β-Glucuronidase and its recombinant expression vector, engineered bacterium, fermenting agent, and method for mass-producing glycyrrhetinic acid

Abstract

To obtain high affinity, enzyme activity, catalytic efficiency, and transformation rate, better reduce 18β-GAMG accumulation, and solve the problem of β-glucuronidase suitable for mass production, the present invention provides a β-glucuronidase, its recombinant expression vector, engineering bacteria, fermentation agent, and a method for mass-producing glycyrrhetinic acid. 【Solution means】It belongs to the fields of enzyme engineering and microbial technology. The β-glucuronidase is selected from β-glucuronidase AcGUS, AcGUS1, AcGUS2, AcGUS3, and AcGUS3 mutants, and AcGUS is an enzyme with specific amino acid mutations. The present invention is simple and efficient, and can produce 18α-GA and 18β-GA on an industrial scale.

Description

Technical Field

[0001] The present invention relates to the fields of enzyme engineering and microbial technology. Specifically, it relates to β-glucuronidase, its recombinant expression vector, engineering bacteria, fermentation agent, and a method for mass-producing glycyrrhetinic acid.

Background Art

[0002] Licorice is a traditional medicinal and edible homologous plant and is widely applied in industries such as medicine, food, and cosmetics. According to historical records, licorice has been used as a medicine for more than four thousand years, and it can be said that "among the court elders in the morning, licorice ranks first among the medicines." Licorice not only has effects such as antipyretic and detoxifying, tonifying the qi of the spleen, moistening the lungs and relieving cough, and harmonizing various medicines, but is also used for symptoms such as throat swelling and pain, spleen and stomach function disorders, and peptic ulcers, and is widely applied in aspects such as anti-inflammatory, liver protection, antioxidant, antiviral, antitumor, and antidiuretic. Licorice or its active ingredients are added to the compound formulations of many modern traditional Chinese medicines.

[0003] Glycyrrhizic acid (GL) and glycyrrhetinic acid (GA) are the main components and active ingredients of licorice, respectively. Currently, the production of glycyrrhetinic acid reported mainly includes chemical methods and biotransformation methods. CN101817867A uses glycyrrhizinate as a raw material, generates acetylglycyrrhetinic acid with sulfuric acid and high-concentration acetic acid as catalysts, and then further deacetylates to obtain glycyrrhetinic acid. A large amount of strong acids, strong bases and organic solvents are required for the reaction, which has problems such as high energy consumption, low yield and large ecological stress. On the other hand, the biotransformation method has the advantages of mild conditions, high yield, and environmental friendliness. It can also provide higher-purity pharmaceutical precursors for the production of different derivatives, and is an industrial production method with development prospects. The applications of GA in anti-inflammatory, liver protection, antioxidant, antiviral, antitumor and antidiuretic effects have been reported in many literatures. According to research, the liver distribution ability of 18α-GA is stronger than that of 18β-GA, and it is more effective in the treatment of hepatitis, with weaker side effects and better safety than 18β-GA.

[0004] In the reports of conventional biotransformation methods, it mainly uses β-glucuronidase to hydrolyze glycyrrhizic acid to produce 18β-GA. After the glycyrrhizic acid substrate undergoes two-step hydrolysis of β-glucuronidase, 18α-glycyrrhetinic acid and 18β-glycyrrhetinic acid (as shown in Figure 1) can be obtained respectively. In the Chinese invention patent CN109628427B, which is the prior research result of the inventor's research group, a recombinant enzyme AtGUS-mix by domain substitution has been reported. When this recombinant enzyme produces 18β-GA under different conditions, the final concentration is up to 16.3 g / L and the reaction cycle is 96 h, but it does not solve the problem of reaction inhibition prone to occur due to the accumulation of the intermediate 18β-GAMG.

[0005] To achieve higher affinity, enzyme activity, catalytic efficiency, and transformation rate, better reduce the accumulation of 18β-GAMG, and further reduce the production cost of glycyrrhetinic acid, it is necessary for this field to develop a new β-glucuronidase for industrial production of glycyrrhetinic acid.

Summary of the Invention

Problems to be Solved by the Invention

[0006] For the purpose of solving the problem of β-glucuronidase with higher affinity, enzyme activity, catalytic efficiency, transformation rate in the conventional technology of this field, better reducing the accumulation of 18β-GAMG, and being suitable for mass production, the present invention provides a β-glucuronidase, its recombinant expression vector, engineering bacteria, fermentation agent, and a method for mass-producing glycyrrhetinic acid.

Means for Solving the Problems

[0007] The technical solution of the present invention is as follows: 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 a mutant of β-glucuronidase AcGUS3, The β-glucuronidase AcGUS is an enzyme in which the first amino acid of the amino acid sequence with GenBank accession number AEK69352.1 is deleted, the 7th alanine is mutated to glycine, the 66th arginine is mutated to lysine, the 256th alanine is mutated to valine, the 270th threonine is mutated to alanine, the 332nd valine is mutated to phenylalanine, the 363rd aspartic acid is mutated to glutamic acid, and the 506th methionine is mutated to valine, The β-glucuronidases AcGUS1, AcGUS2, and AcGUS3 are enzymes obtained by cleaving the 1-39 amino acids at the nitrogen terminus of β-glucuronidase AcGUS. The β-glucuronidase AcGUS3 mutant is selected from a mutant in which glycine at position 461 of AcGUS3 is mutated to cysteine, or a mutant in which glutamine at position 462 of AcGUS3 is mutated to histidine, or a mutant in which isoleucine at position 575 of AcGUS3 is mutated to lysine, or a mutant in which glycine at position 461 of AcGUS3 is mutated to cysteine, glutamine at position 462 is mutated to histidine, and isoleucine at position 575 is mutated to lysine.

[0008] The amino acid sequence of the β-glucuronidase AcGUS is shown in SEQ ID NO.1, Preferably, the gene sequence of the β-glucuronidase AcGUS is shown in SEQ ID NO.2, Preferably, the β-glucuronidase AcGUS1 is an enzyme obtained by cleaving 10 amino acids at the N-terminus of the β-glucuronidase AcGUS, Preferably, the β-glucuronidase AcGUS2 is an enzyme obtained by cleaving 20 amino acids at the N-terminus of the β-glucuronidase AcGUS, Preferably, the β-glucuronidase AcGUS3 is an enzyme obtained by cleaving 30 amino acids at the N-terminus of the β-glucuronidase AcGUS, Preferably, the mutant in which glycine at position 461 of AcGUS3 is mutated to cysteine, glutamine at position 462 is mutated to histidine, and isoleucine at position 575 is mutated to lysine is β-glucuronidase AcGUS3M1.

[0009] A recombinant expression vector, which is ligated to the expression vector of the gene sequence of the above-mentioned β-glucuronidase.

[0010] The expression vector is selected from pET28a, pGAPZαA, pPIC9K, pPICZα, 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.

[0011] An engineered bacterium, selected from the Pichia yeast strain AcGUS, and / or the Pichia yeast strain AcGUS3, and / or the Pichia yeast strain AcGUS3M1, and / or the Pichia yeast strain dG-GA1, The Pichia yeast strain AcGUS can express the β-glucuronidase AcGUS described above, The Pichia yeast strain AcGUS3 can express the β-glucuronidase AcGUS3 described above, The Pichia yeast strain AcGUS3M1 can express the β-glucuronidase AcGUS3M1 described above, The Pichia yeast strain dG-GA1 can simultaneously express the β-glucuronidase AcGUS3M1 described above and the β-glucuronidase AtGUS derived from the Aspergillus terreus strain Li-20.

