QS-7 biosynthesis acetylation method

Abstract

The invention provides a QS-7 biosynthesis acetylation method which comprises the following steps: S1, cloning an SOAP10 coding gene to a prokaryotic expression vector, transforming the SOAP10 coding gene into a competent cell, and performing induced expression; s2, collecting competent cells subjected to induced expression, resuspending, ultrasonically crushing and centrifuging, and taking supernatant, so as to obtain SOAP10 escherichia coli crude enzyme; and S3, mixing the non-acetylated QS-7 and the SOAP10 escherichia coli crude enzyme solution for reaction, so as to complete acetylation modification of the QS-7. According to the method, the acetylation of the non-acetylated QS-7 is successfully realized by adopting the SOAP10 enzyme, the technical blank of in-vitro enzyme catalysis acetylation of the QS-7 is filled, and a key support is provided for in-vitro biosynthesis of the QS-7.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a method for the acetylation of QS-7 biosynthesis. Background Technology

[0002] QS-7 is a triterpenoid saponin derived from the saponin tree (Quillaja saponaria) with unique immunoadjuvant activity, anti-inflammatory and anti-tumor biological functions, making it highly valuable in vaccine development, pharmaceuticals, and biopharmaceuticals. Acetylation is a crucial step in QS-7 biosynthesis, directly affecting its biological activity, stability, and targeting. Therefore, achieving efficient QS-7 acetylation is one of the core technologies for its large-scale preparation. Current QS-7 biosynthesis relies on the traditionally hypothesized ACT1 protein for catalysis. ACT1 is considered a key enzyme mediating QS-7 acetylation, but there are currently no successful reports on in vitro enzymatic catalysis of QS-7 acetylation. Furthermore, the ACT1 protein suffers from severe expression and functional defects; its acetyltransferase activity cannot be detected using conventional expression systems such as E. coli, insect cells, or mammalian cells, hindering the in vitro biosynthesis of QS-7. Summary of the Invention

[0003] In view of this, the present invention provides a method for the acetylation of QS-7 biosynthesis, which successfully achieves the acetylation of non-acetylated QS-7 using the SOAP10 enzyme, filling the technical gap in the in vitro enzyme-catalyzed acetylation of QS-7 and providing key support for its in vitro biosynthesis.

[0004] To achieve the above objectives, the present invention adopts the following technical solution: This invention provides a method for the biosynthetic acetylation of QS-7, comprising the following steps: S1. The SOAP10 encoding gene was cloned into a prokaryotic expression vector, then transformed into competent cells and induced to express. S2. Collect the competent cells after induced expression, resuspend them, sonicate and centrifuge, take the supernatant to obtain SOAP10 E. coli crude enzyme solution; S3. Mix the non-acetylated QS-7 and SOAP10 E. coli crude enzyme solution to complete the acetylation modification of QS-7.

[0005] In the above technical solution, the amino acid sequence of the SOAP10 enzyme expressed by the SOAP10 encoding gene is based on the report in Nature Chemical Biology. GenBank accession information can be found in the corresponding sequence library. This enzyme possesses acetylation modification activity of triterpenoid saponins and can mediate the acetylation reaction of QS-7 precursor substrates (non-acetylated QS-7). It should be noted that the amino acid sequence of SOAP10 is: MGEVNHEEVEIEIISIETIKPSSLLPPKTPPKTITLSHLDQAAPLYYYPLLLYYTNTTTTTPTSQIRVDITSTLKTSLSKTLDKFHPIAGRCVDDSTICCNHQGIPFIETKVDSNILDVMNSPEKMKLLIKFLPHAEFQDVTRPVSDLNHLAFQVNVFRCGGVIIGSYVLHKLLDGISLGTFFKNWSTIANDERVKDDDLVQPDFEATIKAFPPRTATPMLPRNQQLPKAAEKPNNNPVKVLVTKS FVFDISVSLKKMMFMAKSELVPKPTKFETVTGFIWEQTLSTLRNSGVEVEHTSLIIPVNIRPRMSPPLPRGSMGNLLKNAKAQANTSSSNGLQDLVKEIHSSLSQTTQKINTPPPPPPPPPTTTATTIHSSLSQ TTQKINTPPPTTTTIHSSLSQTTQKINTTTTTAEVILTKRKVDNPVTQNREGNYLFTSWCKIGLDEADFGFGKPVWVIPNDGRPPKVRNMIFLTDYRHPETGVEGIAAWITLEEKQMQCLKSNPEFLAFATPN.

[0006] In some embodiments, the reaction formula for QS-7 biosynthetic acetylation is as follows:

[0007] In some embodiments, the acetylation reaction conditions for non-acetylated QS-7 are as follows: final concentration of the reaction system: 100mM Tris-HCl pH7.5, 5mM MgCl2, 1mM non-acetylated QS-7, SOAP10 Escherichia coli crude enzyme solution 3mg / mL, reaction temperature 30℃ for 4-6h.

