β-glucuronidase, recombinant expression vector thereof, engineering bacterium, fermenting agent, and mass production method for glycyrrhetinic acid

Abstract

Provided are a β-glucuronidase, a recombinant expression vector thereof, an engineering bacterium, a fermenting agent, and a mass production method for glycyrrhetinic acid. The provided β-glucuronidase is selected from β-glucuronidase AcGUS, β-glucuronidase AcGUS1, β-glucuronidase AcGUS2, β-glucuronidase AcGUS3, and a β-glucuronidase AcGUS3 mutant. The β-glucuronidase AcGUS is an enzyme obtained in such a way that in the amino acid sequence with the GenBank accession number of AEK69352.1, the amino acid on the 1st site is deleted, the alanine on the 7th site is mutated into glycine, the arginine on the 66th site is mutated into lysine, the alanine on the 256 site is mutated into valine, the threonine on the 270 site is mutated into alanine, the valine on the 332nd site is mutated into phenylalanine, the aspartic acid on the 363rd site is mutated into glutamic acid, and the methionine on the 506th site is mutated into valine. The provided β-glucuronidase can be used for industrial scale production of 18α-GA and 18β-GA.

Description

A beta-glucuronidase, a recombinant expression vector thereof, an engineered bacterium, a fermentation agent and a method for mass production of glycyrrhetinic acid TECHNICAL FIELD

[0001] The present application belongs to the field of enzyme engineering and microbial technology, and particularly relates to a beta-glucuronidase, a recombinant expression vector thereof, an engineered bacterium, a fermentation agent and a method for mass production of glycyrrhetinic acid. BACKGROUND

[0002] Glycyrrhiza uralensis Fisch, as a traditional medicine and food homologous plant, is widely used in medicine, food and cosmetics industries. According to historical records, Glycyrrhiza uralensis Fisch has been used as medicine for more than four thousand years, and is known as "old in China and glycyrrhiza in medicine". Glycyrrhiza uralensis Fisch not only has the effects of clearing heat and resolving toxins, tonifying the spleen and replenishing qi, moistening the lungs and relieving cough, and regulating various drugs, but also can be used for sore throat, spleen and stomach cold, peptic ulcer and other symptoms, and has a wide range of applications in anti-inflammatory, liver protection, antioxidant, antiviral, antitumor and antidiuretic. Glycyrrhiza uralensis Fisch or its active ingredients are added in many modern Chinese medicine compound preparations.

[0003] Glycyrrhizic acid (GL) and glycyrrhetinic acid (GA) are the main component and active component of Glycyrrhiza uralensis Fisch, respectively. At present, the reported production of glycyrrhetinic acid mainly includes chemical method and biological transformation method. CN101817867A reports that acetylglycyrrhetinic acid is generated from glycyrrhizic acid salt under the catalysis of sulfuric acid and high-concentration acetic acid, and then glycyrrhetinic acid is obtained by further deacetylation. However, a large amount of strong acid, strong base and organic solvent are used in the reaction, which has problems such as high energy consumption, low yield and great environmental pressure. The biological transformation method not only has the advantages of mild conditions, high yield and environmental protection, but also can provide higher-purity medicinal precursors for the preparation of different derivatives, and is an extremely promising industrial production method. A large number of literatures have reported the applications of GA in anti-inflammatory, liver protection, antioxidant, antiviral, antitumor and antidiuretic. Studies have shown that the liver distribution ability of 18α-GA is stronger than that of 18β-GA, the therapeutic effect in treating hepatitis is better, the side effect is weaker than that of 18β-GA, and the safety is better.

[0004] In the existing reports of biological transformation method, the production of 18β-GA by hydrolysis of glycyrrhizic acid with beta-glucuronidase is mainly used. After two-step hydrolysis of glycyrrhizic acid substrate by beta-glucuronidase, 18α-glycyrrhetinic acid and 18β-glycyrrhetinic acid can be obtained, respectively (as shown in Figure 1). The previous research results of the research group of the inventors: CN109628427B reports a recombinant enzyme AtGUS-mix obtained based on domain replacement. When the recombinant enzyme produces 18β-GA under different conditions, the final concentration reaches 16.3g / L, and the reaction period is 96h, but the problem of reaction inhibition caused by accumulation of intermediate 18β-GAMG is not solved.

[0005] In order to seek higher affinity, enzyme activity, catalytic efficiency, conversion rate and better reduction of 18β-GAMG accumulation, and further reduce the production cost of glycyrrhetic acid, it is necessary to develop new β-glucuronidase for industrial preparation of glycyrrhetic acid in the field. SUMMARY

[0006] In order to solve the problem of seeking higher affinity, enzyme activity, catalytic efficiency, conversion rate and better reduction of 18β-GAMG accumulation, and suitable mass production of β-glucuronidase in the prior art, the present application provides a β-glucuronidase and its recombinant expression vector, engineering bacteria, fermentation agent and method for mass production of glycyrrhetic acid.

[0007] The technical scheme of the present application is as follows:

[0008] A β-glucuronidase selected from: β-glucuronidase AcGUS, and / or β-glucuronidase AcGUS1, and / or β-glucuronidase AcGUS2, and / or β-glucuronidase AcGUS3, and / or β-glucuronidase AcGUS3 mutant;

[0009] The β-glucuronidase AcGUS is an enzyme obtained by deleting the 1st amino acid, mutating the 7th alanine to glycine, mutating the 66th arginine to lysine, mutating the 256th alanine to valine, mutating the 270th threonine to alanine, mutating the 332nd valine to phenylalanine, mutating the 363rd aspartic acid to glutamic acid, and mutating the 506th methionine to valine of the amino acid sequence of GenBank accession number AEK69352.1;

[0010] The β-glucuronidase AcGUS1, AcGUS2 and AcGUS3 are enzymes obtained by truncating 1-39 amino acids at the nitrogen end of the truncated β-glucuronidase AcGUS;

[0011] The β-glucuronidase AcGUS3 mutant is selected from: a mutant obtained by mutating the 461st glycine of AcGUS3 to cysteine, or a mutant obtained by mutating the 462nd glutamine of AcGUS3 to histidine, or a mutant obtained by mutating the 575th isoleucine of AcGUS3 to lysine, or a mutant obtained by mutating the 461st glycine of AcGUS3 to cysteine, mutating the 462nd glutamine to histidine and mutating the 575th isoleucine to lysine.

[0012] The amino acid sequence of the β-glucuronidase AcGUS is shown in SEQ ID NO. 1;

[0013] Preferably, the gene sequence of the β-glucuronidase AcGUS is as shown in SEQ ID NO. 2;

[0014] Preferably, the β-glucuronidase AcGUS1 is an enzyme obtained by truncating the β-glucuronidase AcGUS at the N-terminal 10 amino acids;

[0015] Preferably, the β-glucuronidase AcGUS2 is an enzyme obtained by truncating the β-glucuronidase AcGUS at the N-terminal 20 amino acids;

[0016] Preferably, the β-glucuronidase AcGUS3 is an enzyme obtained by truncating the β-glucuronidase AcGUS at the N-terminal 30 amino acids;

[0017] Preferably, the mutant obtained by mutating the glycine at position 461 to cysteine, mutating the glutamine at position 462 to histidine, and mutating the isoleucine at position 575 to lysine of the AcGUS3 is the β-glucuronidase AcGUS3M1.

[0018] A recombinant expression vector connected with the expression vector of the gene sequence of the β-glucuronidase.

[0019] The expression vector is selected from pET28a, pGAPZαA, pPIC9K, pPICZα;

[0020] Preferably, the recombinant expression vector is selected from the recombinant expression vector pGAPZαA-AcGUS, and / or the recombinant expression vector pGAPZαA-AcGUS3, and / or the recombinant expression vector pGAPZαA-AcGUS3M1.

[0021] An engineered bacterium selected from the Pichia pastoris strain AcGUS, and / or the Pichia pastoris strain AcGUS3, and / or the Pichia pastoris strain AcGUS3M1, and / or the Pichia pastoris strain dG-GA1;

[0022] The Pichia pastoris strain AcGUS can express the β-glucuronidase AcGUS;

[0023] The Pichia pastoris strain AcGUS3 can express the β-glucuronidase AcGUS3;

[0024] The Pichia pastoris strain AcGUS3M1 can express the β-glucuronidase AcGUS3M1;

[0025] The Pichia pastoris strain dG-GA1 can simultaneously express the β-glucuronidase AcGUS3M1 and the β-glucuronidase AtGUS from Aspergillus terreus strain Li-20.

