Application of Aspergillus ruber isopentenyltransferase EchPT1 in catalyzing isopentenylation of chalcone compounds

Abstract

The application discloses application of Aspergillus ruber isopentenyl transferase EchPT1 in catalyzing isopentenylation of chalcone compounds and belongs to the technical field of biological catalysis and synthesis. The amino acid sequence of the EchPT1 is shown as SEQ ID NO. 2. The application first finds that the EchPT1 can catalyze plant-derived chalcone substrates to generate glycyrrhetin A and glycyrrhetin C in the presence of DMAPP as an isopentenyl donor and Mg 2+ or Mn 2+ The reaction condition is mild and the regioselectivity is good. After optimization, when the reaction system is pH 8.0, 34.8 DEG C, 8 h, DMAPP 50 μM and picraline 50 μM, the total conversion rate of the substrate is increased from about 0.60% to about 5.41%. The application provides a new route for enzyme synthesis of glycyrrhetin active ingredients, can replace plant extraction and chemical synthesis, and has industrial application value.

Description

Technical Field

[0001] This invention relates to the fields of biocatalysis and synthesis technology, and more specifically to the application of Aspergillus erythrorhizon isopentenyltransferase EchPT1 in the catalytic isopentenylation of chalcone compounds. Background Technology

[0002] Glycyrrhizin chalcone A and C are important isopentenylated chalcone active ingredients from Glycyrrhiza genus plants, possessing significant anti-inflammatory, antibacterial, antioxidant, antitumor, and hepatoprotective pharmacological activities. Glycyrrhizin chalcone A, due to its inhibitory effects on tyrosinase and inflammation-related signaling pathways, holds significant value in cosmetic whitening and drug development. Studies have shown that isopentenylation modification can significantly improve the lipophilicity, membrane permeability, target affinity, and metabolic stability of chalcone compounds, making it an important structural modification method for optimizing the drug-likeness of natural products. However, the content of glycyrrhizin chalcone A and C in plants is low, and extraction and separation are limited by plant resources, growth cycle, and production environment, making it difficult to meet the needs of large-scale preparation. Although chemical synthesis methods can construct chalcone skeletons, they suffer from poor selectivity of isopentenylation sites, long reaction steps, cumbersome protective group operations, heavy metal catalyst residues, and high consumption of organic solvents, resulting in high costs and heavy environmental burdens, limiting their large-scale preparation and in-depth development.

[0003] Prenyltransferases (PTs) catalyze the transfer of isopentenyl donors to aromatic substrates, making them highly efficient biocatalysts for the synthesis of isopentenylated natural products. Based on the different chain lengths of the isopentenyl donor, aromatic isopentenyltransferases (PTs) can be classified into DMAPP (C5)-dependent, GPP (C10)-dependent, and FPP (C15)-dependent types. Aromatic PTs typically use DMAPP or GPP as donors, and the regioselectivity of the acceptor substrate determines the diversity of product structures. Most reported aromatic isopentenyltransferases have broad substrate profiles, acting on various aromatic skeletons such as indoles, flavonoids, coumarins, lignans, and phenylpropanoids; however, the number of isopentenyltransferases that specifically recognize chalcone skeletons remains limited, and the regioselective regulatory mechanisms of chalcone substrates and products are not yet fully understood.

[0004] EchPT1 was first in Aspergillus ruberEchPT1 has been identified as an isopentenyl transferase involved in the biosynthesis of echinulin, catalyzing the first step of isopentenylation in the indole-diketopiramate skeleton (Wohlgemuth V, Kindinger F, Xie X, et al. Two Prenyltransferases Govern a Consecutive Prenylation Cascade in the Biosynthesis of Echinulin and Neoechinulin. Organic Letters, 2017, 19(21): 5928-5931). In the prior art, the function of EchPT1 is mainly limited to indole-diketopiramate substrates; its substrate recognition mechanism and catalytic function have not been extended to plant chalcone compounds. To date, no literature reports that EchPT1 can catalyze the formation of glycyrrhizin A and glycyrrhizin C from plant-derived chalcone substrates.