[0012] The Pichia yeast strain AcGUS contains the recombinant expression vector pGAPZαA-AcGUS described above, Preferably, the Pichia yeast strain AcGUS3 contains the recombinant expression vector pGAPZαA-AcGUS3 described above, Preferably, the Pichia yeast strain AcGUS3M1 contains the recombinant expression vector pGAPZαA-AcGUS3M1 described above, Preferably, the Pichia yeast strain dG-GA1 contains the recombinant expression vector pGAPZαA-AcGUS3M1-NrsR in which the recombinant expression vector pGAPZαA-AcGUS3M1 described above is modified, and the recombinant expression vector pGAPZαA-AtGUS in which the β-glucuronidase AtGUS gene sequence derived from the Aspergillus terreus strain Li-20 is ligated. Preferably, the modification is to replace the resistance gene of the original expression vector pGAPZαA of the recombinant expression vector pGAPZαA-AcGUS3M1 with NrsR resistance.

[0013] A ferment containing a fermentation active ingredient, wherein the fermentation active ingredient includes the above-mentioned β-glucuronidase, and / or the above-mentioned recombinant expression vector, and / or the above-mentioned engineered bacterium.

[0014] The above-mentioned ferment further includes an adjuvant.

[0015] A method for mass-producing glycyrrhetinic acid, which uses the above-mentioned β-glucuronidase, and / or the above-mentioned recombinant expression vector, and / or the above-mentioned engineered bacterium to ferment and produce a substrate.

[0016] The above-mentioned substrate is selected from 18α-GL or 18β-GL, Preferably, the above-mentioned glycyrrhetinic acid is selected from 18α-GA or 18β-GA, Preferably, the conditions for the above-mentioned fermentation production include 42.5 °C, pH 5.5, stirring rotation speed of 300 rpm, and charging concentration of 20 g / L.

[0017] According to one aspect of the present invention, a gene fragment encoding the above-mentioned β-glucuronidase AcGUS is provided.

[0018] Based on the above-mentioned gene fragment, a β-glucuronidase AcGUS mutant with significantly improved catalytic efficiency is obtained.

[0019] A method for constructing a β-glucuronidase AcGUS mutant, which performs a rational cleavage operation on the 1-39 amino acids at the N-terminus of β-glucuronidase AcGUS, and the preferred number of amino acid cleavages of the cleavage product AcGUS3 is 30.

[0020] After heterologous expression of the aforementioned truncated AcGUS3 in Pichia pastoris, β-glucuronidase AcGUS3 has a 7-fold improvement in enzyme activity and a 3.74-fold decrease in the accumulation of the intermediate product monoglucuronide glycyrrhetinic acid in the production of glycyrrhetinic acid compared to AcGUS.

[0021] The aforementioned truncated AcGUS3 is mutated by changing glycine at position 461 of AcGUS3 to cysteine, glutamine at position 462 to histidine, and isoleucine at position 575 to lysine to obtain a new mutant AcGUS3M1.

[0022] The aforementioned preferred AcGUS3M1 mutant is characterized by 11.02-fold and 6.10-fold improvements in activity in the hydrolysis of the substrate monoglucuronide glycyrrhetinic acid and glycyrrhetic acid, respectively.

[0023] A method for constructing a strain with improved activity in hydrolyzing 18α-glycyrrhetic acid or 18β-glycyrrhetic acid, which involves introducing the plasmid of the pGAPZαA-AcGUS3M1 mutant into the Pichia pastoris AtGUS host to obtain the Pichia pastoris strain dG-GA1.

[0024] The aforementioned Pichia pastoris strain dG-GA1 can effectively suppress the accumulation of the intermediate product monoglucuronide glycyrrhetinic acid when using glycyrrhetic acid as the substrate, and the proportion of the system in the fermentation process is always lower than 4%.

[0025] The final concentration for producing 18α-glycyrrhetinic acid reaches 41.09 g / L, and the conversion rate is 96.57%. The final concentration for producing 18β-glycyrrhetinic acid reaches 48.73 g / L, and the conversion rate is 97.26%.

Advantages of the Invention

[0026] The present invention provides a coding gene of a β-glucuronidase mutant and its application. By molecular modification and screening of proteins, an efficient β-glucuronidase mutant is obtained, and a method for constructing a strain for efficiently producing glycyrrhetinic acid is further provided. The constructed engineering bacteria are used for industrial-scale production of glycyrrhetinic acid. Different from the conventional β-glucuronidase that prefers hydrolysis of the outer glycoside of substrate GL, the β-glucuronidase AcGUS and its mutants according to the present invention prefer hydrolysis of substrate GAMG (i.e., the inner glycoside of GL) and exhibit extremely excellent activity. Applying the AcGUS mutant to the method for constructing the β-glucuronidase combinatorial engineering bacteria proposed in the present invention, it is verified that when the obtained combinatorial engineering bacteria hydrolyze 18α-GL and 18β-GL in 5L and 1000L fermenters respectively, they all show excellent potential for industrial application, and are found to have advantages such as high catalytic efficiency, strong specificity, short process cycle, and being eco-friendly and environmentally friendly. Thus, it can be seen that the present invention provides a method that is simple and efficient for the field and can produce 18α-GA and 18β-GA on an industrial scale.

Brief Description of the Drawings

[0027]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Modes for Carrying Out the Invention

[0028] Hereinafter, the present invention will be further described by specific examples and experimental examples. It should be understood that the above examples are only for the interpretation and explanation of the present invention, and do not limit the protection scope of the present invention. Unless otherwise specified, the experimental methods used in the following examples and experimental examples are all ordinary methods, and the reagents or materials used can all be obtained by commercial methods.

[0029] Origin and source of biological materials I. The Aspergillus calidoustus CLH-22 strain mentioned in Experimental Example 1 is a known strain reported in Chinese Patent Application CN115786133A.

[0030] II. The E. coli Top10 competent state used in Experimental Examples 2 and 5 of the present invention is commercially available.

[0031] III. The Pichia pastoris GS115 used in Experimental Example 3 of the present invention is commercially available.

[0032] IV. The competent states E. coli BL21(DE3) and JM109(DE3) used in Experimental Example 4 of the present invention are commercially available.

[0033] V. The Pichia pastoris AtGUS used in Experimental Example 6 of the present invention was constructed in the inventor's laboratory. Those skilled in the art can clone from the reported Aspergillus terreus strain Li-20 to obtain β-glucuronidase AtGUS derived from Aspergillus terreus strain Li-20 according to the description in Article (4) of "Reagents and Consumables" of the present invention, ligate it to Pichia pastoris and transform it to obtain the engineered strain Pichia pastoris AtGUS without technical obstacles.

[0034] Reagents and Consumables I. The following are the materials used in the examples: (1) Escherichia coli: Top10, BL21(DE3), JM109(DE3).

[0035] (2) Pichia pastoris: GS115.

[0036] (3) AcGUS is derived from Aspergillus calidoustus strain CLH-22, which is a known strain reported in Chinese Patent Application CN115786133A.

[0037] (4) AtGUS is derived from Aspergillus terreus strain Li-20, which is a known strain reported in Chinese Patent Application CN106047839A, and the GenBank number is JF894133.1.

[0038] (5) AuGUS is derived from Aspergillus ustus strain Li-62, which is a known strain reported in Chinese Patent Application CN106047839A, and the Sequence ID number is JN247805.1.

[0039] (6) Pichia pastoris AtGUS and Pichia pastoris AuGUS are stored in the applicant's laboratory.

[0040] (7) Atgusmix is described in another invention patent of the inventor's project team, "Engineering Bacterium GA108 / PGAPZαA-Atgusmix and Method for Industrially Producing Glycyrrhetinic Acid", and the deposit number of the bacterial strain is CGMCC No. 16731.

[0041] (8) The above (1), (2) and the expression vector are all pGAPZαA and are all commercially available.