[0026] The Pichia pastoris strain AcGUS contains the recombinant expression vector pGAPZαA-AcGUS;

[0027] Preferably, the Pichia pastoris strain AcGUS3 contains the recombinant expression vector pGAPZαA-AcGUS3;

[0028] Preferably, the Pichia pastoris strain AcGUS3M1 contains the recombinant expression vector pGAPZαA-AcGUS3M1;

[0029] Preferably, the Pichia pastoris strain dG-GA1 contains the recombinant expression vector pGAPZαA-AcGUS3M1, the recombinant expression vector pGAPZαA-AcGUS3M1-NrsR modified from the former, and the recombinant expression vector pGAPZαA-AtGUS connected with the β-glucuronidase AtGUS gene sequence from Aspergillus terreus strain Li-20.

[0030] Preferably, the modification refers to replacing the resistance gene of the original expression vector pGAPZαA of the recombinant expression vector pGAPZαA-AcGUS3M1 with NrsR resistance.

[0031] A fermenting agent, comprising a fermenting active ingredient, the fermenting active ingredient comprising the β-glucuronidase, and / or the recombinant expression vector, and / or the engineered bacteria.

[0032] The fermenting agent further comprises an auxiliary material.

[0033] A method for mass production of glycyrrhetic acid, using the β-glucuronidase, and / or the recombinant expression vector, and / or the engineered bacteria to ferment the substrate.

[0034] The substrate is selected from 18α-GL or 18β-GL.

[0035] Preferably, the glycyrrhetic acid is selected from 18α-GA or 18β-GA.

[0036] Preferably, the fermentation conditions include 42.5°C, pH 5.5, stirring speed 300 rpm, and feed concentration 20 g / L.

[0037] The application provides a gene fragment encoding the beta-glucuronidase AcGUS.

[0038] Based on the gene fragment, a beta-glucuronidase AcGUS mutant with significantly improved catalytic efficiency is obtained.

[0039] A method for constructing a beta-glucuronidase AcGUS mutant, wherein 1-39 amino acids at the nitrogen end of the beta-glucuronidase AcGUS are rationally truncated, and the preferred truncated body AcGUS3 has 30 amino acids.

[0040] The truncated body AcGUS3, after heterologous expression of the beta-glucuronidase AcGUS3 in Pichia pastoris, has an enzyme activity that is 7 times that of AcGUS in preparing glycyrrhetic acid, and the accumulation amount of the intermediate product glycyrrhetic acid monoglucuronide is reduced by 3.74 times.

[0041] The truncated body AcGUS3, wherein the 461st glycine of AcGUS3 is mutated into cysteine, the 462nd glutamine is mutated into histidine, and the 575th isoleucine is mutated into lysine, to obtain a new mutant AcGUS3M1.

[0042] The preferred AcGUS3M1 mutant has an activity that is increased by 11.02 times and 6.10 times, respectively, in hydrolyzing the substrates glycyrrhetic acid monoglucuronide and glycyrrhizin.

[0043] A method for constructing a strain with improved activity in hydrolyzing 18alpha-glycyrrhizin or 18beta-glycyrrhizin, wherein the plasmid of the pGAPZalphaA-AcGUS3M1 mutant is introduced into the Pichia pastoris AtGUS host to obtain the Pichia pastoris strain dG-GA1.

[0044] The Pichia pastoris strain dG-GA1 has the characteristics that when glycyrrhizin is used as a substrate, the accumulation of the intermediate product glycyrrhetic acid monoglucuronide can be effectively inhibited, and the system ratio in the fermentation process is always less than 4%.

[0045] The final concentration of 18alpha-glycyrrhetic acid prepared is 41.09 g / L, and the conversion rate is 96.57%.

[0046] The final concentration of 18beta-glycyrrhetic acid prepared is 48.73 g / L, and the conversion rate is 97.26%.

[0047] The application provides a gene encoding a beta-glucuronidase mutant and an application, and through protein molecular modification and screening, a high-efficiency beta-glucuronidase mutant is obtained, and a strain construction method for efficiently preparing glycyrrhetic acid is also provided, and the constructed engineering bacteria are used for industrialized scale production of glycyrrhetic acid. The beta-glucuronidase AcGUS and the mutant thereof provided by the application are different from the existing beta-glucuronidase which preferentially hydrolyzes a substrate GL outside glycoside, and the former is more preferential to hydrolyze a substrate GAMG (i.e. GL inside glycoside) and exhibits excellent activity. The application of the AcGUS mutant to the beta-glucuronidase combination engineering bacteria construction method provided by the application is verified, and it is found that the obtained combination engineering bacteria exhibit excellent industrial application potential when hydrolyzing 18α-GL and 18β-GL in 5L and 1000L fermentors, respectively, and have the advantages of high catalytic efficiency, strong specificity, short process cycle and green environmental protection. Therefore, the application provides a simple and efficient method for industrialized scale preparation of 18α-GA and 18β-GA. BRIEF DESCRIPTION OF DRAWINGS

[0048] Fig. 1 is a schematic diagram of two-step conversion of 18α-glycyrrhizin and 18β-glycyrrhizin by beta-glucuronidase, wherein A: 18α-GL is used to prepare 18α-GA; B: 18β-GL is used to prepare 18β-GA.

[0049] Fig. 2 is an agarose gel electrophoresis verification diagram of gene amplification of AcGUS in the experimental example 2 of the application; wherein lanes 1 and 2 are the PCR product bands of AcGUS, and lane M is a DNA marker.

[0050] Fig. 3 is a liquid phase detection diagram of GAMG accumulation in the 18β-glycyrrhizin conversion process in the experimental example 3 of the application.

[0051] Fig. 4 is a column chart of relative enzyme activity of five beta-glucuronidases: AcGUS, AcGUS1, AcGUS2, AcGUS3 and AcGUS4 respectively hydrolyzing GL to produce GA and GAMG in the eukaryotic expression of Pichia pastoris AcGUS, AcGUS1, AcGUS2, AcGUS3 and AcGUS4 in the experimental example 4 of the application, and the labels of the abscissa respectively correspond to the above-mentioned five beta-glucuronidases.

[0052] Fig. 5 is a column chart of relative enzyme activity of wild type AcGUS3 (WT) and prokaryotic AcGUS3 mutants: AcGUS3 G461C , AcGUS3 Q462H , AcGUS3 I575K, the enzyme activity comparison chart of AcGUS3G461C / I575K, AcGUS3G461C / Q462H / I575K. In which, the test substrates of upper and lower charts are 18β-GL and 18β-GAMG respectively; the labels of abscissa correspond to the wild type and each mutant above respectively.

[0053] Figure 6 is a relative enzyme activity comparison chart of β-glucuronidase expressed by different Pichia pastoris engineering bacteria in the embodiment 7 of the present application. The labels of abscissa are listed as follows: AtGUS refers to the β-glucuronidase AtGUS of the known strain Aspergillus terreus strain Li-20 described in Chinese patent application CN106047839A; AuGUS refers to the β-glucuronidase AuGUS of the known strain Aspergillus ustus strain Li-62 described in Chinese patent application CN106047839A; AtGUS-mix refers to the β-glucuronidase AtGUS-mix described in patent CN109628427B; AcGUS refers to the β-glucuronidase AcGUS described in the present application; AcGUS3M1 refers to the β-glucuronidase AcGUS3M1 described in the present application; and dG-GA1 refers to the β-glucuronidase AcGUS3M1 and the β-glucuronidase AtGUS from Aspergillus terreus strain Li-20 expressed by the Pichia pastoris strain dG-GA1.

[0054] Figure 7 is the enlarged production of GA test of the dG-GA1 combined engineering bacteria in the experimental examples 8 and 9 of the present application. In which, A chart is the substrate and product concentration change chart of converting 18α-GL to prepare 18α-GA in a 5L fermenter; B chart is the substrate and product concentration change chart of converting 18β-GL to prepare 18β-GA in a 1000L fermenter. DETAILED DESCRIPTION

[0055] The present application will be further described by specific examples and experimental examples. It should be understood that the examples are only used to explain and illustrate the present application, and cannot be used to limit the protection scope of the present application. Unless otherwise specified, the experimental methods used in the following examples and experimental examples are conventional methods, and the reagents or materials used can be obtained from commercial channels.

[0056] Sources of biological materials and references

[0057] I. The hot Aspergillus ustus CLH-22 strain mentioned in experimental example 1 is a known strain reported in Chinese patent application CN115786133A.

[0058] II. The E. coli Top10 competent cells used in the experimental example 2 and 5 of the present application are commercially available.