[0005] Therefore, developing new applications of EchPT1 in the isopentenylation of chalcones and establishing efficient, green, and regioselective in vitro enzymatic synthesis routes are of great significance for the preparation of high-value active compounds of glycyrrhizin chalcones and are technical problems that urgently need to be solved by those skilled in the art. Summary of the Invention

[0006] The first objective of this invention is to provide the application of Aspergillus erythrorhizon isopentenyltransferase EchPT1 in the catalytic isopentenylation of chalcone compounds.

[0007] A second objective of this invention is to provide a method for the in vitro synthesis of glycyrrhizin A and / or glycyrrhizin C.

[0008] A third object of the present invention is to provide the use of EchPT1 in the preparation of compositions containing glycyrrhizin A and / or glycyrrhizin C.

[0009] A fourth objective of this invention is to provide the use of engineered bacteria in the synthesis of glycyrrhizin A and / or glycyrrhizin C.

[0010] To achieve the above objectives, this application adopts the following technical solution:

[0011] The primary objective of this application is to provide the use of isopentenyltransferase EchPT1 in catalyzing the isopentenylation reaction of glycyrrhizin chalcone to generate glycyrrhizin A and / or glycyrrhizin C, wherein the amino acid sequence of the isopentenyltransferase EchPT1 is shown in SEQ ID NO.2.

[0012] As a preferred technical solution, the isopentenyltransferase EchPT1 is encoded by the nucleic acid sequence shown in SEQ ID NO.1; the isopentenylation reaction uses dimethylallyl pyrophosphate DMAPP as the isopentenyl donor, in Mg 2+ or Mn 2 + It is performed under the condition that it exists.

[0013] As a preferred technical solution, the isopentenylation reaction has a pH of 7.0-9.0, a reaction temperature of 30-40℃, a reaction time of 6-10 h, a DMAPP concentration of 50-200 μM, and a licorice chalcone concentration of 50-200 μM.

[0014] As a more preferred technical solution, the isopentenylation reaction is carried out at a pH of 8.0, a reaction temperature of 34.8°C, a reaction time of 8 h, a DMAPP concentration of 50 μM, and a licorice chalcone concentration of 50 μM.

[0015] As a preferred technical solution, the isopentenyltransferase EchPT1 is used in the form of purified protein, crude enzyme solution, whole-cell catalyst, immobilized enzyme, or engineered bacteria.

[0016] Another object of this application is to provide: a method for the in vitro synthesis of glycyrrhizin A and / or glycyrrhizin C, wherein, under the catalysis of isopentenyltransferase EchPT1, glycyrrhizin A and / or glycyrrhizin C are generated by isopentenylation reaction using glycyrrhizin as substrate and DMAPP as isopentenyl donor; the amino acid sequence of EchPT1 is shown in SEQ ID NO.2.

[0017] As a preferred technical solution, the reaction system of the isopentenylation reaction contains Mg. 2+ or Mn 2+ As a cofactor, the reaction conditions for isopentenylation were pH 7.0-9.0, 30-40℃, and 6-10 h; the concentration of DMAPP was 50-200 μM, and the concentration of licorice chalcone was 50-200 μM.

[0018] As a more preferred technical solution, the reaction conditions are pH 8.0, 34.8℃, 8 h; DMAPP concentration is 50 μM, and licorice chalcone concentration is 50 μM.

[0019] Another object of this application is to provide: the use of isopentenyltransferase EchPT1 in the preparation of compositions containing glycyrrhizin A and / or glycyrrhizin C, wherein the amino acid sequence of EchPT1 is shown in SEQ ID NO.2; wherein the composition is a pharmaceutical composition or a cosmetic composition.

[0020] Another object of this application is to provide: the use of an engineered bacterium in the synthesis of glycyrrhizin A and / or glycyrrhizin C, wherein the engineered bacterium co-expresses EchPT1 and DMAPP synthesis pathway-related genes, wherein the amino acid sequence of EchPT1 is shown in SEQ ID NO.2; and wherein the engineered bacterium generates DMAPP in situ within cells.

[0021] As can be seen from the above technical solution, compared with the prior art, the present invention has the following beneficial effects: (1) It was discovered for the first time that the known isopentenyltransferase EchPT1 can catalyze plant chalcone substrates, and the in vitro enzymatic synthesis of glycyrrhizin A and glycyrrhizin C was realized, providing an example for the new functional application of old proteins.

