Method for screening endophytic fungi with bioconversion activity of glycyrrhizin and screened strain

Abstract

The present application belongs to the field of biotechnology, and particularly relates to a method for screening endophytic fungi with bioconversion activity of glycyrrhizinic acid and the strains screened by the method. The method of the present application comprises the steps of primary screening and secondary screening. The strains Z6 and Z15 with bioconversion activity of glycyrrhizinic acid are screened by the method, and the registration numbers in the China General Microbiological Culture Collection Center are CGMCC No. 41704 and CGMCC No. 41706 respectively. The present application also obtains GH2 proteins with catalytic β-glucosidase hydrolysis activity from the two strains, which are Z6GH2 protein with the amino acid sequence of SEQ ID No. 4 in the sequence listing and Z15GH2 protein with the amino acid sequence of SEQ ID No. 6 in the sequence listing respectively. The strains Z6 and Z15 can promote the conversion of glycyrrhizinic acid, and have the effect of promoting the growth and quality of licorice.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a method for screening endophytic fungi with biotransformation activity of glycyrrhizic acid and the strains obtained from the screening. Background Technology

[0002] Licorice (Glycyrrhiza uralensis Fisch.) is a perennial herbaceous plant belonging to the legume family. Its roots and rhizomes are used medicinally, possessing properties such as invigorating qi and strengthening the spleen, resolving phlegm and relieving cough, detoxifying, and relieving pain. Records indicate that licorice has been used medicinally for over 2000 years, earning it the reputation of being a "nine out of ten prescriptions using licorice" and a "national elder." Licorice contains abundant flavonoids and triterpenoid saponins, among which glycyrrhizin and glycyrrhizic acid are specified as indicator components for content determination in the Chinese Pharmacopoeia. Glycyrrhizic acid, as an important bioactive component of licorice, possesses anti-inflammatory, antibacterial, antiviral, and anticancer pharmacological activities. Furthermore, recent studies have demonstrated that these plant-produced terpenoid compounds often act as phytoalexins or antibacterial molecules to inhibit the colonization of endophytic fungi.

[0003] Endophytic fungi are a class of microorganisms ubiquitous within plants, primarily acquired vertically from seeds or horizontally from the soil in which they grow. Studies have shown that endophytic fungi not only promote plant growth by producing exogenous hormones and improving nutrient absorption, but also play a crucial role in regulating the synthesis and accumulation of plant secondary metabolites. For example, the endophytic fungi *Chaetomium globosum* D38 and *Trichodermaatroviride* D16 from the roots of *Salvia miltiorrhiza* can promote the accumulation of tanshinone; the endophytic fungus *Penicillium oxalicum* from *Artemisia annua* can promote the synthesis of artemisinin. However, in order to enter the host and establish a stable symbiotic relationship, endophytic fungi must also secrete detoxification enzymes to overcome colonization barriers caused by secondary metabolites from the host. Therefore, screening based on the unique active components of medicinal plants can serve as an effective strategy for obtaining endophytic fungi that interact with host plant components. These component-interacting endophytic fungi may have greater application potential in further regulating the growth and metabolism of host plants. Summary of the Invention

[0004] The technical problem to be solved by this invention is how to promote the growth and improve the quality of the medicinal plant licorice.

[0005] To address the aforementioned technical problems, this invention first provides a method for screening endophytic fungi possessing glycyrrhizic acid biotransformation activity, comprising primary screening and secondary screening steps:

[0006] The initial screening involves culturing candidate endophytic fungi in a solid medium with glycyrrhizic acid as the sole carbon source, and selecting candidate endophytic fungi that show no morphological differences and grow well on a control solid medium with other carbon sources replacing glycyrrhizic acid as candidate endophytic fungi to enter the secondary screening.

[0007] The secondary screening involves liquid fermentation of the candidate endophytic fungi that have entered the secondary screening stage using a liquid culture medium supplemented with glycyrrhizic acid, and selecting candidate endophytic fungi that can convert glycyrrhizic acid in the liquid culture medium as endophytic fungi with biotransformation activity of glycyrrhizic acid.

[0008] In the above method, the glycyrrhizic acid content in the solid culture medium using glycyrrhizic acid as the sole carbon source can be 3 g / L.

[0009] In the above method, the control solid culture medium in which glycyrrhizic acid is replaced by other carbon sources can use glucose with the same concentration as the glycyrrhizic acid being replaced.

[0010] In the above method, glycyrrhizic acid is added during liquid fermentation, and its concentration can be 1 mg / mL after addition.

[0011] To solve the above-mentioned technical problems, the present invention also provides endophytic fungal strains Z6 and / or Z15, which are selected by the above method and have the activity of biotransforming glycyrrhizic acid.

[0012] The strain Z6 provided by this invention is *Aspergillus keveioide*, strain number Z6, and its registration number at the China General Microbiological Culture Collection Center (CGMCC) is CGMCC No. 41704. This strain was deposited at the CGMCC on December 10, 2024, at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China.

[0013] Z6 grew on PDA medium, exhibiting fluffy hyphae with white outer hyphae and brownish-brown inner hyphae, displaying radial striations on the dorsal side. Microscopic observation revealed that Z6 conidia were spherical or elliptical, with smooth, transparent conidiophores and branched hyphae. Strain Z6 possessed an ITS containing the sequence shown in SEQ ID No. 1.

[0014] The strain Z15 provided by this invention is *Aspergillus neoterreus*, strain number Z15, and its registration number at the China General Microbiological Culture Collection Center (CGMCC) is CGMCC No. 41706. This strain was deposited at the CGMCC on December 10, 2024, at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China.

[0015] The hyphae of Z15 are all fluffy, white on the upper surface and white on the outer surface and brownish-yellow on the inner surface with radial striations. Microscopic observation revealed that the conidia of Z15 are dumbbell-shaped or sickle-shaped without obvious septa, and the conidiophores are smooth, transparent, and have branched hyphae. Strain Z15 possesses an ITS containing the sequence shown in SEQ ID No. 2.

[0016] Cultures of Z6 and / or Z15 are also within the scope of protection of this invention.

[0017] In the above text, the culture of Z6 is a substance obtained by culturing Z6 in a microbial culture medium (e.g., Z6 spore liquid, the preparation method of which is: rinsing the spores obtained by culturing Z6 in a microbial culture medium with sterile water to obtain Z6 spore liquid).