[0008] Preferably, in step S1, the SOAP10 encoding gene is derived from spinach; and / or, In step S1, the competent cells are Escherichia coli BL21(DE3).

[0009] Preferably, in step S1, the specific steps for inducing expression include: inoculating the bacterial strain into a culture medium and then adding IPTG to induce expression.

[0010] Preferably, the culture medium is LB medium; and / or, After inoculation into the culture medium, the culture temperature was 37℃, and the culture was carried out until OD... 600 =0.6-0.8.

[0011] Preferably, the final concentration of IPTG is 0.2-1.0 mM; and / or, The induction temperature is 15-25℃, and the induction time is 16-24h.

[0012] Preferably, in step S2, resuspension is performed using a lysis buffer comprising 50 mM Tris-HCl pH 8.0, 10% glycerol, 0.1% Triton X-100, and 1 mM PMSF.

[0013] Preferably, in step S2, the centrifugation conditions are 12000 r / min for 10 min.

[0014] Preferably, in step S2, the final concentration of non-acetylated QS-7 is 0.1-1 mM, and the concentration of SOAP10 Escherichia coli crude enzyme solution is 1-5 mg / mL.

[0015] Preferably, in step S3, the mixing reaction is carried out at a temperature of 30°C for 4-6 hours.

[0016] In some embodiments, the reaction products are detected by high performance liquid chromatography (HPLC) or liquid chromatography-mass spectrometry (LC-MS / MS) and the formation of QS-7 acetylated products is confirmed by comparing the retention time and mass spectrometry characteristics with those of the standard; the acetylated products are separated and purified by column chromatography and other methods.

[0017] In some embodiments, the specific steps for mixing and reacting non-acetylated QS-7 and SOAP10 E. coli crude enzyme solution in step S3 are as follows: add non-acetylated QS-7 (final concentration 0.1-1.0 mM) and SOAP10 E. coli crude enzyme solution (protein concentration 1-5 mg / mL) to the reaction system, and make up the system volume to 50-200 μL with lysis buffer. Incubate at 30°C for 4-6 h to complete the acetylation modification of QS-7.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) Currently, the ACT1 protein, which is synthesized in vivo, shows no detectable acetyltransferase activity in various expression systems such as E. coli, insect cells, and mammalian cells, making it impossible to achieve in vitro acetylation of QS-7. The SOAP10 enzyme selected in this invention can be efficiently expressed in the E. coli BL21(DE3) system, and the crude enzyme solution can efficiently catalyze the acetylation of non-acetylated QS-7 with a high conversion rate, completely breaking through the technical bottleneck of ACT1 lack of enzyme activity. This fills the technical gap in the in vitro acetylation of triterpenoid saponins.

[0019] (2) Conventional acetylation reactions require the addition of exogenous acetyl-CoA (priced at approximately RMB 5,000-8,000 per gram), and its half-life in the reaction system is only 1-2 hours, requiring excessive addition (molar ratio ≥ 5:1), resulting in raw material costs accounting for 60-80%; This invention utilizes the acetyl-CoA generation system of Escherichia coli crude enzyme solution itself, eliminating the need for exogenous addition and significantly reducing costs.

[0020] (3) Conventional acetylation process requires purification of enzyme protein (purification steps ≥ 3 steps) and then additional addition of excess acetyl coenzyme A; the present invention directly uses crude enzyme solution for catalysis, reduces operation steps, has a reaction pH tolerance range of 7.0-8.5, a temperature tolerance range of 25-35℃, high process fault tolerance, and is suitable for industrial mass production.

[0021] (4) Currently, some acyltransferases need to be expressed in mammalian cells, and the culture cost is 20-30 times that of the E. coli system, and the expression period is as long as 7-10 days. The SOAP10 enzyme of the present invention has an expression period of only 16-24 hours in E. coli, with low culture cost. The crude enzyme solution can be used directly without purification, which greatly reduces the cost. Attached Figure Description

[0022] Figure 1 The HPLC detection results provided in Example 1 of this invention; Figure 2 The results are LC-MS / MS detection results provided in Embodiment 1 of the present invention. Detailed Implementation

[0023] The present invention will be further described in detail below with reference to specific embodiments, so that those skilled in the art can more clearly understand the present invention.