[0059] III. The P. pastoris GS115 used in the experimental example 3 of the present application is commercially available.

[0060] IV. The competent E. coli BL21(DE3) and JM109(DE3) used in the experimental example 4 of the present application are commercially available.

[0061] V. The P. pastoris AtGUS used in the experimental example 6 of the present application is constructed in the laboratory of the inventors, and those skilled in the art can clone the β-glucuronidase AtGUS from Aspergillus terreus strain Li-20 according to the description in item (4) of the “Reagents and consumables” of the present application, and obtain the β-glucuronidase AtGUS from Aspergillus terreus strain Li-20 and transform it into P. pastoris to obtain the engineered P. pastoris AtGUS, without technical obstacles.

[0062] Reagents and consumables

[0063] I. The following are the materials used in the examples:

[0064] (1) E. coli: Top10, BL21(DE3), JM109(DE3).

[0065] (2) P. pastoris: GS115.

[0066] (3) AcGUS is derived from Aspergillus calidoustus strain CLH-22, which is a known strain reported in Chinese patent application CN115786133A.

[0067] (4) AtGUS is derived from Aspergillus terreus strain Li-20, which is a known strain reported in Chinese patent application CN106047839A, with GenBank number JF894133.1.

[0068] (5) AuGUS is derived from Aspergillus ustus strain Li-62, which is a known strain reported in Chinese patent application CN106047839A, with Sequence ID number JN247805.1.

[0069] (6) P. pastoris AtGUS and P. pastoris AuGUS are preserved in the laboratory of the applicant.

[0070] (7) Atgusmix, described in another invention patent of the inventors' research group "An engineered bacterium GA108 / PGAPZαA-Atgusmix for industrial production of glycyrrhetinic acid and a method thereof", with the strain preservation number of CGMCC No. 16731.

[0071] (8) The expression vectors of (1), (2) above are all pGAPZαA, which are commercially available.

[0072] II. Culture medium

[0073] LB medium (per liter): weigh 10 g of tryptone, 5 g of sodium chloride, 5 g of yeast extract, add deionized water to 1 L, sterilize at 121°C for 15 min, and for LB solid medium, add 20 g of agar powder. YPD medium (per liter): 20 g of tryptone, 20 g of glucose monohydrate, 10 g of yeast extract, add deionized water to 1 L, sterilize at 115°C for 15 min, and for YPD solid medium, add 20 g of agar powder.

[0074] (2) Preparation of solutions for construction of Pichia pastoris engineering bacteria

[0075] 1M sorbitol solution: weigh 182.17 g of sorbitol, add 900 mL of deionized water, stir to dissolve, and then add deionized water to 1 L, sterilize at 115°C for 20 min. Yeast lysate: weigh 0.372 g of disodium EDTA, 2 g of NaOH, add 5 mL of Triton X-100, dissolve in deionized water and add deionized water to 1 L. Sterile water: measure 200 mL of deionized water, seal in a conical flask with gauze, sterilize at 121°C for 15 min, and store in a 4°C refrigerator.

[0076] (3) Verification solution and reaction buffer

[0077] 4g / L GL-YPD verification solution: 4g glycyrrhizic acid, 20g tryptone, 20g glucose monohydrate, 10g yeast extract were weighed respectively. 5g / L GL of 50mM HAc-NaAc reaction buffer (pH 5.5): 5g glycyrrhizic acid monoammonium salt and 4.1g anhydrous sodium acetate were weighed, 950mL purified water was added, the pH was adjusted to 5.5 with glacial acetic acid, deionized water was added to dissolve and constant volume to 1L, sterilized at 115℃ for 15min, and stored at 4℃. 5g / L GAMG of 50mM HAc-NaAc reaction buffer (pH 5.5): 5g 18β-GAMG and 4.1g anhydrous sodium acetate were weighed, 950mL purified water was added, the pH was adjusted to 5.5 with glacial acetic acid, deionized water was added to dissolve and constant volume to 1L, sterilized at 115℃ for 15min, and stored at 4℃. 10g / L GL of 50mM HAc-NaAc reaction buffer (pH 5.5): 1g glycyrrhizic acid monoammonium salt and 0.41g anhydrous sodium acetate were weighed, 95mL purified water was added, the pH was adjusted to 5.5 with glacial acetic acid, deionized water was added to dissolve and constant volume to 100mL, heated to dissolve at 60-80℃, stirred for 5min, and cooled to room temperature. It was prepared immediately before use.

[0078] Based on the β-glucuronidase AcGUS and its mutants provided by the present application, when the combined engineering bacteria are constructed for GA production by a combination method, different β-glucuronidases are combined, or routine adjustment and selection of fermentation scale fall within the protection scope of the present application.

[0079] Example 1, β-glucuronidase of the present application

[0080] The present example provides a β-glucuronidase. The present example has the following common features: the β-glucuronidase is selected from: β-glucuronidase AcGUS, and / or β-glucuronidase AcGUS1, and / or β-glucuronidase AcGUS2, and / or β-glucuronidase AcGUS3, and / or β-glucuronidase AcGUS3 mutant;

[0081] The β-glucuronidase AcGUS is an enzyme obtained by deleting the 1st amino acid, mutating the 7th alanine to glycine, mutating the 66th arginine to lysine, mutating the 256th alanine to valine, mutating the 270th threonine to alanine, mutating the 332nd valine to phenylalanine, mutating the 363rd aspartic acid to glutamic acid, and mutating the 506th methionine to valine of the amino acid sequence of GenBank accession number AEK69352.1;

[0082] The β-glucuronidase AcGUS1, AcGUS2 and AcGUS3 are enzymes obtained by truncating 1-39 amino acids from the N-terminal of the β-glucuronidase AcGUS;

[0083] The β-glucuronidase AcGUS3 mutant is selected from the group consisting of: a mutant obtained by mutating the glycine at position 461 of AcGUS3 to cysteine, or a mutant obtained by mutating the glutamine at position 462 of AcGUS3 to histidine, or a mutant obtained by mutating the isoleucine at position 575 of AcGUS3 to lysine, or a mutant obtained by mutating the glycine at position 461 of AcGUS3 to cysteine, the glutamine at position 462 of AcGUS3 to histidine and the isoleucine at position 575 of AcGUS3 to lysine.

[0084] In a specific embodiment, the amino acid sequence of the β-glucuronidase AcGUS is as shown in SEQ ID NO. 1.

[0085] Preferably, the gene sequence of the β-glucuronidase AcGUS is as shown in SEQ ID NO. 2.

[0086] Preferably, the β-glucuronidase AcGUS1 is an enzyme obtained by truncating 10 amino acids from the N-terminal of the β-glucuronidase AcGUS.

[0087] Preferably, the β-glucuronidase AcGUS2 is an enzyme obtained by truncating 20 amino acids from the N-terminal of the β-glucuronidase AcGUS.

[0088] Preferably, the β-glucuronidase AcGUS3 is an enzyme obtained by truncating 30 amino acids from the N-terminal of the β-glucuronidase AcGUS.

[0089] Preferably, the mutant obtained by mutating the glycine at position 461 of AcGUS3 to cysteine, the glutamine at position 462 of AcGUS3 to histidine and the isoleucine at position 575 of AcGUS3 to lysine is the β-glucuronidase AcGUS3M1.

[0090] Any act of cloning, amplifying, enriching, expressing, ligating, transforming, synthesizing, culturing, propagating, fermenting, enriching, producing, preparing, using, inoculating, amplifying, transforming, modifying, altering, selling, or offering for sale gene sequences such as β-glucuronidase AcGUS, and / or β-glucuronidase AcGUS1, and / or β-glucuronidase AcGUS2, and / or β-glucuronidase AcGUS3, and / or β-glucuronidase AcGUS3 mutants, and / or the transcription and translation of the amino acid sequence obtained from gene sequences such as β-glucuronidase AcGUS, and / or β-glucuronidase AcGUS1, and / or β-glucuronidase AcGUS2, and / or β-glucuronidase AcGUS3, and / or β-glucuronidase AcGUS3 mutants. The following actions, including but not limited to the production and preparation of pharmacologically active ingredients such as glycyrrhetinic acid, using gene sequences of β-glucuronidase AcGUS, and / or β-glucuronidase AcGUS1, and / or β-glucuronidase AcGUS2, and / or β-glucuronidase AcGUS3, and / or β-glucuronidase AcGUS3 mutants, are within the scope of protection of this invention: the use of other enzymes in combination, and / or the production of pharmaceutically active ingredients using amino acid sequences obtained by transcription and translation of gene sequences of β-glucuronidase AcGUS, and / or β-glucuronidase AcGUS1, and / or β-glucuronidase AcGUS2, and / or β-glucuronidase AcGUS3, and / or β-glucuronidase AcGUS3 mutants.