[0042] II. Medium LB medium (per liter): Weigh 10 g of tryptone, 5 g of sodium chloride, 5 g of yeast extract, add deionized water to make up to 1 L, sterilize at 121 °C for 15 min. For LB solid medium, 20 g of agar powder needs to be added separately. YPD medium (per liter): Weigh 20 g of tryptone, 20 g of glucose monohydrate, 10 g of yeast extract, add deionized water to make up to 1 L, sterilize at 115 °C for 15 min. For YPD solid medium, 20 g of agar powder needs to be added separately.

[0043] (2) Preparation of solutions for constructing Pichia pastoris engineering bacteria 1M sorbitol alcohol solution: Weigh 182.17 g of sorbitol alcohol, add 900 mL of deionized water, stir well to dissolve, then make up to 1 L, and sterilize at 115 °C for 20 min. Yeast lysate buffer: Weigh 0.372 g of disodium EDTA and 2 g of NaOH respectively, add 5 mL of Triton X-100, add deionized water to dissolve, and make up to 1 L. Sterile water: Measure 200 mL of deionized water, seal it in an Erlenmeyer flask with gauze, sterilize at 121 °C for 15 min, and store it in a refrigerator at 4 °C.

[0044] (3) Verification solution and reaction buffer 4 g / L GL - YPD verification solution: Weigh 4 g of glycyrrhizic acid, 20 g of tryptone, 20 g of glucose monohydrate, and 10 g of yeast extract respectively. 5 g / L GL in 50 mM HAc - NaAc reaction buffer (pH 5.5): Weigh 5 g of ammonium monoammonium glycyrrhizinate and 4.1 g of sodium acetate anhydrous, add 950 mL of purified water, adjust the pH to 5.5 with glacial acetic acid, add deionized water to dissolve, make up to 1 L, sterilize at 115 °C for 15 min, and store at 4 °C. 5 g / L GAMG in 50 mM HAc - NaAc reaction buffer (pH 5.5): Weigh 5 g of 18β - GAMG and 4.1 g of sodium acetate anhydrous, add 950 mL of purified water, adjust the pH to 5.5 with glacial acetic acid, add deionized water to dissolve, make up to 1 L, sterilize at 115 °C for 15 min, and store at 4 °C. 10 g / L GL in 50 mM HAc - NaAc reaction buffer (pH 5.5): Weigh 1 g of ammonium monoammonium glycyrrhizinate and 0.41 g of sodium acetate anhydrous, add 95 mL of purified water, adjust the pH to 5.5 with glacial acetic acid, add deionized water to dissolve, make up to 100 mL, heat and dissolve at 60 - 80 °C, then stir for 5 min and cool to room temperature. It can be used after preparation.

[0045] Based on the β - glucuronidase AcGUS and its mutants according to the present invention, when constructing a combined engineering bacterium by a combination method for GA production, combination based on different β - glucuronidases, or normal adjustment and selection for the fermentation scale, all fall within the protection scope of the present invention.

[0046] The first set of examples, the β - glucuronidase of the present invention The examples in this set provide β - glucuronidase. All the examples in this set have the following common features, that is, 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 the mutant of β - glucuronidase AcGUS3. The β-glucuronidase AcGUS is an enzyme in which the first amino acid of the amino acid sequence with GenBank accession number AEK69352.1 is deleted, the seventh alanine is mutated to glycine, the 66th arginine is mutated to lysine, the 256th alanine is mutated to valine, the 270th threonine is mutated to alanine, the 332nd valine is mutated to phenylalanine, the 363rd aspartic acid is mutated to glutamic acid, and the 506th methionine is mutated to valine. The β-glucuronidases AcGUS1, AcGUS2, and AcGUS3 are enzymes obtained by cleaving the 1-39 amino acids at the N-terminus of β-glucuronidase AcGUS. The β-glucuronidase AcGUS3 mutant is selected from a mutant in which the 461st glycine of AcGUS3 is mutated to cysteine, a mutant in which the 462nd glutamine of AcGUS3 is mutated to histidine, a mutant in which the 575th isoleucine of AcGUS3 is mutated to lysine, or a mutant in which the 461st glycine of AcGUS3 is mutated to cysteine, the 462nd glutamine is mutated to histidine, and the 575th isoleucine is mutated to lysine.

[0047] In a specific embodiment, the amino acid sequence of the β-glucuronidase AcGUS is shown in SEQ ID NO.1. Preferably, the gene sequence of the β-glucuronidase AcGUS is shown in SEQ ID NO.2. Preferably, the β-glucuronidase AcGUS1 is an enzyme obtained by cleaving the 10 amino acids at the N-terminus of β-glucuronidase AcGUS. Preferably, the β-glucuronidase AcGUS2 is an enzyme obtained by cleaving the 20 amino acids at the N-terminus of β-glucuronidase AcGUS. Preferably, the β-glucuronidase AcGUS3 is an enzyme obtained by cleaving the 30 amino acids at the N-terminus of β-glucuronidase AcGUS. Preferably, the mutant in which the 461st glycine of AcGUS3 is mutated to cysteine, the 462nd glutamine is mutated to histidine, and the 575th isoleucine is mutated to lysine is β-glucuronidase AcGUS3M1.

[0048] For example, any act of cloning, amplifying, concentrating, expressing, ligating, transforming, synthesizing, culturing, growing, fermenting, producing, manufacturing, using, inoculating, modifying, transforming, selling, or promising to sell the gene sequence 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; and / or any act of using in combination with other enzymes the amino acid sequence transcribed and translated from the gene sequence 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; and / or any act of producing and manufacturing components including but not limited to pharmaceutically active ingredients such as glycyrrhetinic acid using the amino acid sequence transcribed and translated from the gene sequence 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; and / or any act of producing drugs using the amino acid sequence transcribed and translated from the gene sequence 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 are all within the protection scope of the present invention.

[0049] Cloning, amplifying, concentrating, expressing, ligating, transforming, synthesizing, culturing, growing, fermenting, producing, manufacturing, using, inoculating, modifying, altering, selling, promising to sell, and / or any act of, for example, combining the amino acid sequence 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 with other enzymes, and / or producing and manufacturing components including, but not limited to, pharmaceutically active ingredients such as glycyrrhetinic acid using, for example, the amino acid sequence 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, and / or producing drugs using, for example, the amino acid sequence 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 are all within the scope of protection of the present invention.

[0050] The other enzymes include, but are not limited to, α-mannosidase, arabinosidase, β-xylosidase, chitosatriglycosidase, thioglycosidase, α-glucosidase, β-galactosidase, β-ketofuranoside, triosephosphate isomerase, carbonic anhydrase, acetylcholinesterase, catalase, fumarase, β-lactamase, superoxide dismutase, glycogen phosphorylase, hexokinase, lactate dehydrogenase, anhydrase, decarboxylase, carbonic anhydrase, aldolase, citrate synthase, amylase, lipase, phosphatase.

[0051] Those skilled in the art can, according to actual production needs, combine with the ordinary technical means or basic common sense of molecular biology or genetic engineering production processes (for example, "Practical Molecular Biology Operation Guide", "Experimental Operation Guide for Molecular Biology Experimental Techniques", "Molecular Cloning Experimental Guide", "Compiled Molecular Biology Experimental Guide", etc.), reverse-compile the amino acid sequences of the said β-glucuronidase AcGUS, and / or β-glucuronidase AcGUS1, and / or β-glucuronidase AcGUS2, and / or β-glucuronidase AcGUS3, and / or β-glucuronidase AcGUS3 mutant to obtain its gene sequence, design specific amplification primers to obtain its gene sequence, ligate it with an expression vector to obtain a recombinant expression vector capable of expressing 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 transform the recombinant expression vector into recipient cells to obtain a transformant (such as an engineered bacterium) capable of expressing β-glucuronidase AcGUS, and / or β-glucuronidase AcGUS1, and / or β-glucuronidase AcGUS2, and / or β-glucuronidase AcGUS3, and / or β-glucuronidase AcGUS3 mutant, grow and culture it under conditions suitable for the growth of the transformant, so as to efficiently produce the said β-glucuronidase AcGUS, and / or β-glucuronidase AcGUS1, and / or β-glucuronidase AcGUS2, and / or β-glucuronidase AcGUS3, and / or β-glucuronidase AcGUS3 mutant, which has no technical obstacles for those skilled in the art and can be easily carried out.