[0022] (2) The enzyme-catalyzed reaction has good regional selectivity and can simultaneously generate the trans-isoprenylated product glycyrrhizin A and the cis / n-isoprenylated product glycyrrhizin C. The products can be clearly identified by HPLC and mass spectrometry.

[0023] (3) By systematically optimizing the cofactor, donor concentration, pH, temperature, time and substrate concentration, the substrate conversion rate was significantly improved, which has good prospects for synthetic biology applications.

[0024] (4) The reaction conditions are mild and environmentally friendly, and can replace some chemical synthesis steps. It is suitable for the preparation of high-value active compounds of glycyrrhizin chalcone.

[0025] (5) Compared with plant extraction, the present invention does not rely on licorice plant resources, is not limited by season and place of origin, and can achieve controllable production through enzyme catalysis or engineered bacteria fermentation.

[0026] (6) Compared with chemical synthesis, the present invention avoids the use of strong bases, strong acids, protecting groups and heavy metal catalysts, the regioselectivity is determined by the natural enzyme, there are few by-products and the post-processing is simple. Attached Figure Description

[0027] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0028] Figure 1 Here is an SDS-PAGE image of EchPT1 protein expression and purification in Example 2; where M is the protein marker; T1 / T2 is the whole protein, S is the supernatant, F is the flow-through buffer, W is the eluted contaminant protein, E1 is the eluted target protein, and E2 is the desalted and concentrated target protein.

[0029] Figure 2 The following is a sample diagram (A) and HPLC analysis (B) of the reaction formula (A) and HPLC analysis (B) of the EchPT1-catalyzed isopentenylation of glycyrrhizin chalcone in Example 3. The control in B is the reaction system without EchPT1, the EchPT1 is the experimental group with purified EchPT1 added, and the standard is a mixture of glycyrrhizin chalcone, glycyrrhizin chalcone A and glycyrrhizin chalcone C.

[0030] Figure 3 Here are the MS and MS² mass spectra of the EchPT1 enzyme activity product in Example 3.

[0031] Figure 4 The figure shows the results of the single-factor experiment on the optimization of EchPT1 enzymatic reaction conditions in Example 4, with the optimal level of each factor marked by a red star.

[0032] Figure 5 The graph shows the substrate conversion rates of glycyrrhizin catalyzed by EchPT1 before and after optimization in Example 5. The blue column represents glycyrrhizin A (LCA), and the red column represents glycyrrhizin C (LCC). Detailed Implementation

[0033] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. 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.

[0034] Example 1 Construction of EchPT1 prokaryotic expression vector (1) Obtaining the EchPT1 coding sequence and synthesizing the whole gene Using the EchPT1 coding sequence from Aspergillus ruber (GenBank accession number EYE98742) as a template, the gene sequence of the EchPT1 coding region was obtained through whole-genome synthesis, and the amino acid sequence encoding EchPT1 was determined, as follows: EchPT1 gene sequence (1254 bp):

[0035] The amino acid sequence encoded by EchPT1 (417 aa): MPSEVLTSYYDYPTHDQEAWWRDTTGPLFGRFLKGAGYDVHTQYQYLVFFIKNILPSLGPYPARWRSTITPTGLPIEYSLNFQLNSRPLLRIGFEPLSRFSGTPQD PYNKIAAADLLNQLSKLQLHEFDTQLFNHFTNEFELSKSESESLQKQGGINGKSTVRSQTAFGFDLKGGRVAVKGYAFAGLKNRATGTPVGQLISNSIRNLEPQM HCWDSFSILNSYMEESDGWNEYSFVSWDCVDIERSRLKLYGVHNAVTWDKVKEMWTLGGRIENNATIKTGLELLQHMWSLLQINEGDRDYKGGFAADNGGKTLPI IWNYELNKGSPHPAPKFYFPVHGENDLQVSKSISEFFTHLGWQDHARQYPHLLRQIYPNQNISQTERLQAWISFAYNERTGPYLSVYYYSAERPPWGSDQVK, SEQ ID NO.2.

