Glucoside acetyltransferase and application thereof

Abstract

The invention discloses a glucoside acetyltransferase AmAT7-3, which has the application of catalyzing acetylation of 3'and 4 'sites of astragaloside. According to the engineering AmAT7-3 disclosed by the invention, R38F or L290V or A310R or A310G or D311R or A376R or K407W or K407V or K407G can be obtained, and 3'site acetylation of astragaloside can be catalyzed by the R38F or L290V or A310R or A310G or D311R or A376R or K407W or K407V or K407G. According to the engineering AmAT7-3 disclosed by the invention, A310W or A310F or A312F or A312W is obtained, and the A310W or A310F or A312F or A312W can be used for catalyzing acetylation at the 4'site of astragaloside. The invention verifies the application of the AmAT7-3 and the engineered enzyme thereof in the synthesis of acetylated glycoside compounds, including saponin and flavonoid glycoside compounds. The invention provides a feasible method for site-directed acetylation modification of glucoside by an in-vitro enzyme catalysis method.

Description

Technical Field

[0001] The invention relates to the fields of molecular biology and biochemistry, and in particular to glycoside acetyltransferase (AmAT7-3) and its application in synthesizing astragaloside, acetylated triterpenoid saponin and flavonoid glycoside. Background Art

[0002] Astragalus is a widely used common Chinese medicine with the effects of invigorating qi and raising yang, strengthening the exterior and stopping sweating, promoting diuresis and reducing swelling, promoting body fluid and nourishing blood. It is the main ingredient of clinical medicines such as Huangqi Shengmai Granules, Huangqi Jianwei Paste, and Huangqi Granules. The active ingredient of Astragalus is astragaloside, which has cardioprotective, neuroprotective and immunomodulatory activities and is used to treat various cardiovascular and cerebrovascular diseases. In Astragalus, astragaloside I and astragaloside II have high contents and are products of astragaloside IV acetylation. In addition, Astragalus also contains other astragaloside IV acetylated components, including isoastragaloside I, isoastragaloside II, cycloastragaloside II, etc., and there are also literature reports that it has certain pharmacological activities.

[0003] At present, the source of acetylated astragalosides mainly depends on the extraction of traditional Chinese medicine ingredients. However, the extraction conditions are strict, the acyl group is easily hydrolyzed, and it takes a lot of time for the operator. In addition, the growth cycle of medicinal materials is long and the price is expensive. Therefore, opening up the biosynthetic pathway of astragalosides is of great significance to the open source of such compounds. At present, the oxidative squalene cyclase and glycosyltransferase upstream of the biosynthesis of astragalosides have been explored, but the acyltransferase that catalyzes the acetylation of the 3-xylose of astragaloside IV has not yet been discovered. Therefore, finding the acyltransferase in the biosynthetic pathway of astragalosides is critical to opening up the entire biosynthetic pathway.

[0004] In addition, acetylation, as the most commonly used modification method, is widely present in the modification of various drug active molecules, and has a variety of effects on regulating molecular polarity, lipid and water solubility, and pharmacological activity. However, due to the high similarity of the chemical environment of the hydroxyl group on the sugar, it is very difficult to perform site-specific acylation modification on glycoside compounds. There are multiple sites of xylose at the 3rd position of astragaloside IV in Astragalus, and different combinations of acetylated components indicate that acyltransferases have great advantages in site selectivity. Therefore, how to engineer astragaloside IV acetyltransferase to enable it to site-specifically acetylate other glycoside compounds is very necessary for the acylation modification of medicinally active glycosides. Summary of the invention

[0005] In the first aspect, the present invention relates to an isolated enzyme (referred to herein as "AmAT7-3"). The enzyme is an acetyltransferase having the activity of catalyzing the acetylation of the 3' and 4' positions of astragaloside IV. The full-length sequence thereof comprises SEQ ID NO.1, or consists of SEQ ID NO.1:

[0006]

[0007]

[0008] In another aspect, the present invention relates to an isolated enzyme, which comprises or consists of a full-length sequence having a sequence identity of more than 90%, more than 95%, more than 98% or more than 99% to SEQ ID NO. 1. The enzyme is also an acetyltransferase having the activity of catalyzing glycoside acetylation.