[0024] QS-7 is a triterpenoid saponin derived from the saponin tree (Quillaja saponaria) with unique immunoadjuvant activity, anti-inflammatory and anti-tumor biological functions, making it highly valuable in vaccine development, pharmaceuticals, and biopharmaceuticals. Acetylation is a crucial step in QS-7 biosynthesis, directly affecting its biological activity, stability, and targeting. Therefore, achieving efficient QS-7 acetylation is one of the core technologies for its large-scale preparation. Current QS-7 biosynthesis relies on the traditionally hypothesized ACT1 protein for catalysis. ACT1 is considered a key enzyme mediating QS-7 acetylation, but there are currently no successful reports on in vitro enzymatic catalysis of QS-7 acetylation. Furthermore, the ACT1 protein suffers from severe expression and functional defects; its acetyltransferase activity cannot be detected using conventional expression systems such as E. coli, insect cells, or mammalian cells, hindering the in vitro biosynthesis of QS-7.

[0025] To address the aforementioned technical problems, this invention provides a method for the biosynthetic acetylation of QS-7, comprising the following steps: S1. The SOAP10 encoding gene was cloned into a prokaryotic expression vector, then transformed into competent cells and induced to express. S2. Collect the competent cells after induced expression, resuspend them, sonicate and centrifuge, take the supernatant to obtain SOAP10 E. coli crude enzyme solution; S3. Mix the non-acetylated QS-7 and SOAP10 E. coli crude enzyme solution to complete the acetylation modification of QS-7.

[0026] In the above technical solution, the amino acid sequence of the SOAP10 enzyme expressed by the SOAP10 encoding gene is based on the literature reported in Nature Chemical Biology. The relevant information for GenBank accession can be found in the corresponding sequence library. This enzyme has acetylation modification activity of triterpenoid saponins and can mediate the acetylation reaction of QS-7 precursor substrate (non-acetylated QS-7).

[0027] The advantages of the above technical solution are: First, the first in vitro acetylation of QS-7 was achieved: replacing the inactive ACT1 protein, the SOAP10 enzyme was used to successfully acetylate the QS-7 precursor substrate, filling the technological gap in the in vitro enzyme-catalyzed acetylation of QS-7 and providing key support for its in vitro biosynthesis.

[0028] Secondly, SOAP10 enzyme can be functionally expressed in the E. coli system, and the crude enzyme solution can exhibit highly efficient acetylation activity, which solves the core problem of ACT1 having no enzyme activity in various expression systems.

[0029] Third, by utilizing the acetyl-CoA generation system of the crude enzyme solution of E. coli, there is no need to add expensive acetyl-CoA donors, which significantly reduces the cost of raw materials and provides an economical and feasible solution for the large-scale preparation of QS-7.

[0030] Fourth, the catalytic reaction directly uses crude E. coli enzyme solution, eliminating the need for protein purification steps. The reaction conditions are mild (room temperature, conventional buffer system), and the process is simple, making it suitable for industrial scale-up applications.

[0031] Fifth, the SOAP10 enzyme derived from spinach was applied to the QS-7 acetylation reaction for the first time, and the endogenous acetyl-CoA supply mechanism of Escherichia coli crude enzyme solution was discovered, providing a new technical approach for the acetylation modification of triterpenoid saponins.

[0032] In some embodiments, the reaction formula for QS-7 biosynthetic acetylation is as follows:

[0033] In some embodiments, the acetylation reaction conditions for non-acetylated QS-7 are as follows: final concentration of the reaction system: 100mM Tris-HCl pH7.5, 5mM MgCl2, 1mM non-acetylated QS-7, SOAP10 Escherichia coli crude enzyme solution 3mg / mL, and reaction temperature 30℃ for 4-6h.

[0034] Furthermore, in step S1, the SOAP10 encoding gene is derived from spinach; and / or, In step S1, the competent cells are Escherichia coli BL21(DE3).

[0035] Furthermore, in step S1, the specific steps for inducing expression include: inoculating the bacterial strain into the culture medium and then adding IPTG to induce expression.

[0036] Furthermore, the culture medium is LB medium; and / or, After inoculation into the culture medium, the culture temperature was 37℃, and the culture was carried out until OD... 600 =0.6-0.8.

[0037] Furthermore, the final concentration of IPTG is 0.2-1.0 mM; and / or, The induction temperature is 15-25℃, and the induction time is 16-24h.

[0038] Further, in step S2, resuspension is performed using a lysis buffer comprising 50 mM Tris-HCl pH 8.0, 10% glycerol, 0.1% Triton X-100, and 1 mM PMSF.

[0039] Furthermore, in step S2, the centrifugation conditions are 12000 r / min for 10 min.

[0040] Further, in step S2, the final concentration of non-acetylated QS-7 is 0.1-1 mM, and the concentration of SOAP10 E. coli crude enzyme solution is 1-5 mg / mL.

[0041] Furthermore, in step S3, the temperature of the mixing reaction is 30°C, and the time is 4-6 hours.

[0042] In some embodiments, the reaction products are detected by high performance liquid chromatography (HPLC) or liquid chromatography-mass spectrometry (LC-MS / MS) and the formation of QS-7 acetylated products is confirmed by comparing the retention time and mass spectrometry characteristics with those of the standard; the acetylated products are separated and purified by column chromatography and other methods.