[0036] (2) Primer design and synthesis Based on the 5' and 3' sequences of SEQ ID NO.1, homologous recombination primers were designed using the multiple cloning site of the pET28a vector. The primers were synthesized by a biotechnology company, as detailed below: When designing primers, an EcoRI restriction site is introduced at the 5' end: 28a-EchPT1-F: GGTCGCGGATCCGAATTCATGCCCTCTGAAGTATTG, SEQ ID NO.3; An Xho I restriction site is introduced at the 3' end: 28a-EchPT1-R: GTGGTGGTGGTGCTCGAGTTTGACCTGATCACTTCC, SEQ ID NO. 4.

[0037] The amplified product was double-digested with the prokaryotic expression vector pET28a, ligated using a homologous recombinase, and transformed into *E. coli* DH5α competent cells. Positive clones were selected for colony PCR and sequencing verification. The recombinant vector with correct sequencing was named pET28a-EchPT1 and used for subsequent protein expression.

[0038] Example 2 Prokaryotic expression and purification of EchPT1 (1) Transformation and induction of recombinant plasmid expression The correctly sequenced pET28a-EchPT1 plasmid was transformed into *E. coli* BL21(DE3) competent cells and plated on LB agar plates containing ampicillin (50 mg / L), incubated overnight at 37°C. Single colonies were picked and inoculated into 5 mL of LB liquid medium containing ampicillin, incubated at 37°C and 200 rpm for 12 h, then transferred at a 1:100 ratio to 200 mL of the same medium, and cultured until OD600 reached 0.6-0.8. 0.5 mM IPTG was added, and the cells were incubated in an ice-water bath for 30 min, followed by induction culture at 16°C and 180 rpm for 12-16 h.

[0039] (2) Protein affinity purification, desalting and concentration determination Bacterial cells were collected by centrifugation at 4°C, resuspended in pre-cooled Lysis buffer, and sonicated (200 W, 3 s on, 3 s off, total 30 min). The cells were then centrifuged at 4°C and 4000×g for 20 min, and the supernatant (S) and precipitate (P) were collected separately. The supernatant was filtered through a 0.45 μm filter and added to a protein purification column containing 1 mL of Ni-NTA packing material, and incubated at 4°C for 1 h. After incubation, the liquid was allowed to flow out naturally, and the flow-through fluid (F) was collected. The column was then washed with wash buffer containing 30 mM imidazole, and the eluent (W) was collected until the protein concentration in the eluent was very low. Finally, the column was eluted with elution buffer containing 250 mM imidazole, and the target protein eluent (E1) was collected. The target protein eluent (E1) was placed in a 10 kDa ultrafiltration centrifuge tube, concentrated and desalted by centrifugation at 4°C and 4000×g, aliquoted, and stored at -80°C to obtain concentrated desalted protein (E2). Protein concentration was determined using the Bradford method.

[0040] (3) SDS-PAGE analysis Take an appropriate amount of protein sample, add 5×SDS-PAGE loading buffer, boil at 98℃ for 10 min, centrifuge, and take 10 μL for SDS-PAGE electrophoresis.

[0041] Results analysis: such as Figure 1 As shown, the target bands for induced expression were visible in the whole protein T1 / T2 and the supernatant S; after Ni-NTA affinity purification, E1 and E2 showed a single, clear target protein band at approximately 47 kDa, consistent with the theoretical molecular weight of the EchPT1 fusion protein of 47.9 kDa.

[0042] Example 3 EchPT1 in vitro enzyme activity assay Using purified EchPT1 protein as a catalyst, echinatin as a substrate, and DMAPP as an isopentenyl donor, an in vitro enzymatic reaction was carried out in the presence of divalent metal ions. The study aimed to determine whether EchPT1 could catalyze the isopentenylation of echinatin and to identify whether the products were echinatin A (LCA) and echinatin C (LCC). Details are as follows: (1) Reaction system and grouping: The enzyme activation reaction system (100 μL) contained: buffer (100 mM Tris-HCl, pH 8.0), licorice chalcone (200 μM), DMAPP (200 μM), MgCl2 or MnCl2 (1 mM) and purified EchPT1 protein (2 μg).