[0018] In the above text, the culture of Z15 is a substance obtained by culturing Z15 in a microbial culture medium (e.g., Z15 spore liquid, the preparation method of which is: rinsing the sporophytes obtained by culturing Z15 in a microbial culture medium with sterile water to obtain Z15 spore liquid).

[0019] The culture of Z6 and / or the culture of Z15 described above have at least one of the following functions: W1-W4

[0020] W1. Function to promote the conversion of glycyrrhizic acid;

[0021] W2, as a β-glucosidase;

[0022] W3. Functions that promote the growth of licorice;

[0023] W4. Functions to improve the quality of licorice.

[0024] To address the above technical problems, the present invention also provides a product containing a culture of Z6 and / or Z15 and / or a culture of Z6 and / or Z15.

[0025] The product may be a microbial agent or a microecological preparation containing the microbial agent.

[0026] The product may specifically be any of the following products:

[0027] U1, Products that promote the conversion of glycyrrhizic acid;

[0028] U2, Products that promote licorice growth;

[0029] U3, Products that improve the quality of licorice.

[0030] The active ingredients of the above products may be Z6 or / and Z15 or / and cultures of Z6 or / and Z15. The active ingredients of the above products may also contain other biological or non-biological components. Other active ingredients of the above products can be determined by those skilled in the art based on the effects of the products.

[0031] The above products can be liquid or solid microbial agents.

[0032] The product may also include a carrier. The carrier may be a solid carrier or a liquid carrier. The solid carrier may be a mineral material or a biological material; the mineral material may be at least one of peat moss, clay, talc, kaolin, montmorillonite, white carbon, zeolite, silica, and diatomaceous earth; the biological material may be at least one of various crop straws, pine shells, rice straw, peanut shells, corn flour, soybean flour, starch, peat moss, and animal manure; the liquid carrier may be water; in the product, cultures of Z6 and / or Z15 and / or Z6 and / or Z15 may exist in the form of cultured live cells, fermentation broth of live cells, spore liquid, filtrate of cell culture, or a mixture of cells and filtrate.

[0033] The product can be in various dosage forms, such as liquid, emulsion, suspension, powder, granules, wettable powder or water-dispersible granules.

[0034] Depending on the requirements, surfactants (such as Tween 20, Tween 80, etc.), binders, stabilizers (such as antioxidants), pH adjusters, etc. may also be added to the product.

[0035] The application of cultures of Z6 and / or Z15 and / or cultures of Z6 and / or Z15 falls within the scope of protection of this invention, and the application is at least one or more of X1-X3:

[0036] X1. Application in the catalytic hydrolysis of glycyrrhizic acid;

[0037] X2. Application in increasing licorice yield;

[0038] X3. Application in improving the quality of licorice.

[0039] The method for cultivating the Z6 is also within the scope of protection of this invention.

[0040] The method for culturing Z6 provided by the present invention includes the step of culturing Z6 in a culture medium.

[0041] The method for cultivating the Z15 is also within the scope of protection of this invention.

[0042] The method for culturing Z15 provided by the present invention includes the step of culturing Z6 in a culture medium.

[0043] The method for preparing the product is also within the scope of protection of this invention.

[0044] The method for preparing the product provided by the present invention includes the step of using the Z6 or / and Z15 or / and the culture of Z6 or / and the culture of Z15 as the active ingredient of the product to obtain the product, wherein the product is a liquid bacterial agent or a solid bacterial agent.

[0045] To address the above technical problems, the present invention also provides the following applications of substances that enhance the activity of GH2 protein and / or increase the gene expression level of GH2 protein and / or increase the content of GH2 protein:

[0046] Y1. Application in the catalytic hydrolysis of glycyrrhizic acid;

[0047] Y2. Application in increasing licorice yield;

[0048] Y3. Application in improving the quality of licorice;

[0049] The GH2 protein is a protein that is, as shown in A1), A2), or A3):

[0050] A1) The amino acid sequence is that of the protein listed in SEQ ID No. 4 or SEQ ID No. 6;

[0051] A2) A protein obtained by substituting and / or deleting and / or adding amino acid residues of the protein in A1) that has more than 90% identity with the protein shown in A1) and has the same activity.

[0052] A3) is a fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of A1) or A2).

[0053] Of these, SEQ ID No. 4 in the sequence listing consists of 637 amino acid residues and may be derived from Z6. SEQ ID No. 6 in the sequence listing consists of 599 amino acid residues and may be derived from Z15.

[0054] In the above applications, the protein can be synthesized artificially, or its encoding gene can be synthesized first and then expressed biologically.

[0055] In the above applications, the protein tag refers to a polypeptide or protein fused with a target protein using in vitro DNA recombination technology for expression, detection, tracing, and / or purification of the target protein. The protein tag may be a Flag tag, His tag, MBP tag, HA tag, myc tag, GST tag, and / or SUMO tag, etc.

[0056] In the above applications, the identity refers to the identity of amino acid sequences. The identity of amino acid sequences can be determined using homology search sites on the Internet, such as the BLAST page on the NCBI homepage. For example, in Advanced BLAST 2.1, by using blastp as the program, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as the matrix, setting the Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values) respectively, and performing an identity search on a pair of amino acid sequences, the identity value (%) can be obtained.

[0057] In the above applications, the 90% or more of identity can be at least 91%, 92%, 95%, 96%, 98%, 99%, or 100% identity.

[0058] In the above applications, the substance is a biomaterial related to the GH2 protein, and is any one of the following:

[0059] B1) The nucleic acid molecule encoding the GH2 protein;

[0060] B2) An expression cassette containing the nucleic acid molecule described in B1);

[0061] B3) A recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2);

[0062] B4) Recombinant microorganisms containing the nucleic acid molecules described in B1), or recombinant microorganisms containing the expression cassette described in B2), or recombinant microorganisms containing the recombinant vector described in B3).

[0063] The nucleic acid molecule can be DNA, such as cDNA, genomic DNA, or recombinant DNA; the nucleic acid molecule can also be RNA, such as mRNA or hnRNA.

[0064] In the above applications, the nucleic acid molecule encoding the GH2 protein is a nucleic acid molecule whose coding sequence is SEQ ID No. 3 or SEQ ID No. 5.

[0065] In the above application, the expression cassette (GH2 gene expression cassette) containing the nucleic acid molecule described in B1) refers to a nucleic acid molecule capable of expressing GH2 in host cells. This nucleic acid molecule may include not only a promoter to initiate GH2 gene transcription but also a terminator to terminate GH2 transcription. Furthermore, the expression cassette may also include an enhancer sequence.