[0009] In another aspect, the present invention relates to an isolated enzyme, which is engineered (sequence modified) on the basis of SEQ ID NO.1, so as to specifically catalyze the acetylation reaction of the 3′ position of astragaloside IV. In a specific embodiment, the enzyme comprises or consists of a sequence: compared with SEQ ID NO.1, the sequence has a mutation including 38 or 290 or 310 or 311 or 376 or 407 (more specifically R38F or L290V or A310R or A310G or D311R or A376R or K407W or K407V or K407G); more specifically, compared with SEQ ID NO.1, the sequence has a mutation of R38F or L290V or A310R or A310G or D311R or A376R or K407W or K407V or K407G (SEQ ID NO.4-12).

[0010] In another aspect, the present invention relates to an isolated enzyme, which is engineered (sequence modified) based on SEQ ID NO.1, so as to specifically catalyze the acetylation reaction of the 4′ position of astragaloside IV. In a specific embodiment, the enzyme comprises or consists of a sequence: compared with SEQ ID NO.1, the sequence has a mutation including position 310 or position 312 (more specifically A310W or A310F or A312F or A312W); more specifically, compared with SEQ ID NO.1, the sequence has a mutation of A310W or A310F or A312F or A312W (SEQ ID NO.13-16).

[0011] In another aspect, the present invention relates to an isolated enzyme that is engineered (sequence modified) based on SEQ ID NO.1, thereby being able to specifically catalyze site-specific acetylation reactions on sugars of glycoside compounds. In a specific embodiment, the enzyme comprises an engineered mutation at any site compared to SEQ ID NO.1.

[0012] In another aspect, the present invention relates to the use of the enzyme described herein in the preparation of acetylated astragalosides, acetylated glycyrrhetinic acid glucuronides, acetylated ginsenosides, acetylated formononetin and the like.

[0013] In another aspect, the present invention relates to an isolated nucleic acid molecule encoding the enzyme described in the aforementioned aspects. For example, for the enzyme AmAT7-3 whose full-length sequence is SEQ ID NO.1, a nucleic acid sequence encoding the enzyme comprises SEQ ID NO.2 or consists of SEQ ID NO.2:

[0014]

[0015]

[0016] In another aspect, the present invention relates to an E. coli codon-optimized nucleic acid molecule encoding the enzyme described in the above aspects. For example, for the enzyme AmAT7-3 whose full-length sequence is SEQ ID NO.1, a nucleic acid sequence encoding the enzyme includes SEQ ID NO.3 or consists of SEQ ID NO.3:

[0017]

[0018]

[0019] In another aspect, the present invention relates to an expression vector comprising the nucleic acid molecule of the present invention.

[0020] In another aspect, the present invention relates to a host cell having the expression vector of the present invention; the cell is an animal somatic cell, a plant somatic cell or a microbial cell.

[0021] In another aspect, the present invention relates to a method for synthesizing acetylated glycosides, comprising: using the enzyme of the present invention to catalyze the reaction of saponin or flavonoid glycoside and acetyl-CoA to generate an acetylated saponin product.

[0022] In another aspect, the present invention relates to the use of the engineered enzyme described in the present invention in the preparation of acetylated glycosides, wherein the acetylated glycosides include: the product of astragaloside IV being further acetylated at the 3′ position; the product of astragaloside IV being further acetylated at the 4′ position; the product of glycyrrhizic acid 3-O-β-D-glucuronide being further acetylated at the 3′ position; the product of sanguisorbaside II being further acetylated at the 3′ position; the product of sanguisorbaside II being further acetylated at the 4′ position; the product of 20(R) ginsenoside Rh1 being further acetylated at the 6′ position, etc.

[0023] The structural formula of the above product is as follows:

[0024] BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 .Use the AmAT7-3 of the present invention to synthesize astragaloside by in vitro enzymatic method.

[0026] Figure 2 .Use the engineered enzyme of the present invention to synthesize astragaloside by in vitro enzymatic method.

