β-glucuronidase and recombinant expression vector thereof, engineered bacteria, fermentation agent, and method for mass production of glycyrrhetinic acid

Abstract

To provide β-glucuronidase suitable for mass production with high affinity, enzymatic activity, catalytic efficiency, and genetic transformation rate, which effectively reduces 18β-GAMG accumulation, to provide recombinant expression vectors thereof, to provide engineered fungi, to provide fermentation agents, and to provide methods for mass production of glycyrrhetinic acid.SOLUTION: The present invention belongs to the fields of enzyme engineering and microbiology. The β-glucuronidase is selected from β-glucuronidase AcGUS, AcGUS1, AcGUS2, AcGUS3, and AcGUS3 mutants, and AcGUS is an enzyme in which specific amino acids are mutated. The present invention is simple and efficient, and enables industrial-scale production of 18α-GA and 18β-GA.SELECTED DRAWING: Figure 7

Description

[Technical Field]

[0001] The present invention relates to the fields of enzyme engineering and microorganism technology, and more particularly to β-glucuronidase and its recombinant expression vector, engineered bacteria, fermentation agents and methods for mass production of glycyrrhetinic acid. [Background technology]

[0002] Licorice, a traditional herb with the same origins as a medicinal plant, is widely used in the pharmaceutical, food, and cosmetic industries. Historical records indicate that licorice has been used as a medicine for over 4,000 years, and it is said to be "number one among the national elders of the day, and number one among medicines." Licorice not only clears fever and detoxifies, replenishes spleen qi, moistens the lungs, relieves coughs, and harmonizes various medicinal properties, but also treats symptoms such as sore throat, spleen and stomach dysfunction, and peptic ulcers. Its anti-inflammatory, hepatoprotective, antioxidant, antiviral, antitumor, and antidiuretic properties are also widely used. Many modern Chinese herbal formulas incorporate licorice or its active ingredients.

[0003] Glycyrrhizic acid (GL) and glycyrrhetinic acid (GA) are the main and active ingredients in licorice, respectively. Currently, the reported production methods for glycyrrhetinic acid mainly include chemical and biotransformation methods. CN101817867A reports that glycyrrhizinate is used as raw material, and acetylglycyrrhetinic acid is generated using sulfuric acid and high-concentration acetic acid as catalysts, followed by deacetylation to obtain glycyrrhetinic acid. The reaction requires the use of large amounts of strong acids, strong bases, and organic solvents, resulting in high energy consumption, low yield, and significant environmental stress. On the other hand, the biotransformation method not only has the advantages of mild conditions, high yield, and environmental friendliness, but also provides higher-purity medicinal precursors for the production of various derivatives, making it a promising industrial production method. The applications of GA for anti-inflammatory, hepatoprotective, antioxidant, antiviral, antitumor, and antidiuretic purposes have been widely reported. Research has shown that 18α-GA has stronger hepatic distribution ability than 18β-GA, is more effective in treating hepatitis, has fewer side effects than 18β-GA, and is safer.

[0004] Previous reports of biotransformation methods have focused on the use of β-glucuronidase to hydrolyze glycyrrhizinate to produce 18β-GA. After undergoing two hydrolysis steps by β-glucuronidase, the glycyrrhizinate substrate can be converted into 18α-glycyrrhetinic acid and 18β-glycyrrhetinic acid (Figure 1). The inventor's research group's previous research results, Chinese Invention Patent CN109628427B, reported the development of a recombinant enzyme AtGUS-mix via domain substitution. Under different conditions, this recombinant enzyme produced 18β-GA at a maximum final concentration of 16.3 g / L and a reaction cycle of 96 hours. However, it did not address the problem of reaction inhibition, which is prone to occur due to the accumulation of the intermediate 18β-GAMG.

[0005] In order to achieve higher affinity, enzymatic activity, catalytic efficiency, and transformation rate, to better reduce the accumulation of 18β-GAMG, and to further reduce the production costs of glycyrrhetinic acid, the field needs to develop new β-glucuronidases for the industrial production of glycyrrhetinic acid. Summary of the Invention [Problem to be solved by the invention]

[0006] In order to solve the problems of the prior art in this field, such as higher affinity, enzymatic activity, catalytic efficiency, and transformation rate, better reduction of 18β-GAMG accumulation, and the need for a β-glucuronidase suitable for mass production, the present invention provides a β-glucuronidase and its recombinant expression vector, an engineered fungus, a fermentation agent, and a method for mass production of glycyrrhetinic acid. [Means for solving the problem]

[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 β-glucuronidase AcGUS3 mutant; The β-glucuronidase AcGUS is an enzyme in which the first amino acid in the amino acid sequence having GenBank accession number AEK69352.1 is deleted, and the alanine at position 7 is changed to glycine, the arginine at position 66 is changed to lysine, the alanine at position 256 is changed to valine, the threonine at position 270 is changed to alanine, the valine at position 332 is changed to phenylalanine, the aspartic acid at position 363 is changed to glutamic acid, and the methionine at position 506 is changed to valine; The β-glucuronidases AcGUS1, AcGUS2, and AcGUS3 are enzymes obtained by cleaving 1-39 amino acids at the nitrogen terminal 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, a mutant in which glutamine at position 462 of AcGUS3 is mutated to histidine, 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 truncating 10 amino acids at the nitrogen terminal of β-glucuronidase AcGUS, Preferably, the β-glucuronidase AcGUS2 is an enzyme obtained by truncating 20 amino acids at the nitrogen terminal of β-glucuronidase AcGUS, Preferably, the β-glucuronidase AcGUS3 is an enzyme obtained by truncating 30 amino acids at the nitrogen terminal of β-glucuronidase AcGUS, Preferably, the mutant of AcGUS3 in which glycine at position 461 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, in which the gene sequence of β-glucuronidase is ligated into the expression vector.

[0010] the expression vector is selected from pET28a, pGAPZαA, pPIC9K, and 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 fungus 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 Pichia yeast strain AcGUS is capable of expressing the β-glucuronidase AcGUS; The Pichia yeast strain AcGUS3 is capable of expressing the β-glucuronidase AcGUS3; The Pichia yeast strain AcGUS3M1 is capable of expressing the β-glucuronidase AcGUS3M1; The Pichia yeast strain dG-GA1 is capable of simultaneously expressing the β-glucuronidase AcGUS3M1 and the β-glucuronidase AtGUS derived from Aspergillus terreus strain Li-20.

[0012] The Pichia yeast strain AcGUS contains the recombinant expression vector pGAPZαA-AcGUS, Preferably, the Pichia yeast strain AcGUS3 comprises the recombinant expression vector pGAPZαA-AcGUS3; Preferably, the Pichia yeast strain AcGUS3M1 comprises the recombinant expression vector pGAPZαA-AcGUS3M1; Preferably, the Pichia yeast strain dG-GA1 comprises a recombinant expression vector pGAPZαA-AcGUS3M1-NrsR, which is a modified version of the recombinant expression vector pGAPZαA-AcGUS3M1, and a recombinant expression vector pGAPZαA-AtGUS, to which the β-glucuronidase AtGUS gene sequence derived from 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 an NrsR resistance gene.