[0091] Any act of cloning, amplifying, enriching, expressing, linking, transforming, synthesizing, culturing, propagating, fermenting, enriching, producing, preparing, using, inoculating, amplifying, transforming, modifying, engineering, selling, offering for sale, β-glucuronidase AcGUS, and / or β-glucuronidase AcGUS1, and / or β-glucuronidase AcGUS2, and / or β-glucuronidase AcGUS3, and / or β-glucuronidase AcGUS3 mutant amino acid sequence, and / or, the act of using β-glucuronidase AcGUS, and / or β-glucuronidase AcGUS1, and / or β-glucuronidase AcGUS2, and / or β-glucuronidase AcGUS3, and / or β-glucuronidase AcGUS3 mutant amino acid sequence in combination with other enzymes, and / or, the act of producing a pharmaceutical using β-glucuronidase AcGUS, and / or β-glucuronidase AcGUS1, and / or β-glucuronidase AcGUS2, and / or β-glucuronidase AcGUS3, and / or β-glucuronidase AcGUS3 mutant amino acid sequence, including but not limited to: glycyrrhetinic acid and other pharmaceutically active ingredients, and / or, the act of producing a pharmaceutical using β-glucuronidase AcGUS, and / or β-glucuronidase AcGUS1, and / or β-glucuronidase AcGUS2, and / or β-glucuronidase AcGUS3, and / or β-glucuronidase AcGUS3 mutant amino acid sequence, are within the scope of the present invention.

[0092] The other enzymes include but are not limited to: α-mannosidase, arabinosidase, β-xylosidase, chitotriosidase, thioglucosidase, α-glucosidase, β-galactosidase, β-fructosidase, phosphotriose isomerase, carbonic anhydrase, acetylcholinesterase, catalase, fumarase, β-lactamase, superoxide dismutase, glycogen phosphorylase, hexokinase, lactate dehydrogenase, dehydrase, decarboxylase, carbonic anhydrase, aldolase, citrate synthase, amylase, lipase, phosphatase.

[0093] The skilled in the art can obtain the gene sequence of the β-glucuronidase AcGUS, and / or β-glucuronidase AcGUS1, and / or β-glucuronidase AcGUS2, and / or β-glucuronidase AcGUS3, and / or β-glucuronidase AcGUS3 mutant by reverse compiling the amino acid sequence of the β-glucuronidase AcGUS, and / or β-glucuronidase AcGUS1, and / or β-glucuronidase AcGUS2, and / or β-glucuronidase AcGUS3, and / or β-glucuronidase AcGUS3 mutant according to the actual production needs, combining the conventional technical means or basic common sense of the production process in the field of molecular biology or genetic engineering (for example, Practical Molecular Biology Guide, Guide to Molecular Biology Experiment Technology, Guide to Molecular Cloning Experiment, and Guide to Molecular Biology Experiment), designing specific amplification primers to obtain the gene sequence, and linking the gene sequence to an expression vector to obtain a recombinant expression vector capable of expressing the β-glucuronidase AcGUS, and / or β-glucuronidase AcGUS1, and / or β-glucuronidase AcGUS2, and / or β-glucuronidase AcGUS3, and / or β-glucuronidase AcGUS3 mutant, or further transforming the recombinant expression vector into a competent cell to obtain a transformant (for example, an engineering bacterium) capable of expressing the β-glucuronidase AcGUS, and / or β-glucuronidase AcGUS1, and / or β-glucuronidase AcGUS2, and / or β-glucuronidase AcGUS3, and / or β-glucuronidase AcGUS3 mutant, and the skilled in the art can easily and without technical obstacles, under conditions suitable for the growth of the transformant, propagate and culture the transformant to efficiently produce the β-glucuronidase AcGUS, and / or β-glucuronidase AcGUS1, and / or β-glucuronidase AcGUS2, and / or β-glucuronidase AcGUS3, and / or β-glucuronidase AcGUS3 mutant.

[0094] The recombinant expression vector of the present application

[0095] The present embodiment provides a recombinant expression vector. The present embodiment all has the following common features: an expression vector linked with the gene sequence of any one of the β-glucuronidases in the first embodiment.

[0096] In further embodiments, the expression vector is selected from pET28a, pGAPZαA, pPIC9K, pPICZα;

[0097] Preferably, the recombinant expression vector is selected from the recombinant expression vector pGAPZαA-AcGUS, and / or the recombinant expression vector pGAPZαA-AcGUS3, and / or the recombinant expression vector pGAPZαA-AcGUS3M1.

[0098] Any of the following acts of cloning, amplifying, enriching, expressing, connecting, transforming, synthesizing, culturing, propagating, fermenting, enriching, producing, preparing, using, inoculating, amplifying, transforming, modifying, engineering, selling, promising to sell the recombinant expression vector, and / or the act of using the β-glucuronidase expressed by the recombinant expression vector together with other enzymes, and / or the act of producing and preparing the pharmaceuticals including but not limited to the pharmacologically active ingredients such as glycyrrhetinic acid and the like using the β-glucuronidase expressed by the recombinant expression vector, and / or the act of producing the drugs using the β-glucuronidase expressed by the recombinant expression vector all fall within the protection scope of the present application.

[0099] The other enzymes include but are not limited to: α-mannosidase, arabinosidase, β-xylosidase, chitotriosidase, thioglycosidase, α-glucosidase, β-galactosidase, β-fructosidase, phosphotriose isomerase, carbonic anhydrase, acetylcholinesterase, catalase, fumarase, β-lactamase, superoxide dismutase, glycogen phosphorylase, hexokinase, lactate dehydrogenase, dehydrase, decarboxylase, carbonic anhydrase, aldolase, citrate synthase, amylase, lipase, phosphatase.

[0100] The skilled person in the art can express the recombinant expression vector of the β-glucuronidase of the present application according to the actual production needs, combined with the conventional technical means or basic common sense of the production process in the field of molecular biology or genetic engineering (for example, Practical Molecular Biology Operation Guide, Molecular Biology Experiment Technology Operation Guide, Molecular Cloning Experiment Guide, and Concise Molecular Biology Experiment Guide, etc.), or transform the competent cells with the recombinant expression vector to obtain the transformants (for example, engineering bacteria) that can express the β-glucuronidase of the present application, and propagate and culture them under conditions suitable for the growth of the transformants to make them produce the β-glucuronidase of the present application efficiently, which is no technical obstacle for the skilled person in the art and can be and easily done.

[0101] The engineering bacteria of the present application

[0102] The engineering bacteria of the present application

[0103] The Pichia pastoris strain AcGUS can express the β-glucuronidase AcGUS of any one of the embodiments of the second group;

[0104] The Pichia pastoris strain AcGUS3 can express the β-glucuronidase AcGUS3 of any one of the embodiments of the second group;

[0105] The Pichia pastoris strain AcGUS3M1 can express the β-glucuronidase AcGUS3M1 according to any one of the embodiments of Group 2;

[0106] The Pichia pastoris strain dG-GA1 can simultaneously express the β-glucuronidase AcGUS3M1 according to any one of the embodiments of Group 2 and the β-glucuronidase AtGUS from Aspergillus terreus strain Li-20.

[0107] In some embodiments, the Pichia pastoris strain AcGUS contains the recombinant expression vector pGAPZαA-AcGUS according to any one of the embodiments of Group 2;

[0108] Preferably, the Pichia pastoris strain AcGUS3 contains the recombinant expression vector pGAPZαA-AcGUS3 according to any one of the embodiments of Group 2;

[0109] Preferably, the Pichia pastoris strain AcGUS3M1 contains the recombinant expression vector pGAPZαA-AcGUS3M1 according to any one of the embodiments of Group 2;

[0110] Preferably, the Pichia pastoris strain dG-GA1 contains the recombinant expression vector pGAPZαA-AcGUS3M1 according to any one of the embodiments of Group 2, the recombinant expression vector pGAPZαA-AcGUS3M1-NrsR modified from the recombinant expression vector pGAPZαA-AcGUS3M1, and the recombinant expression vector pGAPZαA-AtGUS connected with the β-glucuronidase AtGUS gene sequence from Aspergillus terreus strain Li-20.

[0111] Preferably, the modification refers to replacing the resistance gene of the original expression vector pGAPZαA of the recombinant expression vector pGAPZαA-AcGUS3M1 with NrsR resistance.