[0052] The second set of examples, the recombinant expression vector of the present invention The examples of this set provide a recombinant expression vector. The examples of this set all have the following common features: an expression vector to which the gene sequence of β-glucuronidase described in any one of the first set of examples is ligated.

[0053] In a further embodiment, the expression vector is selected from pET28a, pGAPZαA, pPIC9K, pPICZα, 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.

[0054] For example, any act of cloning, amplifying, concentrating, expressing, ligating, transforming, synthesizing, culturing, growing, fermenting, producing, manufacturing, using, inoculating, modifying, transforming, selling, or promising to sell the recombinant expression vector, and / or an act of binding β-glucuronidase expressed by the recombinant expression vector to other enzymes, and / or an act of producing and manufacturing components including but not limited to pharmaceutically active ingredients such as glycyrrhetinic acid with β-glucuronidase expressed by the recombinant expression vector, and / or an act of producing drugs with β-glucuronidase expressed by the recombinant expression vector are all within the protection scope of the present invention.

[0055] The other enzymes include but are not limited to α-mannosidase, arabinosidase, β-xylosidase, chitosatriglycosidase, thioglycosidase, α-glucosidase, β-galactosidase, β-ketofuranoside, triosephosphate isomerase, carbonic anhydrase, acetylcholinesterase, catalase, fumarase, β-lactamase, superoxide dismutase, glycogen phosphorylase, hexokinase, lactate dehydrogenase, anhydrase, decarboxylase, carbonic anhydrase, aldolase, citrate synthase, amylase, lipase, phosphatase.

[0056] Those skilled in the art can, according to actual production needs, combine with the ordinary technical means or basic common sense of the molecular biology or genetic engineering production process (for example, "Practical Molecular Biology Operation Guide", "Molecular Biology Experiment Technology Experiment Operation Guide", "Molecular Cloning Experiment Guide", "Compiled Molecular Biology Experiment Guide", etc.), express the recombinant expression vector of β-glucuronidase of the present invention with the said recombinant expression vector, or transform the said recombinant expression vector into recipient cells to obtain a transformant (for example, engineered bacteria) capable of expressing the β-glucuronidase of the present invention, grow and culture it under conditions suitable for the growth of the transformant, so as to efficiently produce the β-glucuronidase described in the present invention, which has no technical obstacles for those skilled in the art and can be easily carried out.

[0057] Examples of the third group, engineered bacteria of the present invention Examples of this group provide engineered bacteria. All examples of this group have the following common characteristics: the said engineered bacteria are selected from Pichia yeast strain AcGUS, and / or Pichia yeast strain AcGUS3, and / or Pichia yeast strain AcGUS3M1, and / or Pichia yeast strain dG-GA1, the said Pichia yeast strain AcGUS is capable of expressing β-glucuronidase AcGUS described in any one of the examples of the second group, the said Pichia yeast strain AcGUS3 is capable of expressing β-glucuronidase AcGUS3 described in any one of the examples of the second group, the said Pichia yeast strain AcGUS3M1 is capable of expressing β-glucuronidase AcGUS3M1 described in any one of the examples of the second group, the said Pichia yeast strain dG-GA1 is capable of simultaneously expressing β-glucuronidase AcGUS3M1 described in any one of the examples of the second group and β-glucuronidase AtGUS derived from Aspergillus terreus strain Li-20.

[0058] In some embodiments, the Pichia yeast strain AcGUS contains the recombinant expression vector pGAPZαA-AcGUS described in any one of the second set of embodiments. Preferably, the Pichia yeast strain AcGUS3 contains the recombinant expression vector pGAPZαA-AcGUS3 described in any one of the second set of embodiments. Preferably, the Pichia yeast strain AcGUS3M1 contains the recombinant expression vector pGAPZαA-AcGUS3M1 described in any one of the second set of embodiments. Preferably, the Pichia yeast strain dG-GA1 contains the recombinant expression vector pGAPZαA-AcGUS3M1-NrsR obtained by modifying the recombinant expression vector pGAPZαA-AcGUS3M1 described in any one of the second set of embodiments, and the recombinant expression vector pGAPZαA-AtGUS in which the β-glucuronidase AtGUS gene sequence derived from the Aspergillus terreus strain Li-20 is ligated. Preferably, the modification is to replace the resistance gene of the original expression vector pGAPZαA of the recombinant expression vector pGAPZαA-AcGUS3M1 with NrsR resistance.

[0059] For example, any act of cloning, amplifying, concentrating, expressing, ligating, transforming, synthesizing, culturing, growing, fermenting, producing, manufacturing, using, inoculating, modifying, transforming, selling, or promising to sell the engineering bacteria, and / or the act of binding the β-glucuronidase expressed by the engineering bacteria to other enzymes, and / or the act of producing and manufacturing components including but not limited to pharmaceutically active ingredients such as glycyrrhetinic acid with the β-glucuronidase expressed by the transformant (e.g., engineering bacteria), and / or the act of producing drugs with the β-glucuronidase expressed by the engineering bacteria are all within the protection scope of the present invention.

[0060] The other enzymes include, but are not limited to, α-mannosidase, arabinosidase, β-xylosidase, chitosatriglycosidase, thioglycosidase, α-glucosidase, β-galactosidase, β-ketofuranoside, triosephosphate isomerase, carbonic anhydrase, acetylcholinesterase, catalase, fumarase, β-lactamase, superoxide dismutase, glycogen phosphorylase, hexokinase, lactate dehydrogenase, anhydrase, decarboxylase, carbonic anhydrase, aldolase, citrate synthase, amylase, lipase, phosphatase.

[0061] Those skilled in the art can, according to actual production needs, in combination with the ordinary technical means or basic common sense of molecular biology or genetic engineering production processes (for example, "Practical Molecular Biology Operation Guide", "Molecular Biology Experiment Technology Experiment Operation Guide", "Molecular Cloning Experiment Guide", "Compiled Molecular Biology Experiment Guide", etc.), culture, grow, ferment, concentrate, produce, manufacture, use, and inoculate the engineering bacteria to express, secrete, produce, and obtain β-glucuronidase, which has no technical obstacles for those skilled in the art and can be easily carried out.

[0062] Examples of the fourth group, the ferment of the present invention The examples of this group provide a ferment. All the examples of this group have the following common features: The ferment contains a fermentation active ingredient, and the fermentation active ingredient includes β-glucuronidase described in any one of the examples of the first group, and / or a recombinant expression vector described in any one of the examples of the second group, and / or an engineering bacterium described in any one of the examples of the third group.

[0063] In a further example, the ferment further contains an adjuvant.

[0064] In more specific embodiments, the auxiliary agent is selected from solvents, propellants, solubilizing agents, solubilization aids, emulsifiers, coloring agents, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, suspension aids, coating materials, fragrances, anti-adhesives, chelating agents, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoaming agents, thickeners, inclusion agents, moisturizing agents, absorbents, diluents, flocculants, resolubilizing agents, filter aids, release blockers, and the like.

[0065] Based on the content of the present invention, according to different needs in actual production applications, in combination with ordinary technical means in the field of pharmaceutical manufacturing (such as "Encyclopedia of Pharmaceutical Technology", "Drug Formulation Technology", "Research and Application of Microbial Agent Technology", etc.), those skilled in the art can select and formulate the above pharmaceutically acceptable auxiliary agents, and manufacture the engineering bacteria of the present invention into different dosage forms such as powders, tablets, suppositories, gels, sprays, granules, and the like.