[0013] A fermentation agent comprising a fermentation active ingredient, wherein the fermentation active ingredient comprises the β-glucuronidase, and / or the recombinant expression vector, and / or the engineered bacterium.

[0014] The fermentation agent further comprises an adjuvant.

[0015] A method for mass-producing glycyrrhetinic acid, comprising fermenting a substrate using the β-glucuronidase, and / or the recombinant expression vector, and / or the engineered fungus.

[0016] the substrate is selected from 18α-GL or 18β-GL; Preferably, the glycyrrhetinic acid is selected from 18α-GA or 18β-GA, Preferably, the fermentation conditions include 42.5°C, pH 5.5, agitation speed of 300 rpm, and a feed concentration of 20 g / L.

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

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

[0019] The method for constructing β-glucuronidase AcGUS mutants involves rational truncation of amino acids 1-39 at the nitrogen terminus of β-glucuronidase AcGUS, with the preferred truncated form AcGUS3 having 30 amino acids truncated.

[0020] The truncated AcGUS3 and the heterologously expressed β-glucuronidase AcGUS3 in Pichia yeast showed 7-fold higher enzymatic activity in the production of glycyrrhetinic acid compared to AcGUS, while the accumulation of the intermediate product, monoglucuronide glycyrrhetinic acid, was 3.74-fold lower.

[0021] The truncated AcGUS3 was prepared by mutating the 461st glycine to cysteine, the 462nd glutamine to histidine, and the 575th isoleucine to lysine, to obtain a new mutant, AcGUS3M1.

[0022] The preferred AcGUS3M1 mutant is characterized by an 11.02-fold and 6.10-fold increase in activity in the hydrolysis of the substrates monoglucuronide glycyrrhetinic acid and glycyrrhizinic acid, respectively.

[0023] A method for constructing a strain with improved activity in hydrolyzing 18α-glycyrrhizinic acid or 18β-glycyrrhizinic acid is to introduce the pGAPZαA-AcGUS3M1 mutant plasmid into the Pichia yeast AtGUS host to obtain the Pichia yeast strain dG-GA1.

[0024] When glycyrrhizinic acid is used as a substrate, the Pichia yeast strain dG-GA1 can effectively suppress the accumulation of the intermediate product monoglucuronide glycyrrhetinic acid, and the percentage of this product in the system during the fermentation process is always lower than 4%.

[0025] The final concentration of 18α-glycyrrhetinic acid produced reached 41.09 g / L, and the transformation rate was 96.57%. The final concentration of 18β-glycyrrhetinic acid produced reached 48.73 g / L, and the transformation rate was 97.26%. [Effects of the Invention]

[0026] The present invention provides the coding gene and application of a β-glucuronidase mutant, obtains an efficient β-glucuronidase mutant through protein molecular engineering and selection, and further provides a strain construction method for efficiently producing glycyrrhetinic acid. The constructed engineered strain is used for industrial-scale production of glycyrrhetinic acid. The β-glucuronidase AcGUS and its mutants of the present invention differ from conventional β-glucuronidases, which prefer to hydrolyze the outer glycoside of the substrate GL. The former prefers to hydrolyze the substrate GAMG (i.e., the inner glycoside of GL) and exhibits excellent activity. The AcGUS mutant was applied to the proposed method for constructing a combinatorially engineered β-glucuronidase strain. Testing revealed that the resulting combinatorially engineered strain showed excellent industrial application potential when hydrolyzing 18α-GL and 18β-GL in 5L and 1000L fermenters, respectively, and had advantages such as high catalytic efficiency, strong specificity, short process cycle, and environmental friendliness. It can be seen that the present invention provides the art with a simple and efficient method for producing 18α-GA and 18β-GA on an industrial scale. [Brief explanation of the drawings]

[0027] [Figure 1] Schematic diagram of the two-step transformation of 18α-glycyrrhizinic acid and 18β-glycyrrhizinic acid by β-glucuronidase, where A: 18α-GL produces 18α-GA, and B: 18β-GL produces 18β-GA. [Figure 2] 1 is an agarose gel electrophoresis verification diagram of AcGUS gene amplification in Experimental Example 2 of the present invention, where lanes 1 and 2 are PCR product bands of AcGUS, and lanes M is a DNA marker. [Figure 3] FIG. 10 is a diagram showing liquid-phase detection of GAMG accumulation during 18β-glycyrrhizinic acid transformation in Experimental Example 3 of the present invention. [Figure 4]Five types of β-glucuronidases, AcGUS, AcGUS1, AcGUS2, AcGUS3, and AcGUS4, were expressed in eukaryotes from Pichia pastoris in Experimental Example 4 of the present invention. This bar graph shows the relative enzyme activity of AcGUS, AcGUS1, AcGUS2, AcGUS3, and AcGUS4, respectively, in hydrolyzing GL to produce GA and GAMG. The notations on the abscissa correspond to the five types of β-glucuronidase. [Figure 5] This figure shows a comparison of the enzyme activities of wild-type AcGUS3 (WT) and prokaryotic AcGUS3 mutants: AcGUS3G461C, AcGUS3Q462H, AcGUS3I575K, AcGUS3G461C / I575K, and AcGUS3G461C / Q462H / I575K, in Experimental Example 4 of the present invention, where the test substrates in the upper and lower figures are 18β-GL and 18β-GAMG, respectively, and the abscissa indicates the wild-type and each mutant, respectively. [Figure 6] 1 is a comparative diagram of the relative enzyme activity of β-glucuronidase expressed in different Pichia yeast engineering strains in Experimental Example 7 of the present invention. The abscissa notation is 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 AtGUS of the known strain Aspergillus terreus strain Li-20 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 invention; "AcGUS3M1" refers to the β-glucuronidase AcGUS3M1 described in the present invention; and "dG-GA1" refers to the β-glucuronidase AcGUS3M1 and the β-glucuronidase AtGUS derived from Aspergillus terreus strain Li-20 co-expressed in the Pichia yeast strain dG-GA1 of the present invention. [Figure 7]These are tests on the amplification and production of GA using the dG-GA1 combinatorial engineering strain in Experimental Examples 8 and 9 of the present invention. Here, Figure A shows the change in substrate and product concentrations when 18α-GL is transformed to produce 18α-GA in a 5 L fermenter, and Figure B shows the change in substrate and product concentrations when 18β-GA is produced from 18β-GL in a 1000 L fermenter. DETAILED DESCRIPTION OF THE INVENTION

[0028] The present invention will be further described below with reference to specific examples and experimental examples. It should be understood that the examples are only for the purpose of explaining and illustrating the present invention, and are not intended to limit the scope of protection of the present invention. Unless otherwise specified, the experimental methods used in the following examples and experimental examples are conventional methods, and the reagents or materials used are all commercially available.

[0029] Origin and provenance of biological materials 1. The Aspergillus callidustus CLH-22 strain mentioned in Experimental Example 1 is a known strain reported in Chinese patent application CN115786133A.