[0043] The experiment was set up with the following groups: Experimental group (EchPT1): buffer + glycyrrhizin chalcone + DMAPP + MgCl2 or MnCl2 + purified EchPT1; Control group (inactivated EchPT1): buffer + glycyrrhizin chalcone + DMAPP + MgCl2 or MnCl2 + inactivated purified EchPT1; Standard group: Glycyrrhiza chalcone, glycyrrhiza chalcone A and glycyrrhiza chalcone C standards were dissolved in methanol to prepare mixed standard solutions of appropriate concentrations. After filtration through a 0.22 μm organic filter membrane, the solutions were injected and analyzed under the same HPLC conditions as the experimental group to determine the retention time of each compound. (2) Enzyme-catalyzed reaction operation The above reaction solutions were mixed thoroughly and reacted at 37°C for 4 hours. After the reaction was completed, an equal volume of ethyl acetate was added to each tube to terminate the reaction. The mixture was vortexed to fully extract the product into the organic phase. The product was centrifuged, and the upper organic phase was collected and filtered through a 0.22 μm organic filter membrane. The filtrate was used for subsequent HPLC analysis.

[0044] (3) HPLC analysis The HPLC analysis conditions were as follows: Column: C18 reverse-phase column, 250 mm × 4.6 mm, particle size 5 μm; Mobile phase gradient elution conditions: 0–13 min, 75% formic acid aqueous solution (0.1%), 25% acetonitrile; 13–15 min, 68% formic acid aqueous solution, 32% acetonitrile; 15–40 min, 68% formic acid aqueous solution, 32% acetonitrile; 40–42 min, 75% formic acid aqueous solution, 25% acetonitrile; 42–45 min, 75% formic acid aqueous solution, 25% acetonitrile; Mobile phase rate: 1.0 mL / min; Detection wavelength: 370 nm.

[0045] Results analysis: such as Figure 2 As shown, in the control group, the retention time of glycyrrhizin chalcone was approximately 8.9 min, and no glycyrrhizin A or C was observed. After the addition of EchPT1, the glycyrrhizin chalcone peak significantly decreased, and two new peaks appeared at retention times of approximately 21.3 min and 22.5 min, respectively, consistent with the retention times of glycyrrhizin C (LCC) and glycyrrhizin A (LCA) standards. These results demonstrate that EchPT1 can catalyze the isopentenylation reaction of glycyrrhizin chalcone to generate LCA and LCC.

[0046] Further mass spectrometry identification of the products (the total products of LCC and LCA) yielded the following results: Figure 3 As shown, the quasi-molecular ion peak [M+H]+ of the enzyme active product is 337.1 (m / z 337.14457). The main fragment ions of the secondary mass spectrometry (MS²) include m / z 305.12, 243.10, 175.04, and 120.02, which is consistent with the expected fragmentation pattern of the isopentenylated product.

[0047] Example 4 Optimization of EchPT1 enzyme activity reaction conditions Based on the confirmation that EchPT1 possesses catalytic activity, single-factor experiments were used to optimize the reaction conditions, with two products ( Figure 4 The sum of substrate conversion rates of P2 (LCA) and P3 (LCC) was used as the evaluation index. Single-factor experiments were conducted sequentially to examine cofactor, donor concentration, pH, reaction time, reaction temperature, and substrate concentration. During each optimization, all conditions except the factor under investigation remained consistent.

[0048] (1) Basic reaction system and conversion rate determination: Unless otherwise specified, the basic reaction system is 100 μL, containing 100 mM Tris-HCl (pH 8.0), glycyrrhizin chalcone (200 μM), DMAPP (200 μM), divalent metal ion cofactor (1 mM), and purified EchPT1 protein (2 μg). After mixing the reaction solution, the reaction was carried out at 37℃ for 4 h. The reaction was terminated by adding an equal volume of ethyl acetate, and the product was extracted. After vortexing and centrifugation, the organic phase was filtered through a 0.22 μm organic filter membrane and analyzed by HPLC. The amounts of the two products were calculated based on the peak areas or standard curves of glycyrrhizin chalcone C and glycyrrhizin chalcone A standards, and the sum of the conversion rates of the two products was taken as the total conversion rate. Each experiment was conducted in triplicate, and the average value was taken.