[0066] In specific embodiments, the dosage form of the fermentation agent is selected from one or more of powders, tablets, liquids, and capsules.

[0067] Examples of Group 5, Method for mass-producing glycyrrhetinic acid of the present invention The examples in this group provide a method for mass-producing glycyrrhetinic acid. All the examples in this group have the following common features: fermenting and producing the substrate using β-glucuronidase described in any one item of the examples of Group 1, and / or the recombinant expression vector described in any one item of the examples of Group 2, and / or the engineering bacteria described in any one item of the examples of Group 3.

[0068] In specific embodiments, the substrate is selected from 18α-GL or 18β-GL, Preferably, the glycyrrhetinic acid is selected from 18α-GA or 18β-GA, Preferably, the conditions for the fermentative production include 42.5 °C, pH 5.5, stirring rotation speed of 300 rpm, and charging concentration of 20 g / L.

[0069] Experimental Example 1, Amplification of β-Glucuronidase AcGUS Gene Based on the β-glucuronidase AcGUS sequence, gene amplification primers AcGUS-F and AcGUS-R are designed according to the purpose. Subsequently, a cDNA library of Aspergillus calidoustus CLH-22 strain is constructed. Here, the Aspergillus calidoustus CLH-22 strain is extracted with RNA using a rapid RNA extraction reagent kit, and then the extracted RNA is reverse-transcribed using the RNA reverse transcription reagent PrimeScript TM RT Master Mix. The reverse transcription reaction conditions are: react at 37°C for 15 min, inactivate the enzyme at 85°C for 5 s, and store at 4°C to obtain a cDNA library of Aspergillus calidoustus CLH-22. Furthermore, using the cDNA library of Aspergillus calidoustus CLH-22 strain as a PCR template, PCR amplification is performed by the Novizan Phanta system. The PCR system is: 25 μL of 2×Phanta Max Buffer, 2 μL each of AcGUS-F and AcGUS-R primers, 1 μL of dNTP Mix, 1 μL of cDNA library, 1 μL of Phanta Max Super-Fidelity DNA Polymerase, and supplemented to 50 μL with sterile water. The PCR reaction procedure is: pre-denature at 95°C for 3 min, denature at 95°C for 15 s, anneal at Tm - 5°C for 15 s, extend at 72°C at 2 kb / min, cycle 35 times, then extend at 72°C for 5 min and store at 16°C to obtain the PCR product of AcGUS.

[0070] The following are the PCR primer sequences used in the experimental example:

[0071] [Table 1]

[0072] Experimental Example 2, Construction of AcGUS Clone Vector and Screening of Positive Clones (1) Enzyme Cleavage and Purification of Gene Fragment Perform 1% agarose gel electrophoresis verification on the PCR product described in Experimental Example 1, and show the verification results in Figure 2. That the length of the PCR fragment is consistent with the length of AcGUS indicates that the target gene fragment is initially obtained. Furthermore, perform double digestion with KpnI and NotI, and after a constant temperature reaction at 37°C for 2 h, use the GeneJET Gel Extraction Reagent Kit to purify the digested fragment. Perform a T4 enzyme ligation reaction on the purified AcGUS gene fragment and the cloning vector pGAPZαA. The enzyme ligation reaction system: 1 μL of 10X T4 DNA Ligase Buffer, 1 μL of pGAPZαA plasmid, the target gene fragment (about 500 ng), 1 μL of T4 DNA Ligase, and supplement to 10 μL with sterile water. After instantaneous centrifugation, ligate at a constant temperature of 22°C for 2 h to obtain a ligation product.

[0073] (2) Escherichia coli transformation Add 10 μL of the ligation product to the competent state of Escherichia coli Top10 and incubate in an ice bath for 30 min. Then, perform a heat shock in a 42°C water bath for 90 s, incubate in an ice bath for 2 min again, add 500 μL of LB liquid medium, and incubate at 37°C on a rocking bed at 200 rpm for 1 h. Centrifuge the incubated bacterial solution at 5000 rpm for 3 min, remove 500 μL of the supernatant, uniformly spread it on an LB solid plate containing 50 mg / L of bleomycin, and culture overnight at a constant temperature of 37°C.

[0074] (3) Screening of Escherichia coli positive recombinants Identify the recombinants using colony PCR and plasmid sequencing. E. coli colony PCR system: 7.5 μL of 2×M5 Taq HiFi PCR Mix, 1 μL each of pGAP-F and 3AOX1 primers, the template is a single colony on the LB plate, and supplement to 15 μL with sterile water. PCR reaction procedure: 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, perform 30 cycles, then extend at 72°C for 7 min, and store at 16°C. The recombinants verified to be accurate by colony PCR are sent for DNA sequencing to determine the successful construction of the pGAPZαA-AcGUS plasmid, and the gene sequence of AcGUS is shown in SEQ ID NO.2.

[0075] Experimental Example 3, Construction of Pichia pastoris AcGUS Engineering Bacteria and Verification of Enzyme Activity (1) Construction of Pichia pastoris AcGUS Strain Linearize AcGUS using endonuclease BlnI. The linearization system is: 2 μL of 10X QuickCut Buffer, 1 μL of BlnI, 1 μg of total plasmid, and supplement with sterile water to 20 μL. After instantaneous centrifugation, react at a constant temperature of 37°C for 1 h. Purify and recover the linearized plasmid using the GeneJET Gel Extraction Reagent Kit. Transfer the pGAPZαA-AcGUS linearized plasmid into Pichia pastoris GS115 using the electroporation method commonly used in Pichia pastoris, uniformly coat it on a YPD solid plate containing 100 mg / L bleomycin, and culture at 30°C for 2 - 4 days.

[0076] Identify Pichia pastoris transformants using colony PCR. Select 6 - 12 single colonies from the YPD plate, add 50 μL of yeast lysis buffer, boil in boiling water for 30 min, and add 150 μL of sterile water to the PCR tube to obtain the lysis buffer template. Colony PCR system: 7.5 μL of 2×M5 Taq HiFi PCR Mix, 1 μL each of pGAP-F and 3AOX1 primers, 1 μL of lysis buffer template, and supplement with sterile water to 15 μL. PCR reaction procedure: Pre-denature at 94°C for 10 min, denature at 94°C for 30 s, anneal at 55°C for 30 s, extend at 72°C for 2 min, perform 30 cycles, then extend at 72°C for 10 min and store at 16°C. The PCR products are verified by 1% agarose gel electrophoresis to determine whether the band length meets the requirements and decide whether the construction of the transformant is successful.

[0077] (2) Verification of Enzyme Activity of AcGUS Transformants Verify the enzyme activity expressed eukaryotically by Pichia yeast AcGUS. First, collect the successfully verified AcGUS transformants into a 4 g / L glycyrrhizic acid - YPD verification solution, culture them on a rocking bed at 30 °C for 2 days, and use high - performance liquid chromatography to detect whether the AcGUS strain has the hydrolysis activity of GL. Liquid - phase detection method: The mobile phase is methanol: aqueous acetic acid solution (6‰) = 84:16, the chromatography column is Kromasil 100 - 3.5 - C18, the sample injection volume is 5 μL, the flow rate is 0.8 mL / min, and the detection wavelength is 254 nm. Figure 3 shows the liquid - phase detection situation during substrate transformation. According to the results, AcGUS shows slightly excellent GL hydrolysis activity, and at the same time, the accumulation amount of its intermediate product GAMG is extremely low, indicating that AcGUS prefers higher GUS enzymes by hydrolyzing GAMG. Furthermore, verification was carried out using GL and GAMG buffer substrates (pH 5.5) respectively, and it was found that the rate of AcGUS metabolizing the substrate GAMG is about twice that of GL, which is consistent with the phenotype during the verification of the wild - type strain Aspergillus calidoustus CLH - 22 (deposit number: CGMCC No. 40213). This indicates that AcGUS is the correct target gene and that a Pichia yeast eukaryotic heterologous expression system can be constructed.

