(+)-YjiC-K125I / A322C;

[0043] pET-28a(+)-YjiC-P313W / A322C;

[0044] pET-28a(+)-YjiC-K125I / D142I / N178I;

[0045] pET-28a(+)-YjiC-K125I / N178I / Q189.

[0046] The designed primer sequences are as follows:

[0047] Table 1 Mutation primers

[0048]

[0049] The PCR amplification program was set as follows: first, pre-denaturation at 95°C for 5 min; then 30 cycles of denaturation at 95°C for 30 s, annealing at 72°C for 40 s, extension at 58°C for 3.5 min, and insulation at 4°C. The PCR product was detected by 0.8% agarose gel electrophoresis;

[0050] The recombinant plasmid was treated with Dpn I enzyme in a 37°C water bath for 1 h to remove the template, and then the PCR mixture was transformed into E. coli BL21 competent cells. The transformation solution was spread on LB solid culture medium containing kanamycin (50 μg / mL), and the plasmid was extracted and sequenced. The sequencing work was completed by Shanghai Bioengineering.

[0051] SEQ ID NO.1: MKKYHISMINIPAYGHVNPTLALVEKLCEKGHRVTYATT EEFAPAVQQ AGGEALIYHTSLNIDPKQIREMMEKNDAPLSLLKESLSILPQLEELYKDDQPDLIIYDFVALAGKLFAEKLNVPVIKLCSSYAQNESFQLGNEDML KKIREAEAEFKAYLEQEKLPAVSFEQLAVPEALNIVFMPKSFQIQHETFDDRFCFVGPSLGERKEKESLLIDKDDRPLMLISLGTAFNAWPEFYKMCIKAFRDSSWQVIMSVGKTIDPESLEDI PANFTIRQSVPQLEVLEKADLFISHGGMNSTMEAMNAGVPLVVIPQMYEQELTANRVDELGLGVYLPKEEVTVSSLQEAVQAVSSDQELLSRVKNMQKDVKEAGGAERAAAEIEAFMKKSAVPQ.

[0052]

[0053] Example 2: Expression, Isolation and Purification of Glycosyltransferase BS-YjiC Mutant

[0054] (1) The recombinant plasmids obtained in Example 1 were transformed into E. coli BL21 (DE3) competent cells to prepare genetically engineered bacteria:

[0055] E.coli / pET-28a(+)-YjiC-P313W / A322C;

[0056] E.coli / pET-28a(+)-YjiC-K125I / A322C;

[0057] E.coli / pET-28a(+)-YjiC-K125I / N178I / Q189N;

[0058] E. coli / pET-28a(+)-YjiC-K125I / D142I / N178I.

[0059] (2) inoculating the genetically engineered bacteria prepared in step (1) into 10 mL of LB liquid culture medium containing 50 μg / mL kanamycin, and culturing overnight at 37° C. and 200 rpm to prepare a seed solution;

[0060] The prepared seed solution was transferred to 100 mL of LB liquid medium containing 50 μg / mL kanamycin at an inoculum volume of 2% (v / v), and cultured at 37°C, 200 rpm until OD600 was 0.6-0.9, and IPTG was added at a final concentration of 1 mM, and cultured at 16°C for 20 hours to obtain a fermentation liquid; the prepared fermentation liquid was centrifuged at 12,000 rpm and 4°C for 15 minutes to obtain cell bodies, and the cells were washed 3 times and resuspended with 10 mL of 50 mM Tris-HCl buffer (pH 8.0). The resuspended cells were treated with an ultrasonic disruptor under ice bath conditions for 30 minutes, centrifuged for 30 minutes (8000×g, 4°C), and the supernatant was taken to obtain a crude enzyme solution;

[0061] The supernatant fraction was filtered through a 0.22 μm filter and then further loaded onto a 1 mL Ni affinity column, which was pre-equilibrated with 50 mM wash buffer (20 mM Tris and 250 mM NaCl, pH 8.0), and then the unbound proteins and glycosyltransferase BS-YjiC were eluted with an elution buffer (20 mM Tris, 250 mM NaCl and 500 mM imidazole, pH 8.0) by linear gradient elution; pure enzyme solutions containing wild-type YjiC and pure enzyme solutions containing mutants were prepared, respectively;

[0062] The above pure enzyme solutions were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Figure 1 The results showed that there was an obvious band near 50 kDa, proving that the glycosyltransferase BS-YjiC mutant was expressed.

[0063] In order to test the effect of site-directed mutagenesis on thermal stability, the prepared pure enzyme was subjected to a thermal stability test for preliminary screening as follows:

[0064] The pure enzyme solutions containing wild-type YjiC, P313W / A322C, K125I / A322C, K125I / N178I / Q189N, and K125I / D142I / N178I prepared above were tested respectively, and the results are shown in Table 1.

[0065] After the pure enzymes prepared above were incubated in a 45°C water bath for 90 minutes, 1 mL was taken and the residual enzyme activity of the remaining enzyme was determined according to the glycosyltransferase BS-YjiC enzyme activity determination method. The enzyme activity of the pure enzyme solution that had not been treated at high temperature was used as a blank control to obtain the percentage of residual enzyme activity. The test results are shown in Table 2.