[0030] Second, the E. coli Top10 competent strain used in Experimental Examples 2 and 5 of the present invention is commercially available.

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

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

[0033] 5. The Pichia yeast AtGUS used in Experimental Example 6 of the present invention was constructed in the inventor's laboratory. Those skilled in the art can clone the reported Aspergillus terreus strain Li-20 according to the description in Article (4) of the "Reagents and Consumables" section of the present invention to obtain the β-glucuronidase AtGUS derived from Aspergillus terreus strain Li-20, and then ligate it into Pichia yeast for transformation to obtain the engineered Pichia yeast AtGUS, without any technical obstacles.

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

[0035] (2) Pichia yeast (Pichia pastoris): GS115.

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

[0037] (4) AtGUS is derived from Aspergillus terreus strain Li-20, a known strain reported in Chinese patent application CN106047839A, with GenBank number JF894133.1.

[0038] (5) Augus is derived from the Aspergillus ustus strain Li-62, a known strain reported in Chinese patent application CN106047839A, with sequence ID number JN247805.1.

[0039] (6) Pichia yeast AtGUS and Pichia yeast AuGUS will be preserved in the applicant's laboratory.

[0040] (7) Atgusmix is ​​described in another invention patent of the inventor’s project team, “Engineered fungus GA108 / PGAPZαA-Atgusmix and method for industrially producing glycyrrhetinic acid,” and its strain stock number is CGMCC No. 16731.

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

[0042] 2. Culture medium LB medium (per liter): Measure out 10g of tryptone, 5g of sodium chloride, and 5g of yeast extract, add deionized water to make 1L, and sterilize at 121°C for 15 minutes. For LB solid medium, 20g of agar powder must be added separately. YPD medium (per liter): Measure out 20g of tryptone, 20g of glucose monohydrate, 10g of yeast extract, and add deionized water to make 1L, and sterilize at 115°C for 15 minutes. For YPD solid medium, 20g of agar powder must be added separately.

[0043] (2) Preparation of solutions for constructing Pichia yeast engineering strains 1M sorbic alcohol solution: Weigh out 182.17g of sorbic alcohol, add 900mL of deionized water, stir thoroughly to dissolve, then make a final volume of 1L and sterilize at 115°C for 20 minutes. Yeast lysate buffer: Weigh out 0.372g of EDTA disodium salt and 2g of NaOH, add 5mL of Triton X-100, dissolve with deionized water and make a final volume of 1L. Sterile water: Weigh out 200mL of deionized water, seal in a corn bottle with gauze, sterilize at 121°C for 15 minutes, and store in a refrigerator at 4°C.

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

[0045] When the combination engineering bacteria constructed based on the β-glucuronidase AcGUS and its mutants according to the present invention are used for GA production by a combination method, the combination based on different β-glucuronidases or the usual adjustment and selection for the fermentation scale are all within the scope of protection of the present invention.

[0046] First set of examples, β-glucuronidases of the present invention This set of embodiments provides β-glucuronidases, all of which have the following common feature: 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 a β-glucuronidase AcGUS3 mutant; The β-glucuronidase AcGUS is an enzyme in which the first amino acid in the amino acid sequence having GenBank accession number AEK69352.1 is deleted, and the alanine at position 7 is changed to glycine, the arginine at position 66 is changed to lysine, the alanine at position 256 is changed to valine, the threonine at position 270 is changed to alanine, the valine at position 332 is changed to phenylalanine, the aspartic acid at position 363 is changed to glutamic acid, and the methionine at position 506 is changed to valine; The β-glucuronidases AcGUS1, AcGUS2, and AcGUS3 are enzymes obtained by cleaving 1-39 amino acids at the nitrogen terminal 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, a mutant in which glutamine at position 462 of AcGUS3 is mutated to histidine, 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.

[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 truncating 10 amino acids at the nitrogen terminal of β-glucuronidase AcGUS, Preferably, the β-glucuronidase AcGUS2 is an enzyme obtained by truncating 20 amino acids at the nitrogen terminal of β-glucuronidase AcGUS, Preferably, the β-glucuronidase AcGUS3 is an enzyme obtained by truncating 30 amino acids at the nitrogen terminal of β-glucuronidase AcGUS, Preferably, the mutant of AcGUS3 in which glycine at position 461 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.

[0048] any act of cloning, amplifying, concentrating, expressing, linking, transforming, synthesizing, culturing, propagating, fermenting, producing, manufacturing, using, inoculating, modifying, adapting, selling or offering to sell the genetic sequence of, for example, 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 using the amino acid sequence transcribed and translated from the genetic sequence of, for example, β-glucuronidase AcGUS, and / or β-glucuronidase AcGUS1, and / or β-glucuronidase AcGUS2, and / or β-glucuronidase AcGUS3, and / or β-glucuronidase AcGUS3 mutants in combination with other enzymes, and / or For example, the act of producing ingredients including, but not limited to, pharmaceutically active ingredients such as glycyrrhetinic acid with amino acid sequences transcribed and translated from the 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, and / or the act of producing drugs with amino acid sequences transcribed and translated from the 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 all within the scope of protection of the present invention.

[0049] any act of cloning, amplifying, concentrating, expressing, linking, transforming, synthesizing, cultivating, growing, fermenting, producing, manufacturing, using, inoculating, modifying, adapting, selling, offering to sell 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 mutants, and / or any act of 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 mutants in combination with other enzymes; And / or acts of producing and manufacturing ingredients including, but not limited to, pharmaceutically active ingredients such as glycyrrhetinic acid using the amino acid sequences of, for example, 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 acts of producing drugs using the amino acid sequences of, for example, 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 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, chitosan triglycosidase, thiosidase, α-glucosidase, β-galactosidase, β-actofuranosidase, 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, and phosphatase.

[0051] According to actual production needs, those skilled in the art can, in combination with ordinary technical means or basic common sense in the production processes in the fields of molecular biology or genetic engineering (for example, "Practical Molecular Biology Operation Guide," "Molecular Biology Experimental Techniques Experimental Operation Guide," "Molecular Clone Experimental Guide," "Refined Molecular Biology Experimental Guide," etc.), decompile the amino acid sequences 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 mutants, obtain their gene sequences, design specific amplification primers, obtain their gene sequences, and ligate them into expression vectors to produce β-glucuronidase AcGUS, and / or β-glucuronidase AcGUS1, and / or β-glucuronidase AcGUS2, and / or β-glucuronidase AcGUS3, and / or By obtaining a recombinant expression vector capable of expressing an AcGUS3 β-glucuronidase mutant, or by further transforming the recombinant expression vector into a competent cell to obtain a transformant (e.g., an engineered fungus) capable of expressing AcGUS β-glucuronidase, AcGUS1 β-glucuronidase, AcGUS2 β-glucuronidase, AcGUS3 β-glucuronidase, and / or an AcGUS3 β-glucuronidase mutant, and growing and culturing the transformant under conditions suitable for its growth, the AcGUS β-glucuronidase, AcGUS1 β-glucuronidase, AcGUS2 β-glucuronidase, AcGUS3 β-glucuronidase, and / or an AcGUS3 β-glucuronidase mutant can be efficiently produced, which can be easily performed by a person skilled in the art without any technical obstacles.