[0066] A recombinant expression vector containing the GH2 gene expression cassette can be constructed using existing prokaryotic expression vectors. The prokaryotic expression vector can be pET-32a, etc.

[0067] This invention also protects the GH2 protein.

[0068] The present invention also protects substances that enhance the activity of GH2 protein and / or increase the expression level of the GH2 protein gene and / or increase the content of GH2 protein.

[0069] Compared with the prior art, the present invention has the following advantages:

[0070] (1) This invention provides an effective method for screening endophytic fungi with biotransformation activity of glycyrrhizic acid, and provides a method for verifying their transformation activity.

[0071] (2) The Z6GH2 protein obtained from Z6 and the Z15GH2 protein obtained from Z15 have the activity of catalyzing the hydrolysis of glycyrrhizic acid.

[0072] (3) When the endophytic fungi Z6 and Z15 with glycyrrhizic acid biotransformation activity screened in this invention are used to infect licorice, they can show a significant growth-promoting effect on the growth of medicinal parts of licorice at different growth and development stages.

[0073] (4) Re-infecting licorice with the endophytic fungi Z6 and Z15 with glycyrrhizic acid biotransformation activity screened in this application can significantly increase the content of glycyrrhizin and glycyrrhizic acid active ingredients in licorice root.

[0074] Preservation Instructions

[0075] Z6 Preservation Instructions:

[0076] Classification and nomenclature of biological material: Aspergillus keveioide

[0077] Strain number of the biological material: Z6

[0078] Full name of the depository: China General Microbiological Culture Collection Center, China Microbiological Culture Collection Committee

[0079] Abbreviation of depositary institution: CGMCC

[0080] Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing; Postcode: 100101

[0081] Deposit date: December 10, 2024

[0082] Accession number: CGMCC No. 41704

[0083] Z15 Preservation Instructions:

[0084] Classification and nomenclature of biological materials: Aspergillus neoterreus

[0085] Strain number of the biological material: Z15

[0086] Full name of the depository: China General Microbiological Culture Collection Center, China Microbiological Culture Collection Committee

[0087] Abbreviation of depositary institution: CGMCC

[0088] Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing; Postcode: 100101

[0089] Deposit date: December 10, 2024

[0090] Accession number: CGMCC No. 41706 Attached Figure Description

[0091] Figure 1 This is the screening result of glycyrrhizic acid-converting endophytic fungi in Example 1 of the present invention. Figure 1 A: Growth of Z6 on MM flat plate; Figure 1 B: Growth of Z15 on MM plate; Figure 1 Fermentation images of C:Z6 and Z15 in PDB liquid medium; Figure 1 The conversion of glycyrrhizic acid by D:Z6 and Z15 in PDB liquid medium and MM liquid medium.

[0092] Figure 2 The image shows the GUS staining results of the Z6 and Z15 phenotypes and hyphae induced by different concentrations of glycyrrhizic acid in Example 1 of this invention. Each culture dish and the three small centrifuge tubes below it form a group, with the concentrations of glycyrrhizic acid used for induction from left to right being 0 g / L, 1 g / L, 3 g / L, and 5 g / L respectively.

[0093] Figure 3 This is for the identification of strains Z6 and Z15 in Example 1 of the present invention. Figure 3 A: Schematic diagram of the form of Z6 and Z15 on a PDA tablet; Figure 3 Schematic diagram of microscopic identification of hyphae and spores of B: Z6 and Z15; Figure 3Phylogenetic tree of the C:Z6 and Z15 ITS sequences.

[0094] Figure 4 This invention relates to the cloning, expression, and functional characterization of Z15GH2 and Z6GH2 in Example 1 of the present invention. Figure 4 A: Clonal gel imaging of the Z15GH2 and Z6GH2 genes, where M is the marker, lane 1 is Z15GH2, and lane 2 is Z6GH2; Figure 4 Western blot electrophoresis images of B: Z15GH2 and Z6GH2 expressed proteins, where lane 1 is the marker, lane 2 is Z15GH2, and lane 3 is Z6GH2; Figure 4 HPLC chromatogram of glycyrrhizic acid converted in vitro by C: Z6GH2 and Z15GH2.

[0095] Figure 5 The Z6 and Z15 retrostained licorice phenotypes and related indicators in Example 1 of this invention. Figure 5 A: Schematic diagram of the morphology of licorice plants restained by Z6 and Z15; Figure 5 B: Z6 and Z15 retrograde licorice phenotypic indicators. In the figure, "*" indicates p<0.05, and "**" indicates p<0.01.

[0096] Figure 6 The content of glycyrrhizic acid and glycyrrhizin in Z6-dyed licorice and Z15-dyed licorice in Example 1 of the present invention. Figure 6 A: Glycyrrhizic acid content; Figure 6 B: Glycyrrhizin content. In the figure, "*" indicates p<0.05, and "**" indicates p<0.01. Detailed Implementation

[0097] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0098] Unless otherwise specified, all quantitative experiments in the following examples were performed in triplicate, and the results were averaged. Experimental data were statistically analyzed using Excel and SPSS software.

[0099] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are conventional biochemical reagents and are commercially available.

[0100] The biomaterials used in the following examples are as follows:

[0101] The prokaryotic expression vector pET32a is a prokaryotic expression vector that is routinely stored in the laboratory, or it can be purchased through BioWind International Biological Reagent Logistics Center.

[0102] Ural licorice seeds were purchased from the authentic producing areas of Gansu.

[0103] Example 1

[0104] Endophytic fungi were isolated from Ural licorice using the licorice surface disinfection method, and 28 strains of endophytic fungi were obtained.

[0105] 1. Initial screening of glycyrrhizic acid conversion by endophytic fungi in licorice

[0106] Hyphae of each isolated endophytic fungal strain of licorice were picked and inoculated onto MM solid medium with glycyrrhizic acid as the sole carbon source and control medium (carbon source: glucose), respectively:

[0107] The formula for MM solid medium (MM+GL) with glycyrrhizic acid as the sole carbon source is as follows: glycyrrhizic acid (GL) 3 g / L, ammonium sulfate 1 g / L, anhydrous dimethyl hydrogen phosphate 7 g / L, potassium dihydrogen phosphate 3 g / L, magnesium sulfate heptahydrate 0.1 g / L, sodium citrate 0.5 g / L, and agar 15 g / L.