[0027] Figure 3 . The AmAT7-3 of the present invention and the engineered enzyme were used to synthesize various saponins and flavonoid glycoside acetylation products by in vitro enzymatic method. DETAILED DESCRIPTION OF THE INVENTION

[0029] The term "isolated" as used herein refers to a protein (enzyme) or nucleic acid that exists under conditions other than its natural environment and has a certain degree of purity. In a preferred form, the isolated protein (enzyme) or nucleic acid is substantially free of other substances, for example, in the case of proteins, particularly free of other homologous proteins (i.e., "homologous impurities"). Unless otherwise specified, the isolated enzyme of the present invention refers to a mature enzyme, which does not include a peptide segment that is removed when the proenzyme is activated. DETAILED DESCRIPTION

[0030] In the enzyme (AmAT7-3) of the present invention and its application, for example, by mining the transcriptome information of Astragalus memeranaceus, the gene expressing AmAT7-3 or the gene engineered therefrom can be cloned and heterologously expressed in a vector such as Escherichia coli BL21 (DE3), and the resulting fusion protein with a tag attached to the N-terminus or / and C-terminus can specifically and efficiently catalyze the acetylation of the sugars of the glycoside compounds astragaloside IV, ginsenoside, sanguisorbaside, and formononetin, thereby producing products such as acetylated astragaloside IV, acetylated ginsenoside, acetylated sanguisorbaside, and acetylated formononetin.

[0031] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from conventional biochemical reagent companies and standard product companies (standard products are purchased from Chengdu Munster Biotechnology Co., Ltd. unless otherwise specified).

[0032] Example 1: Saponin acetyltransferase AmAT7-3 from Astragalus

[0033] 1. Preparation of Plant cDNA

[0034] Astragalus membranaceus sterile seedlings were washed with clean water, and the plant surface was wiped with absorbent paper to remove excess water, and then quickly frozen in liquid nitrogen. After grinding, total RNA was extracted using TranZolTM kit (Transgen Biorech, China), and reverse transcribed using FastQuant RT Kit (Transgen Biotech, China) to obtain cDNA.

[0035] 2. AmAT7-3 gene cloning and expression plasmid construction, transformation into E. coli, and induction of protein expression

[0036] By analyzing the transcriptome data of Astragalus, the complete CDS sequence of the AmAT7-3 gene, namely the sequence of SEQID NO.2, was found. After codon optimization, the sequence was cloned into an expression plasmid through the following steps, and then transformed into Escherichia coli to induce protein expression.

[0037] First, primers were designed for ORF amplification. The primer sequences are shown in Table 1. Using cDNA as a template, ORF was amplified. PCR conditions: 94°C pre-denaturation for 3 min, 94°C denaturation for 30 s, 56°C annealing for 30 s, 68°C extension for 1 min 30 s, 40 cycles. Enzyme: TransStart KD Plus DNA Polymerase (Transgen Biotech, China). ORF was recovered and the concentration of the detection DNA should be greater than 50 ng / μL.

[0038] Table 1. AmAT7-3 primer sequence list

[0039]

[0040] The construction of the recombinant vector of astragaloside acetyltransferase (pET28a-AmAT7-3) was carried out by the Quick-change method. The PCR conditions were: 98℃ pre-denaturation for 3min, 98℃ denaturation for 20s, 60℃ annealing for 30s, 72℃ extension for 4min, 40 cycles, and enzyme: Q5 High-fidelity DNA polymerase. The content of the PCR product was ≥250ng, the volume was less than 5μL, the content of the pET28a empty plasmid was between 30 and 50ng, and the PCR reaction volume was 25μL. After the reaction, 2μL of DpnI and 3μL of CutSmart were added, and the enzyme digestion was carried out at 37℃ for 5 hours or overnight. 10μL of the digested solution was added to 100μL of DH5α competent cells for transformation. Spread on LB plates containing kanamycin, culture overnight at 37℃, select single colonies, and screen positive transformants by PCR (using pET28a universal primers).

[0041] The obtained positive single colony was streaked on a new LB plate containing kanamycin and selected colonies were shaken for 5 mL, plasmids were extracted, sequenced, positive plasmids were selected, introduced into BL21 (DE3) competent cells, spread on LB plates containing kanamycin, cultured overnight at 37°C, single colonies were selected, and positive transformants were screened by PCR (using pET28a universal primers). The obtained positive single colony was streaked on a new LB plate containing kanamycin and selected colonies were shaken for 5 mL (containing kanamycin), cultured overnight at 37°C, expanded to 500 mL (containing kanamycin), and cultured to OD 600 ≈0.6, add 0.1mmol / L IPTG, induce at 16℃ for 18-20h, and collect bacteria at 8000rpm×3min.