[0052] Second Set of Examples, Recombinant Expression Vectors of the Invention This set of examples provides recombinant expression vectors. The examples in this set all share the following common feature: an expression vector into which is linked a β-glucuronidase gene sequence as described in any one of the examples in the first set.

[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, linking, transforming, synthesizing, culturing, propagating, fermenting, producing, manufacturing, using, inoculating, modifying, altering, selling, or promising to sell the recombinant expression vector, and / or any act of combining the β-glucuronidase expressed by the recombinant expression vector with other enzymes, and / or any act of producing ingredients, including but not limited to, pharmacologically active ingredients such as glycyrrhetinic acid, using the β-glucuronidase expressed by the recombinant expression vector, and / or any act of producing drugs using the β-glucuronidase expressed by the recombinant expression vector, are all within the scope of protection of the present invention.

[0055] The other enzymes include, but are not limited to, α-mannosidase, arabinosidase, β-xylosidase, chitosan triglycosidase, thiosidase, α-glucosidase, β-galactosidase, β-actofuranosidase, 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, and phosphatase.

[0056] According to actual production needs, a person skilled in the art can express the recombinant expression vector of the β-glucuronidase of the present invention using the recombinant expression vector in combination with ordinary technical means or basic common knowledge in the production processes in the fields of molecular biology or genetic engineering (e.g., Practical Guide to Molecular Biology, Guide to Experimental Techniques in Molecular Biology, Guide to Experimental Techniques in Molecular Cloning, Refined Guide to Experimental Techniques in Molecular Biology, etc.), or transform the recombinant expression vector into a competent cell to obtain a transformant (e.g., an engineered fungus) capable of expressing the β-glucuronidase of the present invention, and then grow and culture the transformant under conditions suitable for its growth, thereby efficiently producing the β-glucuronidase of the present invention, which can be easily performed by a person skilled in the art without any technical obstacles.

[0057] Third Set of Examples, Engineered Fungi of the Invention This set of embodiments provides engineered fungi, all of which have the following common feature: the engineered fungi 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 Pichia yeast strain AcGUS is capable of expressing the β-glucuronidase AcGUS according to any one of the examples in set 2, The Pichia yeast strain AcGUS3 is capable of expressing the β-glucuronidase AcGUS3 described in any one of the examples of the second set, the Pichia yeast strain AcGUS3M1 is capable of expressing the β-glucuronidase AcGUS3M1 described in any one of the examples of set 2; The Pichia yeast strain dG-GA1 is capable of simultaneously expressing the β-glucuronidase AcGUS3M1 described in any one of the examples in the second set and the β-glucuronidase AtGUS from Aspergillus terreus strain Li-20.

[0058] In some embodiments, the Pichia yeast strain AcGUS comprises the recombinant expression vector pGAPZαA-AcGUS described in any one of the second set of embodiments; Preferably, the Pichia yeast strain AcGUS3 comprises the recombinant expression vector pGAPZαA-AcGUS3 described in any one of the examples of the second set; Preferably, the Pichia yeast strain AcGUS3M1 comprises the recombinant expression vector pGAPZαA-AcGUS3M1 described in any one of the examples of the second set; Preferably, the Pichia yeast strain dG-GA1 comprises a recombinant expression vector pGAPZαA-AcGUS3M1-NrsR, which is a modified version of the recombinant expression vector pGAPZαA-AcGUS3M1 described in any one of the Examples in the second set, and a recombinant expression vector pGAPZαA-AtGUS, to which the β-glucuronidase AtGUS gene sequence derived from 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 an NrsR resistance gene.

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

[0060] The other enzymes include, but are not limited to, α-mannosidase, arabinosidase, β-xylosidase, chitosan triglycosidase, thiosidase, α-glucosidase, β-galactosidase, β-actofuranosidase, 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, and phosphatase.

[0061] Those skilled in the art can, according to actual production needs, combine ordinary technical means or basic common sense in the production processes of molecular biology or genetic engineering (e.g., Practical Molecular Biology Operation Guide, Molecular Biology Experimental Techniques Operation Guide, Molecular Cloning Experimental Guide, Refined Molecular Biology Experimental Guide, etc.) to culture, grow, ferment, concentrate, produce, manufacture, use, and inoculate the engineered fungus to express, secrete, produce, and obtain β-glucuronidase, which can be easily done by those skilled in the art without any technical obstacles.

[0062] Fourth set of examples, fermentation agents of the present invention This set of examples provides a fermentation agent, all of which have the following common feature: the fermentation agent comprises a fermentation active component, the fermentation active component comprising a β-glucuronidase as set forth in any one of the examples in set 1, and / or a recombinant expression vector as set forth in any one of the examples in set 2, and / or an engineered fungus as set forth in any one of the examples in set 3.

[0063] In a further embodiment, the fermentation agent further comprises an adjuvant.

[0064] In more specific embodiments, the auxiliary agent is selected from solvents, propellants, solubilizers, solubilization aids, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure adjusters, stabilizers, flow aids, flavorings, preservatives, suspension aids, coating materials, fragrances, anti-adhesion agents, matching agents, penetration enhancers, pH adjusters, buffers, plasticizers, surfactants, foaming agents, anti-foaming agents, thickeners, entrapment agents, moisturizing agents, absorbents, diluents, flocculants, deflocculants, filter aids, release blockers, etc.

[0065] Based on the content of the present invention, in accordance with the different needs of actual production applications, in combination with conventional technical means in the field of formulation manufacturing (e.g., "Encyclopedia of Formulation Technology," "Drug Formulation Technology," "Research and Application of Microbial Agent Technology," etc.), those skilled in the art can select and blend the above-mentioned pharmaceutically acceptable adjuvants to produce the engineered bacteria of the present invention into different dosage forms, such as powders, tablets, suppositories, gels, sprays, granules, etc.

[0066] In a specific embodiment, the dosage form of the fermenting agent is selected from one or more of powder, tablet, liquid, and capsule.

[0067] The fifth embodiment of the present invention is a method for mass-producing glycyrrhetinic acid This set of examples provides a method for mass-producing glycyrrhetinic acid. All examples in this set have the following common feature: a substrate is fermented using a β-glucuronidase described in any one of the examples in set 1, and / or a recombinant expression vector described in any one of the examples in set 2, and / or an engineered fungus described in any one of the examples in set 3.

[0068] In a specific embodiment, the substrate is selected from 18α-GL or 18β-GL; Preferably, the glycyrrhetinic acid is selected from 18α-GA or 18β-GA, Preferably, the fermentation conditions include 42.5°C, pH 5.5, agitation speed of 300 rpm, and a feed concentration of 20 g / L.