[0108] The formula for the control MM solid medium (MM+glu) is as follows: glucose (glu) 3 g / L, ammonium sulfate 1 g / L, anhydrous dimethyl hydrogen phosphate 7 g / L, potassium dihydrogen phosphate 3 g / L, magnesium sulfate heptahydrate 0.1 g / L, sodium citrate 0.5 g / L, and agar 15 g / L.

[0109] After 7 days of growth, the growth was observed. Based on the growth of the endophytic fungi, the strains with no difference in morphology from the control group and good growth were initially identified as endophytic fungi with glycyrrhizic acid conversion activity.

[0110] The results showed that strain Z6 ( Figure 1 strain A) and strain Z15 ( Figure 1 B) showed significantly better growth than the control on MM solid medium with glycyrrhizic acid as the sole carbon source, thus it was preliminarily determined that it has glycyrrhizic acid conversion activity.

[0111] 2. Secondary screening of glycyrrhizic acid transformation by endophytic fungi in licorice

[0112] The spores of endophytic fungus Z6 were rinsed with sterile water and prepared to a concentration of 1×10⁻⁶. 8 Z6 spore suspension at CFU / mL.

[0113] The spores of endophytic fungus Z15 were rinsed with sterile water and prepared to a concentration of 1×10⁻⁶. 8 Z15 spore suspension at CFU / mL.

[0114] The glycyrrhizic acid conversion activities of Z6 and Z15 were further screened through liquid fermentation. Details are as follows:

[0115] MM liquid culture medium formula: ammonium sulfate 1g / L, anhydrous dimethyl hydrogen phosphate 7g / L, potassium dihydrogen phosphate 3g / L, magnesium sulfate heptahydrate 0.1g / L, sodium citrate 0.5g / L.

[0116] 2.1 PDB liquid fermentation

[0117] PDB liquid culture medium formula: Cut potatoes (200g / L) into pieces and boil them in boiling water for 30 minutes to obtain their extract. Then filter the extract through 8 layers of gauze and add glucose (10g / L). Sterilize the extract by high temperature and high pressure steam at 121℃ for later use.

[0118] The following six processing groups were specifically set up (see...). Figure 1 C):

[0119] P+Z6+GL: Add 1 mL of Z6 spore suspension to 50 mL of PDB liquid medium and incubate with shaking at 28 °C and 160 rpm for 48 h. Then, add 50 mg / mL sterile GL solution (prepared as follows: weigh 50 mg of glycyrrhizic acid (GL) powder, dissolve it thoroughly in sterile hot water, and filter it through a 0.22 μm filter membrane to obtain a 50 mg / mL sterile GL solution) to each Erlenmeyer flask, so that the concentration of GL in the reaction system is 1 mg / mL, and continue to incubate with shaking under the same conditions for 5 days.

[0120] P+Z6: Add 1 mL of Z6 spore suspension to 50 mL of PDB liquid medium and incubate with shaking at 28 °C and 160 rpm for 48 h. Then, add sterile water to each conical flask, the same volume as the sterile GL solution in the P+Z6+GL treatment, and continue to incubate with shaking under the same conditions for 5 days.

[0121] P+Z15+GL: Add 1 mL of Z15 spore suspension to 50 mL of PDB liquid medium and incubate with shaking at 28 °C and 160 rpm for 48 h. Then, add 50 mg / mL of sterile GL solution to each Erlenmeyer flask to make the concentration of GL in the reaction system 1 mg / mL, and continue to incubate with shaking under the same conditions for 5 days.

[0122] P+Z15: Add 1 mL of Z15 spore suspension to 50 mL of PDB liquid medium and incubate with shaking at 28 °C and 160 rpm for 48 h. Then, add sterile water to each conical flask, the same volume as the sterile GL solution in the P+Z15+GL treatment, and continue to incubate with shaking under the same conditions for 5 days.

[0123] P+GL: Add 1 mL of sterile water to 50 mL of PDB liquid medium and incubate with shaking at 28 °C and 160 rpm for 48 h. Then, add 50 mg / mL of sterile GL solution to each conical flask to make the concentration of GL in the reaction system 1 mg / mL, and continue to incubate with shaking under the same conditions for 5 days.

[0124] PCK: Add 1 mL of sterile water to 50 mL of PDB liquid medium and incubate with shaking at 28 °C and 160 rpm for 48 h. Then, add sterile water to each conical flask, the same volume as the sterile GL solution in the P+GL treatment, and continue to incubate with shaking under the same conditions for 5 days.

[0125] The fermented bacterial broth was sonicated for 30 min (100 Hz) and filtered through sterile filter paper. 10 mL of the filtrate was added to an equal volume of ethyl acetate, and the mixture was sonicated for another 30 min. The upper organic phase was transferred to a new centrifuge tube. This extraction was repeated twice, and the extracts were combined. The resulting extract was evaporated to dryness using a rotary evaporator, and then reconstituted with 3 mL of methanol (HPLC grade). The resulting solution was filtered through a 0.22 μm filter membrane for subsequent determination of glycyrrhizic acid content.

[0126] The formula for calculating glycyrrhizic acid conversion rate is as follows:

[0127] Glycyrrhizic acid conversion rate = (GL content in control group - GL content in the test solution after fermentation) / GL content in control group × 100%. Liquid fermentation confirmed that both Z6 and Z15 could convert glycyrrhizic acid in PDB liquid medium, with a conversion rate of 100% for both. Figure 1 (D).

[0128] 2.2mm liquid fermentation

[0129] MM liquid culture medium formula: ammonium sulfate 1g / L, anhydrous dimethyl hydrogen phosphate 7g / L, potassium dihydrogen phosphate 3g / L, magnesium sulfate heptahydrate 0.1g / L, sodium citrate 0.5g / L.

[0130] The following six processing groups were specifically set up:

[0131] MM+Z6+GL: Add 1 mL of Z6 spore suspension to 50 mL of MM liquid medium and incubate with shaking at 28 °C and 160 rpm for 48 h. Then, add 50 mg / mL of sterile GL solution to each Erlenmeyer flask to make the concentration of GL in the reaction system 1 mg / mL, and continue to incubate with shaking under the same conditions for 5 days.

[0132] MM+Z6: Add 1 mL of Z6 spore suspension to 50 mL of MM liquid medium and incubate with shaking at 28 °C and 160 rpm for 48 h. Then, add sterile water to each conical flask, the same volume as the sterile GL solution in the P+Z6+GL treatment, and continue to incubate with shaking under the same conditions for 5 days.