[0078] Experimental Example 4, Construction and Verification of Mutants of AcGUS Provide a feasible method for industrially manufacturing GA in order to improve the production efficiency of GA as much as possible and reduce the inhibitory effect caused by the accumulation of the intermediate GAMG. In this experimental example, mutant enzymes with improved activity are selected by combining enzyme engineering, and AcGUS is designed to be a more efficient β - glucuronidase.

[0079] (1) Construction of Prokaryotic Expression of AcGUS in Escherichia coli First, AcGUS was ligated with the pET-28a vector by the commonly used Gibson assembly method to obtain the recombinant vector pET-28a-AcGUS. Subsequently, Escherichia coli transformation (the operation is the same as in Experimental Example 2) was performed to introduce the recombinant plasmid into the recipient states E. coli BL21(DE3) and JM109(DE3) respectively. Positive clone selection was carried out on LB plates containing 50 mg / L kanamycin resistance, and the recombinants verified to be accurate by colony PCR were sent for DNA sequencing. If the sequence was accurate, the construction of recombinant E. coli was successful.

[0080] (2) N-terminal analysis of AcGUS and construction and verification of cleavage products The structural simulation of AcGUS was performed using AlphaFold2, and structural analysis was carried out on the flexible Loop loop at the N-terminus. Since the flexibility of this loop is large and the structural stability is relatively poor, it was considered whether the cleavage of the N-terminus of AcGUS could improve the enzyme activity of heterologous expression. However, for the recombinant N-terminal cleavage products of heterologous expression in Escherichia coli and Pichia pastoris, using pET-28a-AcGUS and pGAPZαA-AcGUS as templates respectively, 10, 20, 30, and 39 amino acids were cleaved from the N-terminus of the gene, and AcGUS1, AcGUS2, AcGUS3, and AcGUS4 expressed in prokaryotes or eukaryotes were obtained correspondingly.

[0081] Subsequently, comparative analysis and verification of GL transformation and intermediate GAMG accumulation were performed for the eukaryotic expression of Pichia pastoris AcGUS, AcGUS1, AcGUS2, AcGUS3, and AcGUS4. First, five types of transformants successfully verified from the score plates were taken out into test tubes containing 5 mL of YPD liquid, OD 600Duplicate into a 250 mL vial containing 4 g / L glycyrrhizic acid - YPD of the same amount at = 0.1, continue culturing in a rocking bed at 30 °C for 4 days, sample once every 12 h, and detect using high - performance liquid chromatography. According to the results, as shown in Figure 4, in the production of the product GA, AcGUS3 has a 7 - fold improvement in enzyme activity compared to AcGUS and has relatively good GL hydrolysis activity. In the accumulation of the intermediate GAMG, the accumulation amount of GAMG, which is an intermediate of AcGUS3, is very small and is 3.74 - fold lower than that of AcGUS. Furthermore, using GL and GAMG as substrates respectively, the pure enzyme was analyzed by measuring the milky dynamics. According to the data, k cat / K m for AcGUS3 to catalyze GL is 2.94 times that of AcGUS, but k cat / K m for AcGUS3 to catalyze GAMG is shown to be 9.06 times that of AcGUS, indicating that the affinity and catalytic efficiency of AcGUS have been significantly improved.

[0082] (3) Construction and verification of mutants of AcGUS By analyzing enzyme engineering methods such as the three-dimensional structure of AcGUS3, sequence alignment analysis, and molecular docking, select G461, Q462, and I575 within a distance of 5 Å in the AcGUS3 pocket as saturation mutation sites (Table 2). Combine and mutate the sites with positive effects. Here, based on the AcGUS3 fragment, using the pET-28a-AcGUS3 plasmid as a template, obtain the PCR product after mutation with plasmid loop P. The loop P system: 25 μL of 2×Phanta Flash Master Mix, 1 μL each of the upstream and downstream primers, 1 - 2 μL of the plasmid template, and supplement to 50 μL with sterile water. PCR reaction procedure: pre-denaturation at 98°C for 30 s, denaturation at 98°C for 10 s, annealing at Tm - 5°C for 5 s, extension at 72°C for 50 s, perform 35 cycles, then extend at 72°C for 1 min, and store at 16°C. Subsequently, digest the PCR product with Dpn I enzyme for 2 h and then perform E. coli transformation, introducing it into the competent state JM109(DE3). Subsequently, verify by colony PCR. The colony PCR system: 7.5 μL of 2×M5 Taq HiFi PCR Mix, 1 μL each of the T7 and T7 term universal primers, the template is a single colony on the LB plate, and supplement to 15 μL with sterile water. PCR reaction procedure: 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, perform 30 cycles, then extend at 72°C for 7 min, and store at 16°C. The recombinant verified to be accurate by colony PCR is sent for DNA sequencing and determined to have successfully constructed the mutant plasmid.

[0083]

Table 2

[0084] To rapidly compare the activities of the mutants, verification and comparison are performed using the crude enzyme solution. First, inoculate the constructed recombinant E. coli into 5 mL of LB medium containing 50 mg / L kanamycin resistance and culture at 37°C for 12 h. Then, duplicate it into 40 mL of LB medium at a 1% duplication amount, and OD 600When it reaches 0.6 - 0.8, IPTG is added for induction. After continuous culturing at 16 °C for 18 h, 2 mL of cells are collected by centrifugation at 12,000 rpm (4 °C) and washed twice with 50 mM HAc - NaAc buffer (pH 5.5). Subsequently, 1 mL of 50 mM HAc - NaAc buffer (pH 5.5) and an appropriate amount of disruption beads are added, and disruption treatment is carried out using a homogenizer. Then, 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 are mixed respectively. After reacting at 42.5 °C for 5 h and 0.5 h respectively, sampling is carried out for detection and analysis by high - performance liquid chromatography (HPLC).

[0085] According to the results in Figure 5, G461, Q462 and I575 are mutant sites with positive effects. Here, single - site mutation AcGUS3 G461C , AcGUS3 Q462H and AcGUS3 I575K showed that the hydrolysis activities of the GAMG substrate were improved by 106.71%, 108.72% and 126.41% respectively compared with the unmutated AcGUS3 (WT), indicating that the mutation policy based on pocket engineering can obtain effective mutant sites.

[0086] Therefore, using the AcGUS3 G461C mutant as the starting strain, iterative saturation mutagenesis was performed with I575. Using AcGUS3 - I575 - F (SEQ ID NO.13) and AcGUS3 - I575 - R (SEQ ID NO.14) as primers, a saturation mutation library of G461C and I575 was constructed. The screening policy and verification were the same as those of the single - site saturation mutation in Example 4, and a mutant strain AcGUS3 G461C / I575K with further improved hydrolysis ability was obtained. AcGUS3 G461C / I575K showed that compared with the unmutated AcGUS3, the hydrolysis GL activity was improved by 124.29% and the hydrolysis GAMG activity was improved by 118.72%.

[0087] Furthermore, AcGUS3 G461C / I575KUsing the mutant as the starting strain, iterative saturation mutagenesis was performed with Q462. Using AcGUS3-Q462N-F (SEQ ID NO.11) and AcGUS3-Q462N-R (SEQ ID NO.12) as primers, a saturation mutation library of G461C / I575K and Q462 was constructed. The screening policy and verification were the same as those for the single-site saturation mutagenesis in Example 4. A mutant strain AcGUS3 with further improved hydrolysis ability was obtained. G461C / Q462H / I575K Compared with the control AcGUS3, the enzyme activities were increased by 138.3% (18β-GL group) and 136.6% (18β-GAMG group), respectively. Here, the optimal combinatorial mutant AcGUS3 G461C / Q462H / I575K was renamed AcGUS3M1.