[0069] Experimental Example 1: Amplification of the β-glucuronidase AcGUS gene Based on the β-glucuronidase AcGUS sequence, gene amplification primers AcGUS-F and AcGUS-R were designed according to the purpose. Then, a cDNA library of Aspergillus calidustus CLH-22 strain was constructed. RNA was extracted from the Aspergillus calidustus CLH-22 strain using an RNA rapid extraction reagent kit, followed by RNA reverse transcription using the PrimeScript reagent. TM The extracted RNA was reverse transcribed using RT Master Mix. The reverse transcription reaction conditions were: 37°C for 15 minutes, enzyme inactivation at 85°C for 5 seconds, and storage at 4°C to obtain a cDNA library of Aspergillus calidustus CLH-22. Furthermore, the cDNA library of Aspergillus calidustus CLH-22 was used as a PCR template for PCR amplification using the Nobyzantine Phanta system. The PCR system consisted of 25 μL of 2x 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 sterile water to make up to 50 μL. The PCR reaction procedure was as follows: pre-denaturation at 95°C for 3 minutes, denaturation at 95°C for 15 seconds, annealing at Tm-5°C for 15 seconds, and extension at 72°C for 2 kb / min. 35 cycles were performed, followed by extension at 72°C for 5 minutes and storage at 16°C to obtain the AcGUS PCR product.

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

[0071] [Table 1]

[0072] Experimental Example 2: Construction of AcGUS clone vector and selection of positive clones (1) Enzymatic digestion and purification of gene fragments The PCR product described in Example 1 was subjected to 1% agarose gel electrophoresis. The results are shown in Figure 2. The PCR fragment length matched that of AcGUS, indicating that the target gene fragment was initially obtained. Further digestion was performed with KpnI and NotI enzymes, followed by incubation at 37°C for 2 hours. The digested fragment was then purified using the GeneJET Gel Recovery Reagent Kit. The purified AcGUS gene fragment and the clone vector pGAPZαA were subjected to T4 enzyme ligation. The enzyme ligation reaction mixture was: 1 μL of 10X T4 DNA Ligase Buffer, 1 μL of pGAPZαA plasmid, the target gene fragment (approximately 500 ng), 1 μL of T4 DNA Ligase, and sterile water to a volume of 10 μL. After instant centrifugation, the ligation product was obtained by incubation at 22°C for 2 hours.

[0073] (2) Colon transformation Add 10 μL of the ligation product to E. coli Top10 recipient cells and incubate for 30 minutes on ice. Then, heat in a 42°C water bath for 90 seconds, then incubate again for 2 minutes on ice. Then, add 500 μL of LB liquid medium and incubate for 1 hour on a rocking bed at 37°C and 200 rpm. After incubation, the culture was centrifuged at 5000 rpm for 3 minutes. Remove 500 μL of the supernatant and spread evenly onto a solid LB plate containing 50 mg / L bleomycin. Incubate overnight at 37°C.

[0074] (3) Selection of E. coli-positive recombinants Recombinants were identified using colony PCR and plasmid sequencing. E. coli colony PCR protocol: 7.5 μL of 2xM5 Taq HiFi PCR Mix, 1 μL each of pGAP-F and 3AOX1 primers. The template was a single colony from an LB plate, supplemented to 15 μL with sterile water. PCR reaction protocol: 94°C pre-denaturation for 4 minutes, 94°C denaturation for 30 seconds, 55°C annealing for 30 seconds, 72°C extension for 2 minutes, 30 cycles, 72°C extension for 7 minutes, and storage at 16°C. Recombinants verified as correct by colony PCR were sent for DNA sequencing to confirm successful construction of the pGAPZαA-AcGUS plasmid. The AcGUS gene sequence is shown in SEQ ID NO. 2.

[0075] Experimental example 3: Construction of Pichia yeast AcGUS engineered strain and verification of enzyme activity (1) Construction of Pichia pastoris AcGUS strain AcGUS was linearized using the endonuclease BlnI. The linearized solution consisted of 2 μL of 10X QuickCut Buffer, 1 μL of BlnI, 1 μg of plasmid, and sterile water (total volume: 20 μL). After instant centrifugation, the mixture was incubated at 37°C for 1 hour. The linearized plasmid was purified and recovered using the GeneJET Gel Recovery Kit. The pGAPZαA-AcGUS linearized plasmid was transferred to Pichia yeast GS115 using the electroporation method commonly used for Pichia yeast. The plasmid was then uniformly plated onto a YPD solid plate containing 100 mg / L bleomycin and cultured at 30°C for 2-4 days.

[0076] Pichia yeast transformants were identified using colony PCR. Six to 12 single colonies were selected from a YPD plate, added to 50 μL of yeast lysate buffer, and boiled for 30 minutes. Add 150 μL of sterile water to a PCR tube to obtain the lysate buffer template. Colony PCR protocol: 7.5 μL of 2x M5 Taq HiFi PCR Mix, 1 μL each of pGAP-F and 3AOX1 primers, 1 μL of lysate buffer template, and topped up to 15 μL with sterile water. PCR reaction protocol: 94°C for 10 minutes of pre-denaturation, 94°C for 30 seconds of denaturation, 55°C for 30 seconds of annealing, 72°C for 2 minutes of extension, 30 cycles, 72°C for 10 minutes of extension, and storage at 16°C. PCR products were then verified by 1% agarose gel electrophoresis to determine whether the required length of the primers met the requirements, confirming successful construction of transformants.

[0077] (2) Verification of enzyme activity in AcGUS transformants The enzyme activity of eukaryotically expressed Pichia yeast AcGUS was verified. First, successfully verified AcGUS transformants were cultured in a 4 g / L glycyrrhizinate-YPD verification solution and cultured for 2 days on a rocking bed at 30°C. High-efficiency liquid chromatography was then used to determine whether the AcGUS strain possessed GL hydrolysis activity. Liquid-phase detection method: The flow phase was methanol:glacial acetic acid solution (6‰) = 84:16, the chromatography column was Kromasil 100-3.5-C18, the sample 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 transformation. The results showed that AcGUS exhibited relatively good GL hydrolysis activity while the accumulation of the intermediate product GAMG was extremely low, indicating that AcGUS prefers the high-performance GUS enzyme for GAMG hydrolysis. Furthermore, when tested using GL and GAMG buffer substrates (pH 5.5), the rate of AcGUS metabolism using the substrate GAMG was found to be approximately twice that of GL, which was consistent with the phenotype of the wild-type strain Aspergillus calidoustus CLH-22 (stock number: CGMCC No. 40213), demonstrating that AcGUS is a valid target gene and that it can be used to construct a Pichia yeast eukaryotic heterologous expression system.

[0078] Experimental Example 4: Construction and verification of AcGUS mutants To maximize GA production efficiency and reduce the inhibitory effects of GAMG accumulation, we provide a viable method for industrially producing GA. In this example, we combined enzyme engineering to select mutant enzymes with improved activity and engineered AcGUS into a more efficient β-glucuronidase.