[0133] MM+Z15+GL: Add 1 mL of Z15 spore suspension to 50 mL of MM liquid medium and incubate with shaking at 28 °C and 160 rpm for 48 h. Then, add 50 mg / mL of sterile GL solution to each Erlenmeyer flask to make the concentration of GL in the reaction system 1 mg / mL, and continue to incubate with shaking under the same conditions for 5 days.

[0134] MM+Z15: Add 1 mL of Z15 spore suspension to 50 mL of MM liquid medium and incubate with shaking at 28 °C and 160 rpm for 48 h. Then, add sterile water to each conical flask, the same volume as the sterile GL solution in the P+Z15+GL treatment, and continue to incubate with shaking under the same conditions for 5 days.

[0135] MM+GL: Add 1 mL of sterile water to 50 mL of MM liquid medium and incubate with shaking at 28 °C and 160 rpm for 48 h. Then, add 50 mg / mL of sterile GL solution to each conical flask to make the concentration of GL in the reaction system 1 mg / mL, and continue to incubate with shaking under the same conditions for 5 days.

[0136] MM: Add 1 mL of sterile water to 50 mL of MM liquid medium and incubate with shaking at 28 °C and 160 rpm for 48 h. Afterward, add sterile water to each conical flask, the same volume as the sterile GL solution in the P+GL treatment, and continue to incubate with shaking under the same conditions for 5 days.

[0137] The fermented bacterial broth was sonicated for 30 min (100 Hz) and filtered through sterile filter paper. 10 mL of the filtrate was added to an equal volume of ethyl acetate, and the mixture was sonicated for another 30 min. The upper organic phase was transferred to a new centrifuge tube. This extraction was repeated twice, and the extracts were combined. The resulting extract was evaporated to dryness using a rotary evaporator, and then reconstituted with 3 mL of methanol (HPLC grade). The resulting solution was filtered through a 0.22 μm filter membrane for subsequent determination of glycyrrhizic acid content.

[0138] Calculate the glycyrrhizic acid conversion rate.

[0139] In MM liquid medium, Z6 achieved a conversion rate of 8.43% for GL, while Z15 achieved a conversion rate of 100% for glycyrrhizic acid. Figure 1 (D).

[0140] Based on the above results, it was determined that Z6 and Z15 endophytic fungi of licorice possess biotransformation activity for glycyrrhizic acid.

[0141] 3. Identification of β-glucosidase activity in endophytic bacteria of Glycyrrhizic acid conversion

[0142] Prepare 1 / 5 PDB liquid culture medium with different glycyrrhizic acid (GL) concentrations:

[0143] 1 / 5PDA liquid culture medium formulation: Dilute PDB five times to obtain 1 / 5PDB liquid culture medium.

[0144] Formula for 1 / 5PDA liquid culture medium (control): Add glycyrrhizic acid powder to 1 / 5PDB liquid culture medium to make the concentration of glycyrrhizic acid in the culture medium 0g / L, and sterilize under high temperature and high pressure.

[0145] Formula for 1 / 5PDA liquid culture medium containing 1g / L glycyrrhizic acid: Add glycyrrhizic acid powder to 1 / 5PDB liquid culture medium to make the concentration of glycyrrhizic acid in the culture medium 1g / L, and sterilize under high temperature and high pressure.

[0146] Formula for 1 / 5PDA liquid culture medium containing 3g / L glycyrrhizic acid: Add glycyrrhizic acid powder to 1 / 5PDB liquid culture medium to make the concentration of glycyrrhizic acid in the culture medium 3g / L, and sterilize under high temperature and high pressure.

[0147] Formula for 1 / 5PDA liquid culture medium containing 5g / L glycyrrhizic acid: Add glycyrrhizic acid powder to 1 / 5PDB liquid culture medium to make the concentration of glycyrrhizic acid in the culture medium 5g / L, and sterilize under high temperature and high pressure.

[0148] Take a sterile 12-well plate and add 2 mL of liquid culture medium (1 / 5 PDA liquid culture medium with the above four different glycyrrhizic acid concentrations) to each well. Take a 5 mm mycelial disc of Z6 or Z15 strain cultured on PDA solid medium and place it in the above 12-well plate. Incubate statically at 28°C in the dark for 5 days. After the culture is completed, collect the mycelium and culture medium mixture into a sterile centrifuge tube, centrifuge at 8000 rpm for 5 min at 4°C, discard the supernatant, and take the mycelium for GUS staining. Prepare the staining solution according to the GUS staining kit (Beijing Cooler Master Technology Co., Ltd.).

[0149] Each strain was repeated 3 times for each liquid culture medium.

[0150] GUS staining results of mycelia after GL induction showed that the mycelia of Z6 and Z15 were stained to varying degrees by GUS under different concentrations of GL induction. Z6 mycelia stained a distinct blue color, clearly different from the control group, under induction with 1 g / L, 3 g / L, and 5 g / L glycyrrhizic acid. Z15 mycelia were almost unstained under induction with no or low concentration (1 g / L) of GL, but were stained by GUS under induction with 3 g / L and 5 g / L GL, with the degree of staining positively correlated with GL concentration. This preliminarily suggests that Z6 and Z15 may express β-glucosidase activity in a glycyrrhizic acid concentration-responsive pattern. Based on staining observation, it is preliminarily inferred that the mycelia stained blue in response to glycyrrhizic acid concentration successfully express β-glucosidase capable of converting glycyrrhizic acid.

[0151] 4. Identification and phylogenetic analysis of target endophytic fungi

[0152] Hyphae of endophytic fungi of licorice that have grown for 7 days were selected and placed on a glass slide. After staining with lactic acid phenol blue solution, the hyphae and spores of the strain were morphologically identified under a microscope (see...). Figure 3 A and Figure 3 (B).

[0153] On PDA medium, Z6 mycelia appear fluffy, with white outer hyphae and brownish-brown inner hyphae, exhibiting radial striations on the dorsal side. Microscopic observation revealed that Z6 conidia are spherical or elliptical, with smooth, transparent conidiophores and branched hyphae.

[0154] On PDA medium, Z15 hyphae are all fluffy, white on the front and white on the back, brownish-yellow on the inside with radial patterns. Microscopic observation revealed that Z15 conidia are dumbbell-shaped or sickle-shaped without obvious septa, and conidiophores are smooth, transparent, and have branched hyphae.