[0049] (2) Cofactor screening: In the basic reaction system, no metal ions (Co-) and Mg- were added respectively. 2+ Mn 2+ Ca2+ Zn 2+ The reaction was carried out in two groups: a metal ion group and an EDTA group. The final concentrations of both groups were 1 mM and 1 mM, respectively. All other conditions were kept consistent. The reaction was terminated at 37℃ for 4 h, and the results were analyzed by HPLC. The experimental results are as follows: Figure 4 As shown.

[0050] The results showed that Mn 2+ The highest overall conversion rate (approximately 0.097%) was achieved under the given conditions. 2+ Next (approximately 0.087%), Ca 2+ Zn 2+ The activity was significantly reduced, and EDTA almost completely inhibited the reaction, indicating that the catalytic activity of EchPT1 depends on divalent metal ions, and Mn 2+ and Mg 2+ It is the most suitable auxiliary factor.

[0051] (3) Donor concentration optimization: Under optimal cofactor conditions, with other conditions fixed, only the DMAPP concentration was changed to 0, 50, 100, 150, and 200 μM, respectively. The reaction was terminated after 4 h at 37℃ and analyzed by HPLC. The experimental results are as follows: Figure 4 As shown.

[0052] The results showed that the total conversion rate reached its highest level (approximately 1.6%) at 50 μM when the DMAPP concentration was in the range of 0-200 μM, and then decreased. Therefore, 50 μM was selected as the optimal donor concentration.

[0053] (4) pH optimization: Under optimal cofactor and DMAPP concentration conditions, and with other conditions fixed, reaction systems were prepared using buffer solutions with pH values ​​of 5.0, 6.0, 7.0, 8.0, 9.0, and 10.0, respectively. The reaction was terminated at 37℃ for 4 h, and the results were analyzed by HPLC. The experimental results are as follows: Figure 4 As shown.

[0054] The results showed that the total conversion rate was highest at pH 8.0 (approximately 0.79%) within the pH range of 5-10, therefore pH 8.0 was selected as the optimal reaction pH.

[0055] (5) Optimization of reaction time: Under optimal cofactor, DMAPP concentration, and pH conditions, while keeping other conditions constant, samples were taken at 2, 4, 6, 8, 10, and 12 h to terminate the reaction, and HPLC analysis was performed. The experimental results are as follows: Figure 4 As shown.

[0056] The results showed that the total conversion rate was highest at 8 hours (approximately 0.96%) within the range of 2-12 hours, and then decreased. Therefore, 8 hours was selected as the optimal reaction time.

[0057] (6) Optimization of reaction temperature: Under optimal conditions of cofactor, DMAPP concentration, pH, and reaction time, and with other conditions fixed, reactions were carried out at 16, 30, 34.8, 43.2, 47.5, and 51.4 °C, respectively. After termination, HPLC analysis was performed, and the experimental results are as follows: Figure 4 As shown.

[0058] The results showed that the total conversion rate was highest at 34.8℃ (approximately 0.89%) within the range of 16-51.4℃, and was close to that at 30℃. Therefore, 34.8℃ was selected as the optimal reaction temperature.

[0059] (7) Substrate concentration optimization: Under optimal conditions of cofactor, DMAPP concentration, pH, reaction time, and temperature, while keeping other conditions constant, only the concentration of chalcone from glycyrrhizin was varied at 0, 50, 100, 150, and 200 μM. After the reaction was terminated, HPLC analysis was performed, and the experimental results are as follows: Figure 4 As shown.

[0060] The results showed that the total conversion rate reached its highest level (approximately 0.61%, marked with a red star in the figure) at 50 μM within the range of 0-200 μM chalcone concentration. The conversion rate was higher at 200 μM, but the substrate consumption was greater. Based on the markings in the figure, 50 μM was selected as the optimal substrate concentration.