[0112] Any cloning, amplification, enrichment, expression, connection, transformation, synthesis, cultivation, propagation, fermentation, enrichment, production, preparation, use, inoculation, amplification, transformation, modification, modification, sale, promise of sale, such as the behavior of the engineered bacteria, and / or the behavior of using the β-glucuronidase expressed by the engineered bacteria with other enzymes, and / or the behavior of using the β-glucuronidase expressed by the transformant (e.g., engineered bacteria) to produce and prepare active ingredients such as glycyrrhizinic acid and other pharmaceutically active ingredients, and / or the behavior of using the β-glucuronidase expressed by the engineered bacteria to produce drugs, all fall within the scope of protection of the present application.

[0113] The other enzymes include, but are not limited to, alpha-mannosidase, arabinosidase, beta-xylosidase, chitotriosidase, thioglucosidase, alpha-glucosidase, beta-galactosidase, beta-fructosidase, phosphotriose isomerase, carbonic anhydrase, acetylcholine esterase, catalase, fumarase, beta-lactamase, superoxide dismutase, glycogen phosphorylase, hexokinase, lactate dehydrogenase, dehydrase, decarboxylase, carbonic anhydrase, aldolase, citrate synthase, amylase, lipase, phosphatase.

[0114] The skilled in the art can culture, proliferate, ferment, enrich, produce, prepare, use, inoculate the engineered bacteria to express, secrete, produce, obtain the beta-glucuronidase according to the actual production needs, combined with the conventional technical means or basic common sense of the production process in the field of molecular biology or genetic engineering (for example, Practical Molecular Biology Operation Guide, Molecular Biology Experiment Technology Operation Guide, Molecular Cloning Experiment Guide, and Concise Molecular Biology Experiment Guide, etc.), which is no technical obstacle for the skilled in the art and can be easily done.

[0115] The fourth group of embodiments, the starter of the present application

[0116] The embodiments of the present application provide a starter. The embodiments of the present application all have the following common features: the starter comprises a fermentation active ingredient, the fermentation active ingredient comprises the beta-glucuronidase according to any one of the first group of embodiments, and / or the recombinant expression vector according to any one of the second group of embodiments, and / or the engineered bacteria according to any one of the third group of embodiments.

[0117] In further embodiments, the starter further comprises: an auxiliary material.

[0118] In more specific embodiments, the auxiliary material is selected from: a solvent, a propellant, a solubilizer, a cosolvent, an emulsifier, a coloring agent, a binding agent, a disintegrating agent, a filling agent, a lubricant, a wetting agent, an osmotic pressure regulator, a stabilizer, a flow aid, a flavoring agent, a preservative, a suspending agent, a coating material, an aromatic agent, an anti-adhesion agent, an integrating agent, a penetration enhancer, a pH regulator, a buffer, a plasticizer, a surfactant, a foaming agent, an antifoaming agent, a thickening agent, an inclusion agent, a humectant, an absorbent, a diluent, a flocculating agent, a deflocculating agent, a filter aid, a release retardant, etc.

[0119] According to the content of the present application, the skilled in the art can select and adjust the above pharmaceutically acceptable auxiliary materials and prepare different dosage forms, such as powder, tablet, suppository, gel, spray, granule, etc., of the engineered bacteria of the present application, in combination with the conventional technical means in the field of preparation preparation (for example, Encyclopedia of Preparation Technology, Pharmaceutical Preparation Technology, and Research and Application of Microbial Agent Technology, etc.), for different needs in actual production application.

[0120] In specific embodiments, the dosage form of the fermenting agent is selected from one or more of the following: a powder, a tablet, a liquid, a capsule.

[0121] Group 5 embodiments, the method for mass production of glycyrrhetic acid according to the present application

[0122] The embodiments of the present application provide a method for mass production of glycyrrhetic acid. The embodiments of the present application all have the following common features: a β-glucuronidase according to any one of the embodiments of group 1, and / or a recombinant expression vector according to any one of the embodiments of group 2, and / or an engineered bacterium according to any one of the embodiments of group 3 is used for fermentation production of a substrate.

[0123] In specific embodiments, the substrate is selected from 18α-GL or 18β-GL.

[0124] Preferably, the glycyrrhetic acid is selected from 18α-GA or 18β-GA.

[0125] Preferably, the fermentation production conditions include 42.5°C, pH 5.5, stirring speed of 300 rpm, and feed concentration of 20 g / L.

[0126] Experimental Example 1, amplification of β-glucuronidase AcGUS gene

[0127] According to the sequence of β-glucuronidase AcGUS, the gene amplification primers AcGUS-F and AcGUS-R are designed. Then, a cDNA library of Aspergillus clavatus CLH-22 strain is constructed. Here, the RNA extraction kit is used to extract RNA from Aspergillus clavatus CLH-22 strain, and then the RNA reverse transcription reagent PrimeScript TMRT Master Mix was used to reverse transcribe the extracted RNA, and the reverse transcription reaction conditions were as follows: reaction at 37℃ for 15 min, enzyme inactivation at 85℃ for 5 s, and storage at 4℃, thereby obtaining the cDNA library of A. thermus CLH-22. Further, the cDNA library of the A. thermus CLH-22 strain was used as a PCR template, and PCR amplification was performed by using the Takara Phanta system, and the PCR system was as follows: 2X Phanta Max Buffer 25 μL, AcGUS-F and AcGUS-R primers each 2 μL, dNTP Mix 1 μL, cDNA library 1 μL, Phanta Max Super-Fidelity DNA Polymerase 1 μL, and sterile water was added to 50 μL. The PCR reaction program was as follows: pre-denaturation at 95℃ for 3 min, denaturation at 95℃ for 15 s, annealing at Tm-5℃ for 15 s, extension at 72℃ for 2 kb / min, 35 cycles, further extension at 72℃ for 5 min, and storage at 16℃, thereby obtaining the PCR product of AcGUS.

[0128] The following are the PCR primer sequences used in the experimental examples:

[0129] Table 1. PCR amplification primer sequence list

[0130] Experimental Example 2, Construction and Positive Clone Screening of AcGUS Cloning Vector

[0131] (1) Enzymatic digestion and purification of the gene fragment

[0132] The PCR product described in Experimental Example 1 was subjected to 1% agarose gel electrophoresis verification, and the verification results are shown in FIG. 2. The length of the PCR fragment was consistent with the length of AcGUS, indicating that the target gene fragment was obtained. Further, KpnI and NotI double digestion was performed, and after constant temperature reaction at 37℃ for 2 h, the GeneJET gel recovery kit was used for purification of the digested fragment. T4 enzyme ligation reaction was performed on the purified AcGUS gene fragment and the cloning vector pGAPZαA, and the ligation reaction system was as follows: 10X T4 DNA Ligase Buffer 1 μL, pGAPZαA plasmid 1 μL, target gene fragment (about 500 ng), T4 DNA Ligase 1 μL, and sterile water was added to 10 μL. After transient centrifugation, ligation was performed at 22℃ for 2 h, thereby obtaining the ligation product.

[0133] (2) E. coli transformation

[0134] The 10 μL ligation product was added to E. coli Top10 competent cells, and incubated in an ice bath for 30 min. Then, the cells were heat shocked at 42°C for 90 s, and then incubated in an ice bath for 2 min. Then, 500 μL of LB liquid medium was added, and the cells were incubated at 37°C and 200 rpm for 1 h. After incubation, the cells were centrifuged at 5000 rpm for 3 min, and 500 μL of supernatant was removed. The cells were uniformly coated on an LB solid plate containing 50 mg / L of bleomycin, and incubated at 37°C overnight.

[0135] (3) Screening of positive recombinants of E. coli

[0136] The recombinants were identified by colony PCR and plasmid sequencing. The E. coli colony PCR system was as follows: 2X M5Taq HiFi PCR Mix 7.5 μL, pGAP-F and 3AOX1 primers each 1 μL, and template was a single colony of LB plate, and sterile water was added to 15 μL. The PCR reaction program was as follows: pre-denaturation at 94°C for 4 min, denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 2 min, 30 cycles, and then extension at 72°C for 7 min, and storage at 16°C. The recombinants that were verified by colony PCR were sent for DNA sequencing to determine that the pGAPZαA-AcGUS plasmid was successfully constructed, and the gene sequence of AcGUS was the same as that shown in SEQ ID NO. 2.