[0079] (1) Construction of prokaryotic expression of AcGUS in E. coli First, AcGUS was ligated into the pET-28a vector using the commonly used Gibson assembly method to obtain the recombinant vector pET-28a-AcGUS. Subsequently, the recombinant plasmid was transformed into competent E. coli BL21(DE3) and JM109(DE3) strains (using the same procedure as in Example 2). Positive clones were selected on LB plates containing 50 mg / L kanamycin resistance. Recombinants verified to be correct by colony PCR were then subjected to DNA sequencing. If the sequence was correct, the recombinant E. coli strain was successfully constructed.

[0080] (2) Analysis of the nitrogen terminal of AcGUS and construction and verification of truncated forms We performed structural simulations of AcGUS using AlphaFold2 and structural analysis of the flexible nitrogen-terminal loop. Because this loop is highly flexible and relatively unstable, we investigated whether truncation of the nitrogen terminus of AcGUS could improve enzymatic activity during heterologous expression. However, recombinant nitrogen-terminal truncations for heterologous expression in E. coli and Pichia pastoris were obtained by truncating 10, 20, 30, and 39 amino acids from the nitrogen terminus of the gene using pET-28a-AcGUS and pGAPZαA-AcGUS, respectively, to obtain AcGUS1, AcGUS2, AcGUS3, and AcGUS4, which were expressed in prokaryotic and eukaryotic organisms, respectively.

[0081] Next, we performed comparative analysis of GL transformation and intermediate GAMG accumulation in the eukaryotic expression of Pichia yeast AcGUS, AcGUS1, AcGUS2, AcGUS3, and AcGUS4. First, five successfully verified transformants were taken from each score plate and placed in a test tube containing 5 mL of YPD liquid. 600The mixture was then duplicated into 250 mL vials containing 4 g / L glycyrrhizin-YPD at 0.1% RI and incubated on a rocking bed at 30°C for 4 days, with samples taken every 12 h and detected using high-efficiency liquid chromatography. As shown in Figure 4, AcGUS3 exhibited a 7-fold improvement in the enzymatic activity of AcGUS in the production of the product GA, demonstrating its relatively good GL hydrolysis activity. The accumulation of the intermediate GAMG by AcGUS3 was significantly lower, 3.74-fold lower than that of AcGUS. Furthermore, the pure enzymes were analyzed by milky dynamics using GL and GAMG as substrates, respectively. The data indicated that AcGUS3 catalyzed GL. cat / K m is 2.94 times that of AcGUS, but the k cat / K m was 9.06 times that of AcGUS, and the above results indicated that the affinity and catalytic efficiency of AcGUS were significantly improved.

[0082] (3) Construction and verification of AcGUS mutants Based on the three-dimensional structure of AcGUS3, sequence matching analysis, and enzyme engineering methods such as molecular docking, G461, Q462, and I575, located within 5 Å of the AcGUS3 pocket, were selected as saturation mutation sites (Table 2). Mutations were then performed on a combination of sites with positive effects. Based on the AcGUS3 truncation, the pET-28a-AcGUS3 plasmid was used as a template to obtain the mutated PCR product using the plasmid loop P. The loop P system consisted of 25 μL of 2x Phanta Flash Master Mix, 1 μL each of upstream and downstream primers, 1-2 μL of plasmid template, and sterile water to a volume of 50 μL. The PCR reaction procedure was as follows: 98°C pre-denaturation for 30 s, 98°C denaturation for 10 s, annealing at Tm-5°C for 5 s, and elongation at 72°C for 50 s. The PCR cycle was repeated 35 times, followed by elongation at 72°C for 1 min and storage at 16°C. The PCR product was then digested with DpnI enzyme for 2 hours, followed by transformation into competent JM109(DE3) colony transformants. The results were then verified by colony PCR. The colony PCR protocol was 7.5 μL of 2x M5 Taq HiFi PCR Mix, 1 μL each of T7 and T7 term universal primers, and the template was a single colony grown on an LB plate, supplemented to 15 μL with sterile water. The PCR reaction procedure was 94°C for 4 minutes of pre-denaturation, 94°C for 30 seconds of denaturation, 55°C for 30 seconds of annealing, and 72°C for 2 minutes of extension, followed by 30 cycles of 72°C extension and storage at 16°C. Recombinants verified as correct by colony PCR were subjected to DNA sequencing to confirm successful construction of the mutant plasmid.

[0083] [Table 2]

[0084] To rapidly compare the activity of the mutants, a validation comparison was performed using crude enzyme solutions. First, the constructed recombinant E. coli was inoculated into 5 mL of LB medium containing 50 mg / L kanamycin resistance and cultured at 37 °C for 12 h. Then, it was duplexed into 40 mL of LB medium at 1% duplexing volume, and the OD 600When the pH reached 0.6-0.8, IPTG was added for induction. After culturing for 18 hours at 16°C, 2 mL of cells were harvested by centrifugation at 12,000 rpm (4°C) and washed twice with 50 mM HAc-NaAc buffer (pH 5.5). Next, 1 mL of 50 mM HAc-NaAc buffer (pH 5.5) and an appropriate amount of disruption beads were added and disrupted using a homogenizer. Then, 200 μL of crude enzyme supernatant was mixed with 800 μL of 50 mM HAc-NaAc buffer (pH 5.5) containing 5 g / L GL or GAMG, respectively, and incubated at 42.5°C for 5 hours and 0.5 hours, respectively, after which samples were taken and analyzed by high-performance liquid chromatography (HPLC).

[0085] According to the results in Figure 5, G461, Q462, and I575 are mutation sites with positive effects, where the single-site mutation AcGUS3 G461C , AcGUS3 Q462H and AcGUS3 I575K demonstrated that the hydrolysis activity of GAMG substrates was improved by 106.71%, 108.72%, and 126.41%, respectively, compared to unmutated AcGUS3 (WT), demonstrating that a pocket-engineered mutation strategy can obtain effective mutation sites.

[0086] Therefore, AcGUS3 G461C Using the mutant as the starting strain, we performed repeated saturation mutagenesis with I575. We used primers AcGUS3-I575-F (SEQ ID NO. 13) and AcGUS3-I575-R (SEQ ID NO. 14) to construct a saturation mutation library for G461C and I575. The selection and verification procedures were the same as those for single-site saturation mutation in Example 4, and the mutant strain AcGUS3 with improved hydrolysis ability was obtained. G461C / I575K AcGUS3 G461C / I575K Compared with the unmutated AcGUS3, the hydrolytic GL activity was improved by 124.29% and the hydrolytic GAMG activity was improved by 118.72%.

[0087] Furthermore, AcGUS3 G461C / I575KUsing the mutant as the starting strain, repeated saturation mutagenesis was performed with Q462. AcGUS3-Q462N-F (SEQ ID NO. 11) and AcGUS3-Q462N-R (SEQ ID NO. 12) were used as primers to construct a saturation mutation library of G461C / I575K and Q462. The selection and verification procedures were the same as those for single-site saturation mutagenesis in Example 4, and the mutant strain AcGUS3 with improved hydrolysis ability was obtained. G461C / Q462H / I575K The enzyme activities of the 18β-GL and 18β-GAMG mutants were improved by 138.3% and 136.6%, respectively, compared to the control AcGUS3. G461C / Q462H / I575K Renamed to AcGUS3M1.