[0066] Table 1 Residual enzyme activities of different enzymes

[0067]

[0068]

[0069] As can be seen from Table 1, the mutants P313W / A322C, K125I / A322C, K125I / N178I / Q189N and K125I / D142I / N178I retained 86%, 89.6%, 94.8% and 99.6% of the relative enzyme activities, respectively, while the wild type and other mutants only retained about 84.5% of the relative enzyme activities; the thermal stability of all mutants was higher than that of the wild type.

[0070] Example 3: Enzymatic property test of glycosyltransferase BS-YjiC mutant

[0071] (1) Thermal stability

[0072] The pure enzyme solution containing wild-type YjiC and the pure enzyme solution containing different mutants prepared in step (2) of Example 2 were placed in a constant temperature water bath at 45°C, and samples were taken every 30 minutes. The residual enzyme activity was measured according to the glycosyltransferase BS-YjiC enzyme activity assay method, and the thermal stability was compared. The test results are shown in FIG. Figure 2 shown.

[0073] Depend on Figure 2It can be seen that the activity of the enzyme decreases with time, and the activity of the triple-mutated enzyme is higher than that of the non-mutated enzyme.

[0074] The half-life of the natural glycosyltransferase BS-YjiC is 3.12 hours, the half-life of the glycosyltransferase BS-YjiC mutant K125I / D142I / N178I / provided by the present invention reaches 20 hours, the half-life of the glycosyltransferase BS-YjiC mutant K125I / N178I / Q189N reaches 10 hours, the half-life of the glycosyltransferase BS-YjiC mutant P313W / A322C reaches 4.16 hours, and the half-life of the glycosyltransferase BS-YjiC mutant K125I / A322C reaches 10.8 hours. Figure 2 It can be seen that the half-life of the triple mutant is much higher than that of the unmutated one.

[0075] 2. Kinetic parameters

[0076] 2 μg of pure enzyme was added to a solution containing 10 mM UDPG, 10 mM MgCl 2 To a 50mM Tris-HCl (pH 8.0) buffer, 50, 100, 200, 300, 400, 500, 600, 800 and 1000 μM protopanaxadiol (PPD) were added respectively, and the reaction was carried out in a 35°C water bath for 30 min. Then, an equal volume of methanol was added to terminate the enzymatic reaction. The supernatant was collected by centrifugation and filtered with a 0.22 μm filter. The remaining amount of protopanaxadiol PPD in the reaction solution was detected by HPLC.

[0077] Table 3 Kinetic parameters

[0078] Enzymes <![CDATA[K m (mM)]]> <![CDATA[V max (μM min -1 )]]> <![CDATA[K cat (s -1 )]]> <![CDATA[k cat / K m (s -1 M -1 )]]> WT 1.3765±0.34 51.095±8.335 14.16.±2.34 <![CDATA[1.028×10 4 ]]> P313W / A322C 1.3525±0.32 53.095±6.42 15.49±1.68 <![CDATA[1.145×10 4 ]]> A322C / K125I 1.3715±0.35 55.345±8.627 15.37±2.4 <![CDATA[1.121×10 4 ]]> K125I / N178I / Q189N 1.3401±0.26 54.92±9.3 15.265±2.533 <![CDATA[1.139×10 4 ]]> D142I / K125I / N178I 1.325±0.29 52.45±7.84 14.56±2.18 <![CDATA[1.09×10 4 ]]>

[0079] The results are shown in Table 3. The Km of the mutants for ginsenoside PPD was lower than that of the wild type, indicating that the affinity of the mutants for the substrate was increased compared with the wild type, and the mutant k cat / K m Both are higher than before mutation, which also shows that the catalytic efficiency of the mutant is improved compared with that before mutation.

[0080] It should be noted that when the claims of the present invention involve numerical ranges, it should be understood that the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. In order to avoid redundancy, the present invention describes a preferred embodiment.

[0081] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

Claims

1. A glycosyltransferase BS-YjiC mutant, characterized in that: The glycosyltransferase BS-YjiC mutant is obtained by mutating the lysine at position 125 of the glycosyltransferase BS-YjiC to isoleucine, the aspartic acid at position 142 to isoleucine, and the asparagine at position 178 to isoleucine; The amino acid sequence of the glycosyltransferase BS-YjiC is shown in SEQ ID NO.

1.

2. The use of the glycosyltransferase BS-YjiC mutant according to claim 1 in the preparation of ginsenosides, characterized in that: The application is to utilize the glycosyltransferase BS-YjiC mutant to catalyze protopanaxadiol to generate non-natural ginsenoside F12 and natural ginsenoside Rh2.

3. A gene encoding the glycosyltransferase BS-YjiC mutant according to claim 1.

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

5. The recombinant expression vector according to claim 4, characterized in that The recombinant expression vector uses pET-28a (+) vector as the original expression vector.

6. A genetically engineered bacterium transformed by the recombinant expression vector according to claim 5.

7. The genetically engineered bacterium according to claim 6, characterized in that The genetically engineered bacteria uses Escherichia coli as a host.

8. The genetically engineered bacterium according to claim 7, characterized in that: The E. coli was BL21 (DE3).

9. Use of the coding gene according to claim 3, the recombinant expression vector according to claim 4 or the genetically engineered bacteria according to claim 6 in the preparation of ginsenosides, characterized in that: The ginsenosides are non-natural ginsenoside F12 and natural ginsenoside Rh2.

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