[0137] Experimental Example 3: Construction of Pichia pastoris AcGUS engineering bacteria and verification of enzyme activity

[0138] (1) Construction of Pichia pastoris AcGUS strain

[0139] The AcGUS was linearized by endonuclease BlnI, and the linearization system was as follows: 10X QuickCut Buffer 2 μL, BlnI 1 μL, total amount of plasmid 1 μg, and sterile water was added to 20 μL. After centrifugation, the reaction was performed at 37°C for 1 h. The linearized plasmid was purified and recovered by using a GeneJET gel recovery kit. The linearized pGAPZαA-AcGUS plasmid was transformed into Pichia pastoris GS115 by using the commonly used electroporation method for Pichia pastoris, and uniformly coated on a YPD solid plate containing 100 mg / L of bleomycin, and incubated at 30°C for 2-4 days.

[0140] Colony PCR was used to identify the Pichia pastoris transformants. 6-12 single colonies were selected from the YPD plate, 50 μL of yeast lysis solution was added, and boiled in boiling water for 30 min. 150 μL of sterile water was added to the PCR tube to obtain the lysis solution template. Colony PCR system: 2xM5 Taq HiFi PCR Mix 7.5 μL, pGAP-F and 3AOX1 primers 1 μL each, lysis solution template 1 μL, and sterile water to 15 μL. PCR reaction program: pre-denaturation 94℃ 10 min, denaturation 94℃ 30 s, annealing 55℃ 30 s, extension 72℃ 2 min, 30 cycles, then extension 72℃ 10 min, 16℃ storage. The PCR product was verified by 1% agarose gel electrophoresis to determine whether the band length met the requirements and whether the transformant construction was successful.

[0141] (2) Enzyme activity verification of AcGUS transformant

[0142] The enzyme activity of the Pichia pastoris AcGUS eukaryotic expression was verified. First, the successfully verified AcGUS transformant was picked into 4 g / L glycyrrhizic acid-YPD verification solution and cultured at 30℃ on a shaker for 2 days. High performance liquid chromatography was used to detect whether the AcGUS strain had GL hydrolysis activity. The liquid phase detection method was as follows: the mobile phase was methanol: glacial acetic acid aqueous solution (6‰) = 84:16, the chromatographic column was Kromasil 100-3.5-C18, the injection volume was 5 μL, the flow rate was 0.8 mL / min, and the detection wavelength was 254 nm. Figure 3 shows the liquid phase detection during substrate conversion. The results show that AcGUS has excellent GL hydrolysis activity, and the accumulation of intermediate product GAMG is extremely low, indicating that AcGUS is a GUS enzyme with higher preference for GAMG hydrolysis. Further, using GL and GAMG buffer substrates (pH 5.5) for verification, it was found that the rate of AcGUS metabolizing GAMG was about 2 times that of GL, which was consistent with the phenotype of wild strain Aspergillus calidoustus CLH-22 (accession number: CGMCC No. 40213). It is shown that AcGUS is the correct target gene and can construct a Pichia pastoris eukaryotic heterologous expression system.

[0143] Experimental Example 4, Construction and verification of AcGUS mutant

[0144] In order to improve the production efficiency of GA as much as possible and reduce the inhibitory effect of intermediate GAMG accumulation, a feasible method for industrial preparation of GA was provided. In this experimental example, the AcGUS was designed to be a more efficient β-glucuronidase by combining enzyme engineering to screen mutant enzymes with improved activity.

[0145] (1) Construction of AcGUS E. coli prokaryotic expression

[0146] First, AcGUS was connected with pET-28a vector by the commonly used Gibson assembly method to obtain the recombinant vector pET-28a-AcGUS. Then, the recombinant plasmid was introduced into competent E. coli BL21(DE3) and JM109(DE3) by E. coli transformation (operation same as experimental example 2), and positive clone screening was performed on LB plates containing 50 mg / L kanamycin resistance. The correct recombinants were verified by colony PCR and sent for DNA sequencing. If the sequencing is correct, the recombinant E. coli is successfully constructed.

[0147] (2) Analysis and construction and verification of N-terminal truncation of AcGUS

[0148] AlphaFold2 was used to simulate the structure of AcGUS, and the structure of the N-terminal flexible Loop ring was analyzed. It was considered that the flexibility of the loop was large and the structural stability was poor, therefore, whether the N-terminal truncation of AcGUS could improve the enzyme activity of heterologous expression was investigated. The N-terminal truncation of 10, 20, 30 and 39 amino acids of the gene was performed by taking pET-28a-AcGUS and pGAPZαA-AcGUS as templates for recombinant N-terminal truncation in E. coli and Pichia pastoris, respectively, and the corresponding prokaryotic or eukaryotic expression of AcGUS1, AcGUS2, AcGUS3 and AcGUS4 was obtained.

[0149] Then, the eukaryotic expression of Pichia pastoris AcGUS, AcGUS1, AcGUS2, AcGUS3 and AcGUS4 was verified by GL transformation and intermediate GAMG accumulation. First, the five kinds of transformants verified successfully were picked from the streak plate to a test tube containing 5 mL YPD liquid, and transferred to a 250 mL flask containing an equal amount of 4 g / L glycyrrhizin-YPD at OD 600 = 0.1, and cultured at 30°C on a shaker for 4 days. Samples were taken every 12 hours, and high performance liquid chromatography was used for detection. The results showed that, as shown in Figure 4, the enzyme activity of AcGUS3 was 7 times higher than that of AcGUS in terms of product GA generation, and had good GL hydrolysis activity. In terms of intermediate GAMG accumulation, the intermediate GAMG accumulation of AcGUS3 was very small, which was 3.74 times lower than that of AcGUS. Further, by measuring the Michaelis kinetics, the data showed that the k cat / K m of AcGUS3 catalyzing GL was 2.94 times that of AcGUS, and the k cat / K m of AcGUS3 catalyzing GAMG was 9.06 times that of AcGUS. The above results showed that the affinity and catalytic efficiency of AcGUS were significantly improved.

[0150] (3) Mutant construction and verification of AcGUS

[0151] By analyzing the three-dimensional structure of AcGUS3, sequence alignment analysis and molecular docking, AcGUS3 pocket G461, Q462, I575 as saturation mutation sites (Table 2). And the site with positive effect is combined mutation. Here, based on AcGUS3 truncation, pET-28a-AcGUS3 plasmid as template, the PCR product after mutation is obtained by plasmid loop P, loop P system: 2xPhanta Flash Master Mix 25μL, 1μL of each upstream and downstream primers, 1-2μL of plasmid template, 50μL of sterile water. PCR reaction program: pre-denaturation 98℃ 30s, denaturation 98℃ 10s, annealing Tm-5℃ 5s, extension 72℃ 50s, 35 cycles, then re-extension 72℃ 1min, 16℃ storage. Then, the PCR product is digested with Dpn I enzyme for 2h and then transformed into competent JM109(DE3). Then, the colony PCR is used for verification, colony PCR system: 2xM5 Taq HiFi PCR Mix 7.5μL, 1μL of each T7 and T7 term universal primer, template is LB plate single colony, supplemented with 15μL of sterile water. PCR reaction program: pre-denaturation 94℃ 4min, denaturation 94℃ 30s, annealing 55℃ 30s, extension 72℃ 2min, 30 cycles, then re-extension 72℃ 7min, 16℃ storage. The correct recombinants verified by colony PCR are sent for DNA sequencing to determine that the mutant plasmid construction is successful.

[0152] Table 2. AcGUS3 saturation mutation primer sequence list

[0153] In order to quickly compare the activity of the mutants, crude enzyme solution is used for verification and comparison. First, the constructed recombinant E. coli is inoculated in 5mL LB medium containing 50mg / L kanamycin resistance, and cultured at 37℃ for 12h. Then, 40mL of LB medium is transferred to 40mL of LB medium at 1% transfer amount, and when OD 600When the OD600 reached 0.6-0.8, IPTG was added for induction. After 18 h of continuous culture at 16℃, 2 mL of cells were collected by centrifugation at 12000 rpm (4℃), and washed twice with 50 mM HAc-NaAc buffer (pH 5.5). Then, 1 mL of 50 mM HAc-NaAc buffer (pH 5.5) and an appropriate amount of beads were added, and the cells were broken by a homogenizer. Subsequently, 200 μL of crude enzyme supernatant and 800 μL of 50 mM HAc-NaAc buffer (pH 5.5) containing 5 g / L GL or GAMG were mixed, respectively. After 5 h and 0.5 h of reaction at 42.5℃, respectively, samples were taken and analyzed by high performance liquid chromatography (HPLC).

[0154] The results of FIG. 5 show that G461, Q462 and I575 are mutation sites with positive effects, in which single-point mutation AcGUS3 G461C , AcGUS3 Q462H and AcGUS3 I575K has hydrolysis activity on GAMG substrate, which is increased by 106.71%, 108.72% and 126.41% compared with the unmutated AcGUS3 (WT), respectively, indicating that the mutation strategy based on pocket engineering has obtained effective mutation sites.