[0043] Example This embodiment provides a method for QS-7 biosynthetic acetylation, including the following steps: 1) Construction of a prokaryotic expression vector for SOAP10 enzyme: The SOAP10 encoding gene was cloned into the prokaryotic expression vector pET-28b and transformed into Escherichia coli competent cells BL21(DE3).

[0044] 2) Induction of expression: The positive transformant bacteria were inoculated into LB medium and cultured at 37°C until OD500 was reached. 600 When the concentration was 0.7, IPTG was added to a final concentration of 0.8 mM, and expression was induced at 20°C for 24 h.

[0045] 3) Preparation of crude enzyme solution: Collect induced E. coli cells, resuspend them in lysis buffer (50mM Tris-HCl pH 8.0, 10% glycerol, 0.1% Triton X-100, 1mM PMSF), sonicate them, centrifuge at 12000r / min for 10min, and take the supernatant as SOAP10 E. coli crude enzyme solution, which can be used directly for catalytic reaction without purification.

[0046] 4) Add non-acetylated QS-7 to the reaction system at a final concentration of 1.0 mM, SOAP10 E. coli crude enzyme solution at a concentration of 3 mg / mL, 100 mM Tris-HCl at pH 7.5, and 5 mM MgCl2. Make up the volume of the system to 200 μL with lysis buffer and react at 30 °C for 6 h to complete the acetylation modification of QS-7.

[0047] 5) Product detection and separation: The reaction products were detected using high-performance liquid chromatography (HPLC) or liquid chromatography-mass spectrometry (LC-MS / MS). The formation of the QS-7 acetylated product was confirmed by comparing retention times and mass spectrometric characteristics with standards. The acetylated product was separated and purified using column chromatography and other methods. Results are as follows: Figure 1-2 As shown.

[0048] Figure 1 The data are from high-performance liquid chromatography (HPLC) detection. Peak 1 is QA-C3-GlcA-Xyl-Gal-C28-Fuc-Rha-Xyl (substrate). This curve is the control group curve and is used for substrate localization. Peak 2 is QA-C3-GlcA-Xyl-Gal-C28-Fuc-Rha-Xyl-Ac (product). This curve is the experimental group curve. There is no substrate peak on this curve. Compared with the experimental data of the control group, a new peak appears and is identified as the product peak.

[0049] Figure 2 The LC-MS / MS detection results show that the mass spectrometry response data 1422 is [MH]-, which is consistent with the target molecular mass of 1423 for QA-C3-GlcA-Xyl-Gal-C28-Fuc-Rha-Xyl-Ac (product), and is in line with the experimental results.

[0050] Unless otherwise specified, all raw materials used in this invention are existing substances that can be purchased directly from the market.

[0051] The above are merely preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for QS-7 biosynthetic acetylation, characterized in that, Includes the following steps: S1. The SOAP10 encoding gene was cloned into a prokaryotic expression vector, then transformed into competent cells and induced to express. S2. Collect the competent cells after induced expression, resuspend them, sonicate and centrifuge, take the supernatant to obtain SOAP10 E. coli crude enzyme solution; S3. Mix the non-acetylated QS-7 and SOAP10 E. coli crude enzyme solution to complete the acetylation modification of QS-7.

2. The method according to claim 1, characterized in that, In step S1, the SOAP10 encoding gene is derived from spinach; and / or, In step S1, the competent cells are Escherichia coli BL21(DE3).

3. The method according to claim 1, characterized in that, In step S1, the specific steps for inducing expression include: inoculating the bacterial strain into the culture medium and then adding IPTG to induce expression.

4. The method according to claim 3, characterized in that, The culture medium is LB medium; and / or, After inoculation into the culture medium, the culture temperature was 37℃, and the culture was carried out until OD... 600 =0.6-0.

8.

5. The method according to claim 3, characterized in that, The final concentration of IPTG is 0.2-1.0 mM; and / or, The induction temperature is 15-25℃, and the induction time is 16-24h.

6. The method according to claim 1, characterized in that, In step S2, resuspension is performed using a lysis buffer comprising 50 mM Tris-HCl pH 8.0, 10% glycerol, 0.1% Triton X-100, and 1 mM PMSF.

7. The method according to claim 1, characterized in that, In step S2, the centrifugation conditions are 12000 r / min for 10 min.

8. The method according to claim 1, characterized in that, In step S2, the final concentration of non-acetylated QS-7 is 0.1-1 mM, and the concentration of SOAP10 E. coli crude enzyme solution is 1-5 mg / mL.

9. The method according to claim 1, characterized in that, In step S3, the temperature of the mixing reaction is 30°C and the time is 4-6 hours.

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