[0155] Under aseptic conditions, hyphae were picked and placed in 2 ml centrifuge tubes to extract genomic DNA from the target endophytic fungus. The fungal ITS sequence was amplified using universal fungal primers ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3'), and detected by agarose gel electrophoresis. The target fragment was sequenced by Songon Biotech (Shanghai, China).

[0156] The ITS sequence of strain Z6 (as shown in SEQ ID No. 1).

[0157] SEQ ID No.1

[0158] GGGCAGTCTGCCCCCGGGCAGGCCTAACCTCCCACCCGTGAATACCTGACCAACGTTGCTTCGGCGGTGCGCCCCCCCGGGGGTAGCCGCCGGAGACCACATTGAACCTCTTGTCTTTAGTGTTGTCTGAGCTTGATAGCAAACCTATTAAAACTTTCAACAATGGATCTCTTGGTTCCGGCATCGATGAAGAACGCAGCGAACTGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAGTCTTTGAACGCACATTGCGCCCCCTGGCATTCCGGGGGGCATGCCTGTCCGAGCGTCATTGCTGCCCTTCAAGCCCGGCTTGTGTGTTGGGTCGTCGTCCCCCCCGGGGGACGGGCCCGAAAGGCAGCGGCGGCACCGCGTCCGGTCCTCGAGCGTATGGGGCTTTGTCACCCGCTCGATTAGGGCCGGCCGGGCGCCAGCCGGCGTCTCCAACCTTCTATTTTACCAGGTTGACCTCGGATCAGGTAGGGATACCCGCTGAACTTAAGCATATCAAAAGCCGGAGGAA

[0159] ITS sequence of strain Z15 (shown in SEQ ID No.2).

[0160] SEQ ID No.2

[0161] GATTACCGGAGTGCGGGTCTTTATGGCCCAACCTCCCACCCGTGACTATTGTACCTTGTTGCTTCGGCGGGCCCGCCAGCGTTGCTGGCCGCCCGGGGGGCGTCTCGCCCCCGGGCCCGTGCCCGCCGGAGACCCCAACATGAACCC TGTTCTGAAAGCTTGCAGTCTGAGTGTGATTCTTTGCAATCAGTTAAAACTTTCAACAATGGATCTCTTGGTTCCGGCATCGATGAAGAACGCAGCGAAATGCGATAACTAATGTGAATTGCAGAATTCAGTGAATCATCGAGTCTT TGAACGCACATTGCGCCCCCTGGTATTCCGGGGGGCATGCCTGTCCGAGCGTCATTGCTGCCCTCAAGCCCGGCTTGTGTGTTGGGCCCTCGTCCCCCGGCTCCCGGGGGACGGGCCCGAAAGGCAGCGGCGGCACCGCGTCCGGTC CTCGAGCGTATGGGGCTTCGTCTTCCGCTCCGTAGGCCCGGCCGGCGCCCGCCGACGCATTTGTTTGCAACTTGTTTTTTTCCAGGTTGACCTCGGATCAGGTAGGGATACCCGCTGAACTTAAGCATATCAATAAGGCCGGAGGAA

[0162] The species information of the target strain was identified by searching the nucleotide sequence database using NCBI's BLAST program. Sequence data of relevant species were retrieved and downloaded from NCBI, and a phylogenetic tree was constructed using Neighbor-joining (NJ) in MEGA 11.0.

[0163] The results showed that Z6 and Z15 clustered together with closely related species in the genus *Aspergillus*, but were further classified into two different taxa. Z6 was more closely related to *Aspergillus keveioides*, with a similarity of 89%, while Z15 was most closely related to *Aspergillus neoterreus*, with a similarity of 64%. Therefore, Z6 was identified as *Aspergillus keveioides*, and Z15 as *Aspergillus neoterreus*.

[0164] Z6 was deposited on December 10, 2024, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China. This bacterium is *Aspergillus keveioide*, strain number Z6, and its registration number at CGMCC is CGMCC No. 41704.

[0165] Z15 was deposited on December 10, 2024, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China. The strain is *Aspergillus neoterreus*, strain number Z15, and its registration number at CGMCC is CGMCC No. 41706.

[0166] Cloning and prokaryotic expression of the 5Z6 and Z15 glycyrrhizic acid conversion-active β-glucosidase genes

[0167] Genomic DNA was extracted from Z6 and Z15, and after passing purity and concentration tests, libraries were constructed and sequenced using Illumina HiSwq. β-glucosidases with GL transformation activity are known to be distributed in the GH2 and GH79 families. Based on annotation results, BLAST analysis was performed in the Carbohydrate-Active enZYmes Database (CAZy) to identify GH2 and GH79 family-related glycoside hydrolases (GHs) in Z6 and Z15. Further bioinformatics analysis identified Z6A08613 as a candidate gene for Z6GH2 and Z15A5430 as a candidate gene for Z15GH2.

[0168] 5.1 Cloning and prokaryotic expression of the Z6GH2 gene

[0169] RNA was extracted from Z6 and cDNA was obtained through reverse transcription. Primers were designed based on the CDS sequence of the Z6GH2 candidate gene, and homologous arm sequences were added to the 5' end. The primer sequences are as follows:

[0170] Z6GH2-F: gccatggctgatatcggatccATGAAGTTCTCAAAAGCCCTGTACC (where the sequence marked with lowercase letters is the homologous arm sequence of pET32a, and the sequence marked with uppercase letters is the same as the sequence of positions 1-25 of SEQ ID No. 3);

[0171] Z6GH2-R: acggagctcgaattcggatccTCACTCACTGCCCAACTGCG (where the lowercase letter-tagged sequence is the homologous arm sequence of pET32a, and the uppercase letter-tagged sequence is the reverse complementary sequence of positions 1895-1914 of SEQ ID No. 3).

[0172] Candidate genes were amplified by PCR using 2×Phanta Flash Master Mix (Vazyme, China), and gel imaging was performed as shown in the figure. Figure 1 Lane 2 of A was used to recover the amplification product, yielding the Z6GH2 gene fragment.

[0173] The Z6GH2 gene fragment was further inserted into the prokaryotic expression vector pET32a using seamless cloning (ClonExpress II One Step Cloning Kit, Vazyme, China) to obtain a recombinant plasmid expressing the Z6GH2 gene, named pET32a-Z6GH2. The recombinant plasmid pET32a-Z6GH2 was transformed into *E. coli* competent cells Trans1-T1 Phage Resistant Chemically Competent Cell (TransGen, China) and sequenced. The CDS sequence of the Z6GH2 gene is shown in SEQ ID No. 3 (1914 bp), and its encoded amino acid sequence is shown in SEQ ID No. 4 (composed of 637 amino acid residues).