[0155] Therefore, further taking AcGUS3 G461C mutant as the starting strain, iterative saturation mutation was performed on I575, and the saturation mutation library of G461C and I575 was constructed by using AcGUS3-I575-F (SEQ ID NO. 13) and AcGUS3-I575-R (SEQ ID NO. 14) as primers. The screening strategy was the same as that in verification and example 4, and the mutant strain AcGUS3G461C / I575K with further improved hydrolysis capacity was obtained. Compared with the unmutated AcGUS3, the hydrolysis activity of AcGUS3G461C / I575K on GL was increased by 124.29%, and the hydrolysis activity on GAMG was increased by 118.72%.

[0156] Further, the AcGUS3G461C / I575K mutant was used as a starting strain, and iterative saturation mutation was performed with Q462. The saturation mutation library of G461C / I575K and Q462 was constructed by using AcGUS3-Q462N-F (SEQ ID NO. 11) and AcGUS3-Q462N-R (SEQ ID NO. 12) as primers, and the screening strategy was the same as that in verification and example 4. The mutant strain AcGUS3G461C / Q462H / I575K with further improved hydrolysis capacity was obtained. Compared with the control AcGUS3, the enzyme activity was increased by 138.3% (18β-GL group) and 136.6% (18β-GAMG group), respectively. Here, the optimal combination mutant AcGUS3G461C / Q462H / I575K was renamed as AcGUS3M1.

[0157] In addition, the kinetic parameters were further determined for activity characterization, and the results showed that the kcat of the mutant AcGUS3M1 for catalyzing GL was 6.1 times that of the wild-type AcGUS, and the kcat for catalyzing GAMG was 11.02 times that of AcGUS, indicating that the affinity and catalytic efficiency were further improved. The significant improvement of the enzyme activity of AcGUS3M1 provides stronger support for the construction of the β-glucuronidase combination engineering strain in the next step. cat / K m is 6.1 times that of the wild-type AcGUS, and the k cat / K m for catalyzing GAMG is 11.02 times that of AcGUS, indicating that the affinity and catalytic efficiency are further improved. The significant improvement of the enzyme activity of AcGUS3M1 provides stronger support for the construction of the β-glucuronidase combination engineering strain in the next step.

[0158] Experimental Example 5, Construction of Pichia pastoris AcGUS3M1

[0159] Firstly, the pET-28a-AcGUS3M1 plasmid verified in experimental example 4 was used as a template, and the AcGUS3M1 gene was connected with the pGAPZαA vector by the Gibson assembly method to obtain the recombinant vector pGAPZαA-AcGUS3M1 plasmid. Then, the recombinant plasmid was introduced into competent Top10 by heat shock method, and positive clone screening was performed on an LB plate containing 100 mg / L of blasticidin resistance. The correct recombinants were verified by colony PCR and sent for DNA sequencing. If the sequencing is correct, the recombinant pGAPZαA-AcGUS3M1 is successfully constructed. Further, the construction and verification method of Pichia pastoris in experimental example 3 was referred to, and the Pichia pastoris AcGUS3M1 strain was obtained.

[0160] Experimental Example 6, Construction of β-glucuronidase combination engineering strain

[0161] (1) Construction of pGAPZαA-AcGUS3M1-NrsR vector

[0162] In order to facilitate the subsequent screening and construction of the β-glucuronidase combination engineering bacteria, the pGAPZαA-AcGUS3M1 plasmid in Example 5 was replaced with the nourseothricin resistance according to the operation instruction of the Gibson seamless ligation kit of the NEB company, and the blasticidin resistance fragment in the pGAPZαA-AcGUS3M1 plasmid was replaced with the nourseothricin resistance to obtain a new resistant pGAPZαA-AcGUS3M1-NrsR recombinant plasmid.

[0163] (2) Construction and verification of dG-GA1 combination engineering bacteria

[0164] The pGAPZαA-AcGUS3M1-NrsR recombinant plasmid was linearized by endonuclease BlnI, and the linearization system was as follows: 10X QuickCut Buffer 2 μL, BlnI 1 μL, total amount of plasmid 1 μg, and sterile water was added to 20 μL. After transient centrifugation, the reaction was carried out at 37°C for 1 h. The linearized plasmid was purified and recovered using the GeneJET gel recovery kit. The pGAPZαA-AcGUS3M1-NrsR recombinant plasmid linearized plasmid was transformed into Pichia pastoris AtGUS by electroporation, and was uniformly coated on YPD solid plates containing 100 mg / L blasticidin and 100 mg / L nourseothricin, and was incubated at 30°C for 2-3 days.

[0165] The single colonies on the above resistant plates were picked and inoculated in 5 mL of YPD liquid medium containing 100 mg / L blasticidin and 100 mg / L nourseothricin, and were cultured at 30°C and 200 rpm for 2 days. Then, the culture was transferred to YPD medium containing 4 g / L GL at a transfer amount of 1%, and was further cultured for 2 days, and was sampled every 6 h for HPLC detection. It was found that the intermediate product GAMG was always less than 4%, which indicated that the construction of the Pichia pastoris dG-GA1 combination engineering bacteria was successful.

[0166] The recombinant expression vector contained in the engineering bacteria dG-GA1 is pGAPZαA-AtGUS and pGAPZαA-AcGUS3M1-NrsR, wherein NrsR refers to the resistance gene in the original plasmid being replaced with NrsR resistance.

[0167] Experimental Example 7, Comparison of enzyme activities of different Pichia pastoris engineering bacteria for producing GA

[0168] In order to meet the actual production needs, the enzyme activity of different Pichia pastoris used for producing GA is compared by using crude enzyme liquid and high concentration GL for reaction. Here, the examples used for analysis and comparison include AtGUS-mix disclosed in the patent CN109628427B developed by the inventors in the early stage, AcGUS, AcGUS3M1 and dG-GA1 disclosed in the present application, and other comparative examples of the research group of the inventors: AtGUS and AuGUS disclosed in the patent CN109628427B (“Properties and structures of β-glucuronidases with different transformation types of glycyrrhizin” reported the sequences of AuGUS and AtGUS).

[0169] Firstly, single colonies of the above strains were picked from the resistant plates and inoculated into 5 mL YPD medium containing 100 mg / L zeocin, and cultured in a shaker at 30°C and 200 rpm for 48 h. Then, the inoculation amount was 0.1 OD, and the inoculation was transferred to 100 mL YPD medium, and the culture was continued for 48 h. Then, 200 μL of crude enzyme supernatant was mixed with 800 μL of 10 g / L GL reaction buffer (pH 5.5), and reacted at 40°C for 1 h, and then sampled for HPLC detection. 600 =0.1 of the transfer amount, and the transfer was to 100 mL YPD medium, and the culture was continued for 48 h. Then, 200 μL of crude enzyme supernatant was mixed with 800 μL of 10 g / L GL reaction buffer (pH 5.5), and reacted at 40°C for 1 h, and then sampled for HPLC detection.

[0170] The results of FIG. 6 show that under the condition of high concentration GL substrate, the combined engineering strain dG-GA1 performs best, has the highest GA production efficiency, and can maintain a very low accumulation amount of intermediate product GAMG. The comparative example AuGUS has no enzyme activity, which indicates that it cannot be expressed heterologously, which is consistent with the result that AuGUS has been reported to be able to be expressed only in wild-type strains. In addition, AtGUS and AtGUSmix have a high accumulation of GAMG, and the conversion efficiency is much weaker than that of dG-GA1. It is worth noting that in the present application, the mutant AcGUS3M1 expressed by the recognized safe production strain Pichia pastoris is still 13.18 times higher than the activity of the wild-type AcGUS, which again indicates the effectiveness of the rational design mutant described in experimental example 4, which can not only be expressed in prokaryotic Escherichia coli, but also be successfully expressed in eukaryotic Pichia pastoris. Therefore, the combined engineering strain dG-GA1 is considered to be the best GA production strain in the enzyme activity comparison.