[0061] Example 5 Catalytic conversion of EchPT1 under optimized conditions Under optimized conditions (pH 8.0, 34.8℃, 8 h, DMAPP 50 μM, glycyrrhizin chalcone 50 μM, Mg...), 2+ As a cofactor with a final concentration of 1 mM and 2 μg of purified EchPT1 protein, the conversion rates of EchPT1-catalyzed glycyrrhizin chalcone to glycyrrhizin A and glycyrrhizin C were determined and compared with the reaction conditions before optimization (pH 8.0, 37℃, 4 h, DMAPP 200 μM, glycyrrhizin chalcone 200 μM, Mg...). 2+ As a cofactor, the final concentration was 1 mM, and 2 μg of purified EchPT1 protein was compared.

[0062] The results are as follows Figure 5As shown, the total substrate conversion before optimization was approximately 0.60% (of which glycyrrhizin A was approximately 0.35% and glycyrrhizin C was approximately 0.25%). After optimization, the total conversion increased to approximately 5.41% (of which glycyrrhizin A was approximately 3.20% and glycyrrhizin C was approximately 2.21%), an improvement of approximately 9.0 times compared to before optimization. These results indicate that optimizing reaction conditions can significantly improve the catalytic efficiency of EchPT1 for glycyrrhizin.

[0063] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0064] 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. The application of Aspergillus rubrum isopentenyltransferase EchPT1 in the catalytic isopentenylation of chalcone compounds, characterized in that, The chalcone compound is glycyrrhizin chalcone; the isopentenylation product is glycyrrhizin chalcone A and / or glycyrrhizin chalcone C; the amino acid sequence of the isopentenyltransferase EchPT1 is shown in SEQ ID NO.

2.

2. The application according to claim 1, characterized in that, The isopentenyltransferase EchPT1 is encoded by the nucleic acid sequence shown in SEQ ID NO. 1; the isopentenylation reaction uses dimethylallyl pyrophosphate (DMAPP) as the isopentenyl donor, in Mg... 2+ or Mn 2+ It is performed under the condition that it exists.

3. The application according to claim 1 or 2, characterized in that, The isoprenylation reaction was carried out at a pH of 7.0-9.0, a reaction temperature of 30-40℃, a reaction time of 6-10 h, a DMAPP concentration of 50-200 μM, and a licorice chalcone concentration of 50-200 μM.

4. The application according to claim 3, characterized in that, The isoprenylation reaction was carried out at a pH of 8.0, a reaction temperature of 34.8℃, a reaction time of 8 h, a DMAPP concentration of 50 μM, and a licorice chalcone concentration of 50 μM.

5. The application according to any one of claims 1-4, characterized in that, The isopentenyltransferase EchPT1 is used in the form of purified protein, crude enzyme solution, whole-cell catalyst, immobilized enzyme, or engineered bacteria.

6. A method for the in vitro synthesis of glycyrrhizin A and / or glycyrrhizin C, characterized in that, Catalyzed by the isopentenyltransferase EchPT1, glycyrrhizin chalcone was used as a substrate and DMAPP as an isopentenyl donor for isopentenylation reaction to generate glycyrrhizin chalcone A and / or glycyrrhizin chalcone C; the amino acid sequence of EchPT1 is shown in SEQ ID NO.

2.

7. The method according to claim 6, characterized in that, The reaction system of the isopentenylation reaction contains Mg 2+ or Mn 2+ As a cofactor, the reaction conditions for isopentenylation were pH 7.0-9.0, 30-40℃, and 6-10 h; the concentration of DMAPP was 50-200 μM, and the concentration of licorice chalcone was 50-200 μM.

8. The method according to claim 7, characterized in that, The reaction conditions were pH 8.0, 34.8℃, and 8 h; the concentration of DMAPP was 50 μM, and the concentration of licorice chalcone was 50 μM.

9. The use of isopentenyltransferase EchPT1 in the preparation of compositions containing glycyrrhizin A and / or glycyrrhizin C, wherein the amino acid sequence of EchPT1 is shown in SEQ ID NO.2; the composition is a pharmaceutical composition or a cosmetic composition.

10. The application of an engineered bacterium in the synthesis of glycyrrhizin A and / or glycyrrhizin C, characterized in that, The engineered bacteria co-express EchPT1 and genes related to the DMAPP synthesis pathway. The amino acid sequence of EchPT1 is shown in SEQ ID NO.

2. The engineered bacteria generate DMAPP in situ within the cells.

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