[0088] In addition, the kinetic parameters were further measured and the activity was characterized. According to the results, the k cat / K m of the mutant AcGUS3M1 for catalyzing GL was 6.1 times that of the wild-type AcGUS, while the k cat / K m for catalyzing GAMG was 11.02 times that of AcGUS, indicating that the affinity and catalytic efficiency were further improved. The significant improvement in the enzyme activity of AcGUS3M1 provided strong support for the construction of the next-step β-glucuronidase combinatorial engineering bacteria.

[0089] Experimental Example 5, Construction of Pichia pastoris AcGUS3M1 First, using the pET-28a-AcGUS3M1 plasmid verified in Experimental Example 4 as a template, AcGUS was ligated with the 3M1 gene and the pGAPZαA vector by the Gibson assembly method to obtain the recombinant vector pGAPZαA-AcGUS3M1 plasmid. Subsequently, the recombinant plasmid was introduced into competent Top10 using the heat shock method, and positive clones were screened on an LB plate containing 100 mg / L bleomycin resistance. The recombinants verified to be accurate by colony PCR were sent for DNA sequencing. If the sequence was accurate, the construction of recombinant pGAPZαA-AcGUS3M1 was successful. Further referring to the Pichia pastoris construction and verification method in Experimental Example 3, the Pichia pastoris AcGUS3M1 strain was obtained.

[0090] Experimental Example 6, Construction of β-Glucuronidase Combinatorial Engineering Bacteria (1) Construction of the pGAPZαA-AcGUS3M1-NrsR Vector To facilitate the subsequent screening and construction of β-glucuronidase combinatorial engineering bacteria, the pGAPZαA-AcGUS3M1 plasmid in Example 5 was used to replace the bleomycin resistance fragment in the pGAPZαA-AcGUS3M1 plasmid with noramycin resistance according to the instruction manual of the NEB Gibson seamless ligation reagent kit to obtain a new resistant pGAPZαA-AcGUS3M1-NrsR recombinant plasmid.

[0091] (2) Construction and Verification of the dG-GA1 Combinatorial Engineering Bacteria The pGAPZαA-AcGUS3M1-NrsR recombinant plasmid was linearized using endonuclease BlnI. The linearization system was as follows: 2 μL of 10X QuickCut Buffer, 1 μL of BlnI, a total of 1 μg of plasmid, and supplemented with sterile water to 20 μL. After instantaneous centrifugation, the reaction was carried out at a constant temperature of 37 °C for 1 h. The linearized plasmid was purified and recovered using the GeneJET Gel Extraction Reagent Kit. The pGAPZαA-AcGUS3M1-NrsR recombinant plasmid linearized plasmid was transferred to Pichia pastoris AtGUS using the electroporation method, uniformly coated on a YPD solid plate containing 100 mg / L bleomycin and 100 mg / L noromycin, and cultured at a constant temperature of 30 °C for 2 - 3 days.

[0092] A single colony from the above resistance plate was collected, inoculated into 5 mL of YPD liquid medium containing 100 mg / L bleomycin and 100 mg / L noromycin, and cultured for 2 days on a rocking bed at 30 °C and 200 rpm. Then, it was subcultured into YPD medium containing 4 g / L GL at a subculturing amount of 1%, and the subculturing continued for 2 days. Sampling was performed once every 6 h for HPLC detection. According to the detection, it was found that the intermediate product GAMG was always at a ratio of 4% or less, indicating the successful construction of the Pichia pastoris dG-GA1 combinatorial engineering bacterium.

[0093] The recombinant expression vectors contained in this engineering bacterium dG-GA1 are pGAPZαA-AtGUS and pGAPZαA-AcGUS3M1-NrsR. Here, NrsR means that the resistance gene in the original plasmid is replaced with NrsR resistance.

[0094] Experimental Example 7: Comparison of Enzyme Activities of Different Pichia pastoris Engineering Bacteria for Producing GA. To meet the actual production needs, the comparison format of the enzyme activities of different Pichia pastoris for producing GA reacts with a relatively high concentration of GL using crude enzyme solution. Here, the examples for analysis and comparison include AtGUSmix described in the patent CN109628427B developed by the inventor in the previous stage, AcGUS, AcGUS3M1, and dG-GA1 described in the present invention, and other comparative examples of the project team to which the inventor belongs: AtGUS and AuGUS described in the patent CN109628427B (“Properties and structures of β-glucuronidases with different transformation types of glycyrrhizin ", Xiaoyan Wang, Yanli Liu, Chao Wang, Xudong Feng and Chun Li, RSC Adv., 2015, 5, 68345 - 68350, Fig. 3 ) which reported the sequences of AuGUS and AtGUS).

[0095] First, collect single colonies of the above strains from the resistant plates and place them in 5 mL of YPD medium containing 100 mg / L zeocin, and culture them at 30 °C and 200 rpm on a rocking bed for 48 h. Then, duplicate them into a medium with a liquid content of 100 mL of YPD at a duplicating amount of OD 600 = 0.1, and continue culturing for 48 h. Then, take 200 μL of each crude enzyme supernatant, mix it with 800 μL of 10 g / L GL reaction buffer (pH 5.5), react at 40 °C for 1 h, and then sample for HPLC detection.

[0096] According to the results of Figure 6, it is shown that in the case of a relatively high concentration of GL substrate, the combined engineering bacterium dG-GA1 appears the best, its GA production efficiency is the highest, and it can maintain an extremely low accumulation amount of the intermediate product GAMG. In contrast, Comparative Example AuGUS is shown to have no enzyme activity and cannot be heterologously expressed, which is consistent with the reported result that it can only be expressed in AuGUS and wild strains. Also, AtGUS and Atgusmix have relatively high GAMG accumulation, and their transformation efficiency is much weaker than that of dG-GA1. It should be noted that in the present invention, the mutant AcGUS3M1 in the recognized safe production bacterium Pichia pastoris expression is still 13.18 times higher than the activity of the AcGUS wild type, which shows again that the effectiveness of the mutant obtained by the rational design described in Experimental Example 4 can not only be prokaryotically expressed in Escherichia coli, but also be successfully expressed in the eukaryotic Pichia pastoris. Therefore, the combined engineering bacterium dG-GA1 is recognized as the optimal GA production bacterium in the enzyme activity comparison.

[0097] Experimental Example 8, Amplified production of 18α-GA in a 5L fermenter Verify the ability of dG-GA1 to produce 18α-GA in a 5L fermenter. Inoculate dG-GA1 from a scribed YPD plate into a 5 mL YPD liquid medium test tube and activate it at 30 °C in a 200 rpm rocking bed for 36 h. OD 600 = 0.1, duplicate the seed solution into a 100 mL YPD liquid medium vial and culture it at 30 °C in a 200 rpm rocking bed for 24 h. Duplicate it into a 5L fermenter containing 3L of YPD liquid medium. Set the culture conditions to a fermentation temperature of 30 °C, pH 5.5, and a rotation speed of 200 rpm. Use batch-fed fermentation, the length of the fermentation stage process is about 60 h, and glucose is replenished twice. During fermentation, the strain growth OD 600Monitor the situation in real time. However, the reaction conditions of the substrate-catalyzed process are set at 42.5 °C, pH 5.5 and a stirring rotation speed of 300 rpm. The charging concentration of 18α-GL each time is 20 g / L, and a total of 4 times are charged. At this stage, detect the concentration changes of the substrate 18α-GL, the intermediate product GAMG and 18α-GA in the reaction system in real time, accurately evaluate the catalytic effect of dG-GA1, and the results are shown in Figure 7A. According to the results, in the 5L fermenter during batch feeding, the transformation process takes 20 h, the final concentration of 18α-GA reaches 41.09 g / L, the conversion rate reaches 96.57%, indicating that it has good potential for industrial application in the transformation of 18α-GL.