[0042] 3. Purification of AmAT7-3

[0043] Add 15 mL of lysis buffer to the bacterial precipitate, fully suspend the bacteria, sonicate on ice (200 W, 10 s, 10 s, 10 min), centrifuge at 10,000 rpm and 4 ° C for 30 min, take the supernatant, add the pre-balanced Ni-NTA Resin (the column is first rinsed with lysis buffer), pass the supernatant through the column three times, rinse the impurities with 30 mM imidazole until there is no obvious blue color in the Coomassie Brilliant Blue test, rinse the target protein with 300 mM imidazole, and collect the fractions until there is no obvious blue color in the Coomassie Brilliant Blue test. Use AmiconUltra-15 Ultracel-30K centrifugal concentrator (Merck Millipore) for centrifugal concentration to obtain the target protein.

[0044] 4. Functional Characterization of AmAT7-3

[0045] Functional characterization of AmAT7-3: see Figure 1The catalytic reaction system included 100 μL of phosphate buffer solution (pH 6.0), substrate (0.1 mmol / L): astragaloside I, acetyl CoA (Zhenzhun Biotechnology): 0.5 mmol / L, DTI (Merck): 10 mmol / L, and appropriate amount of AmAT7-3. The reaction was carried out at 30°C for 30 min, and 200 μL of methanol was added to terminate the reaction. The reaction was centrifuged and detected by UPLC / MS. Detection method: Vanquish UHPLC was connected to a Q-Exactive quadrupole-orbitrap mass spectrometer (ThermoFisherScientific, USA), a Waters T3 chromatographic column (2.1×100 mm, 1.8 μm), 0.1% (v / v, A) of formic acid in water and acetonitrile (B) as the mobile phase, the mobile phase gradient was: 0-1 min, 8% B; 1-19 min, 8%-47% B; 10-16 min, 47%-65% B; 16-19 min, 65%-100% B; 19-22 min, 100% B; 22-25 min, 8% B, flow rate was 0.3 mL / min, and the injection volume was 2 μL. MS detection method: positive and negative ion mode, spray voltage ±3.5kV, ion transfer tube temperature 350℃, sheath gas pressure 45arb, auxiliary gas pressure 10arb, probe heating temperature 400℃, primary mass spectrometry resolution 70000FWHM, secondary mass spectrometry resolution 17500FWHM, scanning range m / z 100-1200, NCE: 35eV. Compared with isoastragaloside II, cycloastragaloside II, 3′,4′-diacetylastragaloside IV standards, AmAT7-3 has the activity of catalyzing the acetylation of 3′ and 4′ positions of astragaloside IV, with a conversion rate of 3′ position of 35.1%, a conversion rate of 4′ of 19.8%, a conversion rate of 3′ and 4′ diacetylation of 3′ and 4′ positions of 30.5%, and a total conversion rate of 85.3% (conversion rate (%) = product peak area / (product peak area + substrate peak area)).

[0046] Example 2: Engineering enzymes A310G and A310W

[0047] 1. Engineering A310G, A310W gene cloning and expression plasmid construction, transformation into Escherichia coli, and induction of protein expression First, primers were designed for mutation of AmAT7-3. The primer sequences are shown in Table 2.

[0048] Table 2. List of engineered gene primer sequences (1)

[0049]

[0050]

[0051] The construction of the engineered recombinant vector (pET28a-A310G, pET28a-A310W) was carried out by the Quick-change method, PCR conditions: 98℃ pre-denaturation for 3min, 98℃ denaturation for 20s, 60℃ annealing for 30s, 72℃ extension for 4min, 25 cycles, enzyme: Fastpfu Fly mix. The content of pET28a-AmAT7-3 plasmid was between 30 and 50ng, and the PCR reaction volume was 25μL. After the reaction, DMT digestion was performed and enzyme digestion was performed at 37℃ for 20min. 10μL of the solution after enzyme digestion was added to 100μL of DH5α competent cells for transformation. Spread on LB plate containing kanamycin, culture overnight at 37℃, select single colonies, and PCR screen positive transformants (using pET28a universal primers).