[0174] SEQ ID No. 3

[0175]

[0176] SEQ ID No. 4

[0177] MKFSKALYLLSLLPSTLSLPSTHHETRDLSSTQTPLLIQLKPKITPTRELISLDGLWSFALAQEGNTTAKPWTAPLPKGLECPVPASYNDIFVDRNIRDHVGWVYYQREVTVPRGWSEERYLVHVEAATHEGRIYVNDDLIAEHVGGYTPFEADITSLV AAGEKFRLTIAVNNELTMQTIPPGTIEVSEITGRRVQTYRHDFYNYAGLARSVWLYSVPKEQFIRDRPSLWQLVQETGVIRYNITTSEGTTGDVRIAVVDEEGNTVATTAGVNGTATIESVHLWQPGAAYLYDFRVTIVDSSNTTIDSYTLPVGIRTVS INGTQLLINNTPFYFTGFGKHEDSPIRGKGHDQAYMIHDFQLLDWIGANSFRTSHYPYAEEVLEFADRHGIVVIDETAAVGMAFSLSAGAAAASSSSLTFSPEGINNETRRNHAQAIRELVARDKNHASVVLWSIANEPASFEEGARGYFEPLVQVAREA DPAGRPVAFVNVGQATYETDKISDLFDVLCLNRYYGWYSQTGVLEEAEVALEEELRGWVDLYDKPIIITEYGADTVAGLHSVMAVPWSEEFQVQFLDMYHRVFDRFDAIVGEHVWNFADFQTSMGIIRVDGNKKGVFTRDRRPKAAAHKLRERWTQLGSE

[0178] The recombinant plasmid pET32a-Z6GH2 with the correct sequence was extracted and transformed into BL21(DE3)pLysS ChemicallyCompetent Cell. Positive bacterial cultures identified by PCR were used as the target culture medium for induction. The culture was then transferred to LB liquid medium (containing 100 μg / mL ampicillin) and cultured at 37°C with shaking at 180 rpm until OD600. 600The concentration was approximately 0.6. 0.2 mM IPTG was added, and the mixture was incubated at 16°C and 180 rpm for 16 h. The cultured bacterial solution was centrifuged at 4°C and 4000 × g for 15 min, the supernatant was discarded, and the bacterial pellet was washed twice with 1×PBS. The pellet was resuspended in 1×PBS containing lysis buffer and protease inhibitor, and the cells were sonicated on ice for 15 min (30% amplitude, 5 s interval between pulses). The mixture was then centrifuged (4°C, 8000 rpm) for 10 min, and the supernatant was collected to obtain the protease solution.

[0179] The results of Western blot electrophoresis are shown below. Figure 4 Lane 3 of swim 3 in B indicates that Z6GH2 was successfully expressed, and the obtained protease solution is the Z6GH2 protein solution.

[0180] 5.2 Cloning and prokaryotic expression of the Z15GH2 gene

[0181] RNA was extracted from Z15 and cDNA was obtained through reverse transcription. Primers were designed based on the CDS sequence of the Z15GH2 candidate gene, and homologous arm sequences were added to the 5' end. The primer sequences are as follows:

[0182] Z15GH2-F: ccgaattcgagctccgtcgacATGCTGAAGCCTCGACAAAC (where the sequence marked with lowercase letters is the homologous arm sequence of pET32a, and the sequence marked with uppercase letters is the same as the sequence of positions 1-20 of SEQ ID No. 5);

[0183] Z15GH2-R: tgcggccgcaagcttgtcgacTCAGTGGTCCTCATCCATCT (where the sequence marked with lowercase letters is the homologous arm sequence of pET32a, and the sequence marked with uppercase letters is the reverse complementary sequence of positions 1781-1800 of SEQ ID No. 5).

[0184] Candidate genes were amplified by PCR using 2×Phanta Flash Master Mix (Vazyme, China), and gel imaging was performed as shown in the figure. Figure 1 Lane 1 of A was used to recover the amplification product, yielding the Z15GH2 gene fragment.

[0185] The Z15GH2 gene fragment was further inserted into the prokaryotic expression vector pET32a using seamless cloning (ClonExpress II One Step Cloning Kit, Vazyme, China) to obtain a recombinant plasmid expressing the Z15A543 gene, named pET32a-Z15A543. The recombinant plasmid pET32a-Z15A543 was transformed into *E. coli* competent cells Trans1-T1 PhageResistant Chemically Competent Cell (TransGen, China) and sequenced. The CDS sequence of the Z15GH2 gene is shown in SEQ ID No. 5 (1800 bp), and its encoded amino acid sequence is shown in SEQ ID No. 6 (599 amino acid residues) of the Z15GH2 protein.

[0186] SEQ ID No. 5

[0187]

[0188] SEQ ID No. 6

[0189] MLKPRQTPFRDLISLDGLWKFDLESGNNATAAPWTGPLTTDLECPPVPASYNDIFVDRQIRDHVGWVYYQREAIVPRGWSQQQYLVRVDAATHQGRIYINDNLVAEHRGGYTPFEADITGLVSAGDSFRLTIAVNNELTHETIPPGRIEV EEYTGKKVQVYQHDFFNYAGLARSVWLYSVPQQHIQDIKVVTHVEGPAGLIDYLVMVSNTTTGRVKIHVIDEDGTTVAEASGTRGTVTIPSVKLWQPGAAYLYQFRVSIVGLNNSILDTYCVETGVRTVEVSGNRFLINDKPFYFTGFGK HEDSAVRGKGYDPAYMVHDFQLMNWMGANSFRTSHYPYAEEVMEFADRHGIVVIDETPAVGLSFSIGTGVSSENSPQTFTPEGINNNTREAHKQAIRELIARDKNHASVVMWSIANEPASQETGAREYFAPLVDLAHELDPTRPVCFANY GDATYEVDRISDMFDVLCLNRYFGWYSQTGEIGEEAAALEKELLGWEGKYEKPIVITEYGADTMAGLHSVLALPWSEEFQVQLLDMYHRVFDRIKSVVGEHVWNFADFQTAVGIIRVDGNKKGVFTRERKPKAAAHTLKTRWSGKMDEDH

[0190] The recombinant plasmid pET32a-Z15A543 with the correct sequence was extracted and transformed into BL21(DE3)pLysSChemically Competent Cell. PCR-positive bacterial culture was used as the induction culture and transferred to LB liquid medium (containing 100 μg / mL ampicillin). The culture was incubated at 37°C with shaking at 180 rpm until OD200. 600The concentration was approximately 0.6. 0.2 mM IPTG was added, and the mixture was incubated at 16°C and 180 rpm for 16 h. The cultured bacterial solution was centrifuged at 4°C and 4000 × g for 15 min, the supernatant was discarded, and the bacterial pellet was washed twice with 1×PBS. The pellet was resuspended in 1×PBS containing lysis buffer and protease inhibitor, and the cells were sonicated on ice for 15 min (30% amplitude, 5 s interval between pulses). The mixture was then centrifuged (4°C, 8000 rpm) for 10 min, and the supernatant was collected to obtain the protease solution.