[0098] Experimental Example 9, Amplification Production of 18β-GA in a 1000L Fermenter While further verifying the large-scale production capacity of dG-GA1 and characterizing the transformation ability of 18β-GL, it was verified in a 1000L fermenter. In the fermentation stage, first, pick up the Pichia yeast dG-GA1 combined engineering bacteria from the scribed YPD plate, inoculate a single colony into 100 mL of liquid YPD medium, and culture at 30 °C for about 24 h. Then, the inoculum is amplified step by step in 10L, 100L and 1000L fermenters containing YPD medium, and the whole fermentation process is maintained under the culture conditions of a temperature of 30 °C, pH 5.5 and a stirring rotation speed of 200 rpm. Here, the seed liquids of the 10L fermenter and the 100L fermenter are both cultured for about 24 h. In addition, the culture is cultured in a 1000L fermenter for about 48 h, and then 20 g / L of glucose is supplemented every 12 h, and a total of 2 times are supplemented. During the 1000L fermentation culture process, the growth OD of the strain 600 is monitored in real time. However, in the substrate-catalyzed stage, the reaction conditions are set at 42.5 °C, pH 5.5 and a stirring rotation speed of 300 rpm. The charging concentration of 18β-GL each time is 20 g / L, and the concentration changes of the substrate 18β-GL, the intermediate product GAMG and 18β-GA are monitored in real time, and the results are shown in Figure 7B.

[0099] According to the results, the entire substrate catalysis stage takes 24 hours, and a total of 6 times of substrate 18β-GL supplementation are carried out. At 24 hours, the GA concentration is 48.73 g / L, and the GL conversion rate is shown to reach 97.26%. At the same time, throughout the entire process of the amplification test, the proportion of the accumulation amount of 18β-GAMG in the reaction system is always lower than 4%, and the GAMG concentration at 24 hours is less than 1.43 g / L. It meets the needs to solve the above engineering problems, and the efficiency and production volume are also the highest among the currently reported processes.

[0100] From the above results, it can be seen that the overall performance of the dG-GA1 combination engineering bacteria is excellent, explaining the significant effect of AcGUS and its mutants in hydrolyzing GAMG and the effectiveness of the β-glucuronidase combination policy. It is further shown that the present invention has advantages such as high catalytic efficiency, short fermentation cycle, and environmentally friendly process, and has the prospect of producing glycyrrhetinic acid on an industrial scale.

Claims

1. A β-glucuronidase, which is selected from β-glucuronidase AcGUS, β-glucuronidase AcGUS1, β-glucuronidase AcGUS2, β-glucuronidase AcGUS3, or a β-glucuronidase AcGUS3 mutant; said β-glucuronidase AcGUS is an enzyme in which the first amino acid of the amino acid sequence of SEQ ID NO. 1 is deleted, the 7th alanine is mutated to glycine, the 66th arginine is mutated to lysine, the 256th alanine is mutated to valine, the 270th threonine is mutated to alanine, the 332nd valine is mutated to phenylalanine, the 363rd aspartic acid is mutated to glutamic acid, and the 506th methionine is mutated to valine; said β-glucuronidase AcGUS1, AcGUS2, and AcGUS3 are enzymes in which 10, 20, and 30 amino acids from the nitrogen terminus of β-glucuronidase AcGUS are cleaved, respectively, and the amino acid sequence of β-glucuronidase AcGUS is as shown in SEQ ID NO. 1; said β-glucuronidase AcGUS3 mutant is selected from a mutant in which the 461st glycine of AcGUS3 is mutated to cysteine, a mutant in which the 462nd glutamine of AcGUS3 is mutated to histidine, a mutant in which the 575th isoleucine of AcGUS3 is mutated to lysine, or a mutant in which the 461st glycine of AcGUS3 is mutated to cysteine, the 462nd glutamine is mutated to histidine, and the 575th isoleucine is mutated to lysine; a β-glucuronidase, characterized by the above.

2. The gene sequence of said β-glucuronidase AcGUS is as shown in SEQ ID NO. 2; a mutant in which the 461st glycine of AcGUS3 is mutated to cysteine, the 462nd glutamine is mutated to histidine, and the 575th isoleucine is mutated to lysine is β-glucuronidase AcGUS3M1. The β-glucuronidase according to Claim 1.

3. A recombinant expression vector in which the gene sequence of the β-glucuronidase according to Claim 1 is ligated characterized by the above.

4. said expression vector is selected from pET28a, pGAPZαA, pPIC9K, or pPICZα The recombinant expression vector according to Claim 3.

5. The recombinant expression vector is selected from the recombinant expression vector pGAPZαA-AcGUS3 or the recombinant expression vector pGAPZαA-AcGUS3M1 The recombinant expression vector according to claim 3

6. An engineered bacterium, selected from the Pichia yeast strain AcGUS3, the Pichia yeast strain AcGUS3M1, or the Pichia yeast strain dG-GA1, the Pichia yeast strain AcGUS3 can express the β-glucuronidase AcGUS3 described in claim 1, the Pichia yeast strain AcGUS3M1 can express the β-glucuronidase AcGUS3M1 described in claim 2, the Pichia yeast strain dG-GA1 can simultaneously express the β-glucuronidase AcGUS3M1 described in claim 2 and the β-glucuronidase AtGUS derived from the Aspergillus terreus strain Li-20 An engineered bacterium characterized by the above.

7. The Pichia yeast strain AcGUS3 contains the recombinant expression vector pGAPZαA-AcGUS3 described in claim 5 The engineered bacterium according to claim 6

8. The Pichia yeast strain AcGUS3M1 contains the recombinant expression vector pGAPZαA - AcGUS3M1 described in claim 5 The engineered bacterium according to claim 6

9. The Pichia yeast strain dG-GA1 contains the recombinant expression vector pGAPZαA-AcGUS3M1-NrsR obtained by modifying the recombinant expression vector pGAPZαA-AcGUS3M1 described in claim 5, and the recombinant expression vector pGAPZαA-AtGUS in which the β-glucuronidase AtGUS gene sequence derived from the Aspergillus terreus strain Li-20 is ligated. The modification refers to replacing the resistance gene of the original expression vector pGAPZαA of the recombinant expression vector pGAPZαA-AcGUS3M1 with NrsR resistance The engineered bacterium according to claim 6

10. A ferment containing a fermentation active ingredient, wherein the fermentation active ingredient contains the β-glucuronidase described in claim 1 or 2, or the recombinant expression vector described in any one of claims 3 to 5, or the engineered bacterium described in any one of claims 6 to 9 A ferment characterized by the above.

11. Further containing auxiliary materials The ferment according to claim 10

12. A method for mass-producing glycyrrhetinic acid, which uses the Pichia yeast strain AcGUS3M1 or the Pichia yeast strain dG-GA1 in the engineering bacteria according to any one of claims 6 to 9 to ferment and produce a substrate, wherein the substrate of the Pichia yeast strain AcGUS3M1 is 18β-GL, and the substrate of the Pichia yeast strain dG-GA1 is selected from 18α-GL or 18β-GL A method for mass-producing glycyrrhetinic acid, characterized by the above.

13. When the substrate is 18β-GL, the glycyrrhetinic acid is 18β-GA; when the substrate is 18α-GL, the glycyrrhetinic acid is 18α-GA The method for mass-producing glycyrrhetinic acid according to claim 12.

14. The conditions for the fermentation production include 42.5 °C, pH 5.5, stirring rotation speed 300 rpm, and charging concentration 20 g / L The method for mass-producing glycyrrhetinic acid according to claim 12.

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