[0088] Furthermore, kinetic parameters were further measured and activity characterization was performed. The results showed that mutant AcGUS3M1 catalyzed GL. cat / K m is 6.1 times higher than that of wild-type AcGUS, but the k cat / K m The activity of AcGUS3M1 was 11.02-fold higher than that of AcGUS, indicating further improvements in affinity and catalytic efficiency. This significant improvement in the enzymatic activity of AcGUS3M1 provided strong support for the next step, the construction of a combinatorially engineered β-glucuronidase strain.

[0089] Experimental Example 5: Construction of Pichia yeast AcGUS3M1 First, using the pET-28a-AcGUS3M1 plasmid verified in Experimental Example 4 as a template, the AcGUS3M1 gene was ligated to the pGAPZαA vector using the Gibson assembly method to obtain the recombinant vector pGAPZαA-AcGUS3M1 plasmid. The recombinant plasmid was then introduced into the competent Top10 strain using thermal excitation, and positive clones were selected on LB plates containing 100 mg / L bleomycin resistance. Recombinants verified as correct by colony PCR were subjected to DNA sequencing. If the sequence was correct, the recombinant pGAPZαA-AcGUS3M1 was successfully constructed. Further referencing the Pichia yeast construction and verification methods in Experimental Example 3, the Pichia yeast AcGUS3M1 strain was obtained.

[0090] Experimental Example 6: Construction of β-glucuronidase combinatorially engineered bacteria (1) Construction of pGAPZαA-AcGUS3M1-NrsR vector To facilitate the subsequent selection and construction of β-glucuronidase recombinant strains, the bleomycin resistance fragment in the pGAPZαA-AcGUS3M1 plasmid from Example 5 was replaced with normycin resistance fragment according to the instructions of the Gibson Seamless Ligation Reagent Kit (NEB) to obtain the new resistant pGAPZαA-AcGUS3M1-NrsR recombinant plasmid.

[0091] (2) Construction and validation of dG-GA1 combinatorially engineered bacteria The pGAPZαA-AcGUS3M1-NrsR recombinant plasmid was linearized using the endonuclease BlnI. The linearization system was as follows: 2 μL of 10X QuickCut Buffer, 1 μL of BlnI, 1 μg of plasmid, and supplemented with sterile water to a volume of 20 μL. After instantaneous centrifugation, the mixture was incubated at 37°C for 1 hour. The linearized plasmid was purified and recovered using the GeneJET Gel Recovery Reagent Kit. The linearized pGAPZαA-AcGUS3M1-NrsR recombinant plasmid was transferred to Pichia yeast AtGUS using electroporation, and the resulting mixture was spread evenly onto a YPD solid plate containing 100 mg / L bleomycin and 100 mg / L normocycin. The plate was then incubated at 30°C for 2-3 days.

[0092] A single colony from the resistant plate was inoculated into 5 mL of YPD liquid medium containing 100 mg / L bleomycin and 100 mg / L normocycin and grown at 30°C for 2 days at 200 rpm on a rocking bed. The medium was then duplexed into YPD medium containing 4 g / L GL at a 1% duplexing volume and grown for 2 days, sampling every 6 hours and performing HPLC analysis. The intermediate product GAMG was consistently found to be below 4%. This demonstrates the successful construction of the Pichia yeast dG-GA1 recombinant engineered strain.

[0093] The recombinant expression vectors contained in this engineered bacterium dG-GA1 are pGAPZαA-AtGUS and pGAPZαA-AcGUS3M1-NrsR, where NrsR refers to the replacement of the resistance gene in the original plasmid with NrsR resistance.

[0094] Experimental Example 7: Comparison of enzyme activities of different Pichia yeasts engineered to produce GA To meet actual production needs, the enzyme activity comparison format for different Pichia yeast strains for GA production involves using a crude enzyme solution with a relatively high concentration of GL. Examples for analytical comparison include the AtGUS-mix described in Patent CN109628427B, which was previously developed by the inventor, the AcGUS described in the present invention, AcGUS3M1, and dG-GA1, and other comparative examples from the project team to which the inventor belongs: AtGUS and AuGUS described in Patent CN109628427B (the sequence of AuGUS and AtGUS is reported in the article "Properties and structures of β-glucuronidases with different transformation types of glycyrrhizin").

[0095] First, a single colony of the strain was picked from the resistant plate and placed in 5 mL of YPD medium containing 100 mg / L zeocin, and cultured at 30°C and 200 rpm on a rocking bed for 48 hours. 600 The mixture was duplexed into 100 mL YPD medium with a duplexing volume of 0.1 and cultured for 48 hours. 200 μL of crude enzyme supernatant was then taken and mixed with 800 μL of 10 g / L GL reaction buffer (pH 5.5). The mixture was incubated at 40°C for 1 hour, after which it was sampled and subjected to HPLC detection.

[0096] The results in Figure 6 show that the combinatorially engineered strain dG-GA1 is most effective at relatively high concentrations of GL substrate, exhibiting the highest GA production efficiency and maintaining extremely low levels of intermediate GAMG accumulation. In contrast, the comparative example AuGUS lacked enzyme activity and could not be heterologously expressed, consistent with reported results showing that it can only be expressed in AuGUS and wild-type strains. Furthermore, AtGUS and Atgusmix exhibited relatively high GAMG accumulation, but their transformation efficiencies were significantly lower than those of dG-GA1. It should be noted that the activity of the mutant AcGUS3M1, expressed in the safe Pichia coli strain, was still 13.18-fold higher than that of the wild-type AcGUS. This again demonstrates that the mutants obtained by the rational design described in Example 4 are not only capable of prokaryotic expression in E. coli, but can also be successfully expressed in eukaryotic Pichia yeast. Therefore, the combinatorially engineered strain dG-GA1 is considered the optimal GA producer in terms of enzyme activity comparison.

[0097] Experimental Example 8: Amplification of 18α-GA in a 5L fermenter The ability of dG-GA1 to produce 18α-GA was verified in a 5 L fermenter. dG-GA1 was inoculated from the scribed YPD plate into a 5 mL YPD liquid medium test tube and activated at 30°C in a rocking bed at 200 rpm for 36 h. OD 600 The seed solution was duplexed into a 100 mL YPD liquid medium vial at 0.1 and cultured for 24 hours at 30°C and 200 rpm on a rocking bed. The seed solution was duplexed into a 5 L fermenter containing 3 L of YPD liquid medium. The culture conditions were set at a fermentation temperature of 30°C, pH 5.5, and a rotation speed of 200 rpm. Batch fermentation was used, and the fermentation process lasted approximately 60 hours, with glucose being replenished twice. The strain growth OD during fermentation was monitored. 600The progress was monitored in real time. The reaction conditions for the substrate catalytic process were set at 42.5°C, pH 5.5, and agitation speed of 300 rpm. The 18α-GL concentration was 20 g / L each time, for a total of four feedings. The concentrations of the substrate 18α-GL, intermediate products GAMG, and 18α-GA in the reaction system were monitored in real time during this stage to accurately evaluate the catalytic effect of dG-GA1. The results are shown in Figure 7A. The results showed that in a 5L fermenter, the transformation process took 20 hours during batch feeding, with the final 18α-GA concentration reaching 41.09 g / L and a conversion rate of 96.57%, demonstrating good potential for industrial application in 18α-GL transformation.