[0171] Experimental Example 8: 5L Fermentor Scale-up Preparation of 18α-GA

[0172] The ability of dG-GA1 to produce 18a-GA was verified in a 5L fermenter. dG-GA1 was inoculated from a streaked YPD plate into a test tube containing 5mL of YPD liquid medium and activated at 30°C for 36h on a 200rpm shaker. The seed liquid was then inoculated into a 100mL YPD liquid medium at OD 600 = 0.1 and incubated at 30°C for 24h on a 200rpm shaker. The seed liquid was then inoculated into a 5L fermenter containing 3L of YPD liquid medium. The culture conditions were set at 30°C, pH 5.5 and 200rpm stirring speed. The fermentation was carried out in a fed-batch mode and the fermentation stage lasted for about 60h. The strain growth OD 600 was monitored in real time. The substrate catalysis stage was carried out at 42.5°C, pH 5.5 and 300rpm stirring speed. The 18a-GL was fed at a concentration of 20g / L each time and the reaction was monitored in real time. The results are shown in Figure 7A. The results show that the conversion process lasted for 20h and the final concentration of 18a-GA reached 41.09g / L with a conversion rate of 96.57%. This demonstrates that dG-GA1 has good industrial application potential in the conversion of 18a-GL.

[0173] Experimental Example 9: Preparation of 18b-GA in a 1000L Fermenter

[0174] To further verify the production capacity of dG-GA1 and to characterize the ability to convert 18b-GL, a 1000L fermenter was used. In the fermentation stage, dG-GA1 was inoculated from a streaked YPD plate into a 100mL YPD liquid medium and incubated at 30°C for about 24h. The seed liquid was then inoculated into a 10L, 100L and 1000L fermenter containing YPD medium in sequence. The fermentation was carried out at 30°C, pH 5.5 and 200rpm stirring speed. The seed liquid of the 10L fermenter and the 100L fermenter were incubated for about 24h. In addition, the culture was incubated in the 1000L fermenter for about 48h and then 20g / L of glucose was fed every 12h for a total of two times. The strain growth OD 600 was monitored in real time. In the substrate catalysis stage, the reaction conditions were set at 42.5°C, pH 5.5 and 300rpm stirring speed. The 18b-GL was fed at a concentration of 20g / L each time and the concentrations of the substrate 18b-GL, the intermediate product GAMG and 18b-GA were monitored in real time. The results are shown in Figure 7B.

[0175] The results show that the whole substrate catalysis stage consumes 24 h, and 6 times of substrate 18β-GL feeding are performed. At 24 h, the GA concentration is 48.73 g / L, and the GL conversion rate reaches 97.26%. At the same time, during the whole amplification test process, the accumulation amount of 18β-GAMG accounts for less than 4% in the reaction system, and the GAMG concentration at 24 h is less than 1.43 g / L. It meets the demand of solving the foregoing engineering problem, and the efficiency and yield are the highest in the currently reported processes.

[0176] From the above results, it can be seen that the dG-GA1 combined engineering bacteria have excellent comprehensive performance, which proves the significant effect of AcGUS and its mutant in hydrolyzing GAMG and the effectiveness of the β-glucuronidase combination strategy. Further, it is proved that the present application has the advantages of high catalytic efficiency, short fermentation period and environmentally friendly process, and has the prospect of industrialized production of glycyrrhetic acid.

Claims

1. A β-glucuronidase, characterized in that, which is selected from the group consisting of: β-glucuronidase AcGUS, and / or β-glucuronidase AcGUS1, and / or β-glucuronidase AcGUS2, and / or β-glucuronidase AcGUS3, and / or β-glucuronidase AcGUS3 mutant; the β-glucuronidase AcGUS is an enzyme obtained by deleting the 1st amino acid, mutating the 7th alanine to glycine, mutating the 66th arginine to lysine, mutating the 256th alanine to valine, mutating the 270th threonine to alanine, mutating the 332nd valine to phenylalanine, mutating the 363rd aspartic acid to glutamic acid, and mutating the 506th methionine to valine of the amino acid sequence of GenBank Accession No. AEK69352.1; the β-glucuronidase AcGUS1, AcGUS2, and AcGUS3 is an enzyme obtained by truncating the 1-39 amino acids at the N-terminus of the β-glucuronidase AcGUS; the β-glucuronidase AcGUS3 mutant is selected from the group consisting of: a mutant obtained by mutating the 461st glycine to cysteine of AcGUS3, or a mutant obtained by mutating the 462nd glutamine to histidine of AcGUS3, or a mutant obtained by mutating the 575th isoleucine to lysine of AcGUS3, or a mutant obtained by mutating the 461st glycine to cysteine, the 462nd glutamine to histidine, and the 575th isoleucine to lysine of AcGUS3.

2. The β-glucuronidase according to claim 1, characterized in that, the amino acid sequence of the β-glucuronidase AcGUS is shown as SEQ ID NO. 1; and / or, the gene sequence of the β-glucuronidase AcGUS is shown as SEQ ID NO. 2; the β-glucuronidase AcGUS1 is an enzyme obtained by truncating the 10 amino acids at the N-terminus of the β-glucuronidase AcGUS; the β-glucuronidase AcGUS2 is an enzyme obtained by truncating the 20 amino acids at the N-terminus of the β-glucuronidase AcGUS; the β-glucuronidase AcGUS3 is an enzyme obtained by truncating the 30 amino acids at the N-terminus of the β-glucuronidase AcGUS; the mutant obtained by mutating the 461st glycine to cysteine, the 462nd glutamine to histidine, and the 575th isoleucine to lysine of AcGUS3 is β-glucuronidase AcGUS3M1.

3. A recombinant expression vector, characterized in that, an expression vector linked with the gene sequence of the β-glucuronidase according to claim 1 or 2.

4. The recombinant expression vector according to claim 3, wherein, the expression vector is selected from the group consisting of pET28a, pGAPZαA, pPIC9K, pPICZα; and / or, the recombinant expression vector is selected from the group consisting of recombinant expression vector pGAPZαA-AcGUS, and / or recombinant expression vector pGAPZαA-AcGUS3, and / or recombinant expression vector pGAPZαA-AcGUS3M1.

5. An engineered bacterium, characterized in that, Pichia pastoris strain AcGUS, and / or Pichia pastoris strain AcGUS3, and / or Pichia pastoris strain AcGUS3M1, and / or Pichia pastoris strain dG-GA1; The Pichia pastoris strain AcGUS can express the β-glucuronidase AcGUS as claimed in claim 1 or 2; The Pichia pastoris strain AcGUS3 can express the β-glucuronidase AcGUS3 as claimed in claim 1 or 2; The Pichia pastoris strain AcGUS3M1 can express the β-glucuronidase AcGUS3M1 as claimed in claim 1 or 2; The Pichia pastoris strain dG-GA1 can express both the β-glucuronidase AcGUS3M1 as claimed in claim 1 or 2 and the β-glucuronidase AtGUS from Aspergillus terreus strain Li-20.

6. The engineered bacterium of claim 5, wherein the bacterium is Escherichia coli. The Pichia pastoris strain AcGUS contains the recombinant expression vector pGAPZαA-AcGUS as claimed in claim 4; The Pichia pastoris strain AcGUS3 contains the recombinant expression vector pGAPZαA-AcGUS3 as claimed in claim 4; The Pichia pastoris strain AcGUS3M1 contains the recombinant expression vector pGAPZαA-AcGUS3M1 as claimed in claim 4; The Pichia pastoris strain dG-GA1 contains the recombinant expression vector pGAPZαA-AcGUS3M1 as claimed in claim 4, the recombinant expression vector pGAPZαA-AcGUS3M1-NrsR after modification of the former, and the recombinant expression vector pGAPZαA-AtGUS with the β-glucuronidase AtGUS gene sequence from Aspergillus terreus strain Li-20 connected thereto; The modification refers to replacing the resistance gene of the original expression vector pGAPZαA of the recombinant expression vector pGAPZαA-AcGUS3M1 with NrsR resistance.

7. A leavening agent comprising a fermenting active ingredient, characterized in that, The fermentation active ingredient includes a β-glucuronidase as claimed in claim 1 or 2, and / or a recombinant expression vector as claimed in claim 3 or 4, and / or an engineered bacterium as claimed in claim 5 or 6.

8. A fermenting agent according to claim 7, characterised in that, Also included are: Auxiliary materials.

9. A method of mass production of glycyrrhetinic acid, characterized by, The fermentation production of the substrate is carried out using a β-glucuronidase as claimed in claim 1 or 2, and / or a recombinant expression vector as claimed in claim 3 or 4, and / or an engineered bacterium as claimed in claim 5 or 6.

10. The process for mass production of glycyrrhetinic acid according to claim 9, wherein, The substrate is selected from 18α-GL or 18β-GL; The glycyrrhetinic acid is selected from 18α-GA or 18β-GA; The fermentation production conditions include 42.5℃, pH 5.5, stirring speed 300 rpm, and feed concentration 20 g / L.

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