[0052] The operation procedures of transformation into Escherichia coli and inducing protein expression are the same as those in "Example 1, item 2".

[0053] 2. Purification of engineered enzymes A310G and A310W

[0054] The operation process is the same as that of "Example 1, Article 3".

[0055] 3. Functional characterization of engineered enzymes A310G and A310W

[0056] Functional characterization of engineered enzymes: see Figure 2 The catalytic reaction system includes 100 μL of phosphate buffer solution (pH 6.0), substrate (0.1 mmol / L): astragaloside IV, acetyl-CoA (0.5 mmol / L), and appropriate amount of A310G or A310W. The reaction was carried out at 37°C for 30 min, and 200 μL of methanol was added to terminate the reaction, and the reaction was centrifuged. The detection method was the same as that in "Example 1, Item 4". It was determined by comparison with the standard that A310G has the activity of catalyzing the acetylation of astragaloside IV at the 3′ position. A310W has the activity of catalyzing the acetylation of astragaloside IV at the 4′ position.

[0057] Example 3: Acetylation of other saponins using AmAT7-3 and its engineered enzymes

[0058] 1. The gene cloning and expression plasmid construction of AmAT7-3 and its engineered enzyme, transformation into Escherichia coli, and induction of protein expression are the same as the operation procedures of "Example 1, Item 2" and "Example 1, Item 3".

[0059] 2. Functional characterization of AmAT7-3 and its engineered enzymes on other saponin compounds

[0060] Functional characterization of AmAT7-3: see Figure 3The catalytic reaction system includes 100 μL of phosphate buffer solution (pH 6.0), substrate (0.1 mmol / L): compound 1-8, acetyl coenzyme A: 0.5 mmol / L, DTT: 10 mmol / L, and appropriate amount of AmAT7-3. The reaction was carried out at 30°C for 30 min, and 200 μL of methanol was added to terminate the reaction, centrifuged, and detected by UPLC / MS. The detection method is the same as "Example 1, item 4". The results showed that AmAT7-3 could catalyze the acetylation products of astragaloside VII (2), sanguisorbaside II (3), bufotoxin 3-O-β-D-glucoside (4), cinobufagin 3-O-β-D-glucoside (5), 20(R)-ginsenoside Rh1 (6), glycyrrhetinic acid 3-O-β-D-glucuronide (7), ginsenoside Iva (8) and formononetin (9), among which it had multi-site acetylation activity for compounds 1-5 and specific site acetylation activity for compounds 6-9. The characterization results of the engineered enzyme activity showed that A310G had specific site acetylation activity for compounds 1, 2, 3, and 5, and A310W had specific site acetylation activity for compounds 1, 2, and 4.

Claims

1. An isolated enzyme, which is an acetyltransferase, comprising the amino acid sequence of SEQ ID NO.

1.

2. An isolated enzyme, which is an acetyltransferase, comprising a full-length sequence having a sequence identity of 90% or more, 95% or more, 98% or more, or 99% or more to SEQ ID NO.

1.

3. An isolated enzyme that can specifically catalyze the acetylation of the 3' position of astragaloside IV, wherein compared with SEQ ID NO.1, the enzyme sequence has a mutation at position 38, position 290, position 310, position 311, position 376, or position 407.

4. An isolated enzyme capable of specifically catalyzing the acetylation of the 4' position of astragaloside IV, wherein the enzyme sequence comprises a mutation at position 310 or 312 compared with SEQ ID NO.

1.

5. The isolated enzyme according to claims 3-4, comprising the amino acid sequence of any one of SEQ ID NOs. 4-16.

6. An isolated nucleic acid molecule encoding the enzyme according to any one of claims 1-5.

7. The isolated nucleic acid molecule according to claim 6, wherein the nucleic acid sequence thereof comprises SEQ ID NO. 2-3.

8. An expression vector comprising the nucleic acid molecule according to claim 6 or 7.

9. A host cell comprising the expression vector according to claim 8.

10. Use of the enzyme according to any one of claims 1 to 5 in the preparation of acetylated glycosides.

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