[0098] Experimental Example 9: Amplification of 18β-GA in a 1000L fermenter To further verify the scalable production potential of dG-GA1 and characterize its transformation potential with 18β-GL, a 1000L fermenter was used. During the fermentation process, the Pichia yeast dG-GA1 recombinant engineered strain was first harvested from a scribed YPD plate, and a single colony was inoculated into 100mL of YPD liquid medium and cultured at 30°C for approximately 24 hours. The inoculum was then amplified in 10L, 100L, and 1000L fermenters containing YPD medium. The entire fermentation process was maintained under the following conditions: temperature 30°C, pH 5.5, and agitation speed 200 rpm. The seed liquid in both the 10L and 100L fermenters was cultured for approximately 24 hours. After culturing the culture in the 1000L fermenter for approximately 48 hours, 20g / L glucose was replenished every 12 hours, for a total of two replenishments. During the 1000L fermentation process, the strain growth OD was measured. 600 However, in the substrate catalysis stage, the reaction conditions were set at 42.5°C, pH 5.5, and agitation speed of 300 rpm. The 18β-GL concentration was 20 g / L each time. The changes in the concentrations of the substrate 18β-GL and the intermediate products GAMG and 18β-GA were monitored in real time. The results are shown in Figure 7B.

[0099] The results showed that the entire substrate catalysis step took 24 hours, with a total of six 18β-GL substrate supplements. At 24 hours, the GA concentration was 48.73 g / L, and the GL transformation rate reached 97.26%. Meanwhile, throughout the entire amplification test, the proportion of 18β-GAMG accumulated in the reaction system was always below 4%, and the GAMG concentration at 24 hours was less than 1.43 g / L. This process not only meets the needs for solving the above engineering problems, but also boasts the highest efficiency and yield of any currently reported process.

[0100] The above results demonstrate that the dG-GA1 combined engineering strain exhibited excellent overall expression, demonstrating the remarkable effects of AcGUS and its mutants in hydrolyzing GAMG and the effectiveness of the β-glucuronidase combination strategy. This further demonstrates that the present invention has the advantages of high catalytic efficiency, a short fermentation cycle, and an environmentally friendly process, making it promising for the industrial-scale production of glycyrrhetinic acid.

Claims

1. β-glucuronidase, beta-glucuronidase AcGUS, beta-glucuronidase AcGUS1, beta-glucuronidase AcGUS2, beta-glucuronidase AcGUS3 or a beta-glucuronidase AcGUS3 mutant; The β-glucuronidase AcGUS is an enzyme in which the first amino acid in the amino acid sequence having GenBank accession number AEK69352.1 is deleted, and the seventh alanine is changed to glycine, the sixty-sixth arginine is changed to lysine, the twenty-fifth alanine is changed to valine, the twenty-seventh threonine is changed to alanine, the thirty-second valine is changed to phenylalanine, the thirty-third aspartic acid is changed to glutamic acid, and the fifth methionine is changed to valine; The β-glucuronidases AcGUS1, AcGUS2, and AcGUS3 are enzymes obtained by cleaving 10, 20, and 30 amino acids from the nitrogen terminus of β-glucuronidase AcGUS, respectively, and the amino acid sequence of the β-glucuronidase AcGUS is as shown in SEQ ID NO.

1. The β-glucuronidase AcGUS3 mutant is selected from a mutant in which glycine at position 461 of AcGUS3 is mutated to cysteine, a mutant in which glutamine at position 462 of AcGUS3 is mutated to histidine, 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. β-glucuronidase characterized by:

2. The gene sequence of the β-glucuronidase AcGUS is shown in SEQ ID NO.

2. The mutant of AcGUS3 in which glycine at position 461 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. The β-glucuronidase according to claim 1.

3. An expression vector to which the gene sequence of β-glucuronidase according to claim 1 or 2 is ligated. A recombinant expression vector characterized by:

4. The expression vector is selected from pET28a, pGAPZαA, pPIC9K, or pPICZα. The recombinant expression vector of 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 of claim 3.

6. An engineered fungus, selected from Pichia yeast strain AcGUS3, Pichia yeast strain AcGUS3M1, or Pichia yeast strain dG-GA1; The Pichia yeast strain AcGUS3 is capable of expressing the β-glucuronidase AcGUS3 according to claim 1 or 2, The Pichia yeast strain AcGUS3M1 is capable of expressing the β-glucuronidase AcGUS3M1 according to claim 2; The Pichia yeast strain dG-GA1 is capable of simultaneously expressing the β-glucuronidase AcGUS3M1 according to claim 2 and the β-glucuronidase AtGUS derived from Aspergillus terreus strain Li-20. An engineered fungus characterized by:

7. The Pichia yeast strain AcGUS3 comprises the recombinant expression vector pGAPZαA-AcGUS3 according to claim 5. The engineered fungus of claim 6.

8. The Pichia yeast strain AcGUS3M1 comprises the recombinant expression vector pGAPZαA-AcGUS3M1 according to claim 5. The engineered fungus of claim 6.

9. The Pichia yeast strain dG-GA1 comprises a recombinant expression vector pGAPZαA-AcGUS3M1-NrsR, which is a modified version of the recombinant expression vector pGAPZαA-AcGUS3M1 described in claim 5, and a recombinant expression vector pGAPZαA-AtGUS, to which the β-glucuronidase AtGUS gene sequence derived from Aspergillus terreus strain Li-20 is ligated, where the modification refers to replacing the resistance gene of the original expression vector pGAPZαA with NrsR resistance in the recombinant expression vector pGAPZαA-AcGUS3M1. The engineered fungus of claim 6.

10. A fermentation agent containing a fermentation active component, wherein the fermentation active component comprises the β-glucuronidase according to claim 1 or 2, and / or the recombinant expression vector according to any one of claims 3 to 5, and / or the engineered bacterium according to any one of claims 6 to 9. A fermentation agent characterized by:

11. Further includes auxiliary materials The fermentation agent according to claim 10.

12. A method for mass-producing glycyrrhetinic acid, comprising fermenting a substrate using the engineered fungus Pichia yeast strain AcGUS3M1 or Pichia yeast strain dG-GA1 according to any one of claims 6 to 9, wherein the substrate for the Pichia yeast strain AcGUS3M1 is 18β-GL, and the substrate for the Pichia yeast strain dG-GA1 is selected from 18α-GL and 18β-GL. A method for mass-producing glycyrrhetinic acid.

13. When the substrate is 18β-GL, the glycyrrhetinic acid is 18β-GA, and 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 fermentation conditions include 42.5°C, pH 5.5, agitation speed of 300 rpm, and a feed concentration of 20 g / L. The method for mass-producing glycyrrhetinic acid according to claim 12.

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