[0191] The results of Western blot electrophoresis are shown below. Figure 4 Lane 2 of swim bladder B indicates that Z15GH2 was successfully expressed, and the obtained protease solution is the Z15GH2 protein solution.

[0192] Screening of in vitro catalytic activity of 6Z6GH2 and Z15GH2

[0193] To further determine whether the Z6GH2 and Z15GH2 candidate proteins possess catalytic activity for the hydrolysis of glycyrrhizic acid, in vitro catalytic experiments were conducted using glycyrrhizic acid as a substrate. The detection method is as follows:

[0194] A 1 mg / mL glycyrrhizic acid solution was prepared using HAc-NaAc buffer (pH 5.0). In a 1.5 mL centrifuge tube, 100 μL of the glycyrrhizic acid solution was added as substrate, followed by 100 μL of the target protein solution (either the Z6GH2 protein solution obtained in 5.1 or the Z15GH2 protein solution obtained in 5.2). The reaction was carried out at 37 °C for 12 h. The reaction was terminated by adding 200 μL of methanol to the resulting solution. The solution was filtered through a 0.22 μm filter and used for HPLC analysis to identify its transforming activity and transformation mechanism.

[0195] HPLC analysis results showed that ( Figure 4 (C) After reacting with glycyrrhizic acid substrates, the glycyrrhizic acid chromatographic peaks of Z6GH2 and Z15GH2 proteins showed a significant decrease or disappearance. Specifically, after reacting at 37℃ for 12 h, only glycyrrhetinic acid was detected after conversion of Z6GH2, while both glycyrrhetinic acid monoglucuronide and glycyrrhetinic acid were present in the conversion product of Z15GH2. Therefore, it was further determined that Z6GH2 and Z15GH2 proteins possess catalytic activity for GL hydrolysis, although their catalytic methods are not entirely consistent.

[0196] 7. Effects of endophytic fungi Z6 and Z15 on licorice growth

[0197] The licorice seeds were retrostained using the bacterial spore solution soaking method, with Z6 and Z15 as follows:

[0198] Z6 fungal spore solutions were prepared separately using sterile water and adjusted to a concentration of 1×10⁻⁶. 6cfu / mL was used as the stock solution for Z6 spores.

[0199] Z15 fungal spore solutions were prepared separately using sterile water and adjusted to a concentration of 1×10⁻⁶. 6 cfu / mL was used as the stock solution for Z15 spores.

[0200] Configure the following three processes:

[0201] Z6 retrofection group (Z6): Select plump Ural licorice seeds and soak them overnight in a 50-fold diluted Z6 spore stock solution. Then transfer the seeds to a petri dish and add a 500-fold diluted Z6 spore stock solution to culture until the seeds show signs of sprouting.

[0202] Z15 retrofection group (Z15): Select plump Ural licorice seeds and soak them overnight in a 50-fold diluted Z15 spore stock solution. Then transfer the seeds to a petri dish and add a 500-fold diluted Z15 spore stock solution to culture until the seeds show white sprouts.

[0203] Control group (CK): Select plump Ural licorice seeds and soak them in sterile water overnight. Then transfer the seeds to a petri dish and add sterile water to incubate until the seeds show signs of sprouting.

[0204] After the seeds in each group showed signs of germination, they were transplanted into a potting mix (nutrient substrate: vermiculite: sand, 2:1:1) with approximately 50 seeds per pot and allowed to grow under natural conditions. Each treatment was replicated five times.

[0205] Licorice plants from the control group, Z6 re-staining group, and Z15 re-staining group were collected and photographed at 2 months, 5 months, and 12 months of age. The phenotypic values ​​of taproot length, taproot diameter, fresh root weight, and dry root weight were measured to clarify the effects of Z6 and Z15 re-staining on licorice growth.

[0206] See results Figure 5 Phenotypically, at 2 months of age, the Z6 and Z15 re-inoculation of licorice showed no significant effect on promoting taproot length, taproot diameter, fresh root weight, and dry weight. At 5 months of age, the taproot diameter, fresh root weight, and dry weight of the Z6 and Z15 re-inoculated licorice were all higher than those of the control licorice. By 12 months of age, the taproot diameter, fresh root weight, and dry weight of the Z6 and Z15 re-inoculated licorice significantly exceeded those of the control licorice. This indicates that re-inoculation with Z6 and Z15 can effectively promote the growth of the medicinal parts of licorice, especially showing a stronger growth-promoting effect in the middle and late stages of the re-inoculated licorice growth.

[0207] 8. Effects of endophytic fungi Z6 and Z15 on licorice quality

[0208] Further qualitative and quantitative analysis was conducted on the indicative components such as glycyrrhizic acid and glycyrrhizin in the restained licorice roots of Z6 and Z15.

[0209] Licorice root samples from 2-month-old, 5-month-old, and 12-month-old plants (as described in Part 7 of this example) were dried in a 60°C oven and powdered. The powder was then passed through a No. 3 sieve according to the Chinese Pharmacopoeia standard. 0.2 g of the powder was accurately weighed into a 150 mL stoppered conical flask, and 100 mL of 70% ethanol was accurately added. After weighing, the mixture was sonicated for 30 min, cooled, and the weight loss was made up with 70% ethanol. The filtrate was filtered through a 0.22 μm filter membrane, and the contents of glycyrrhizic acid and glycyrrhizin, the indicative components, in the roots of the control and experimental groups were determined by HPLC.

[0210] The results show (see) Figure 6 After re-staining Z6 and Z15, the levels of glycyrrhizic acid and glycyrrhizin in the roots of 2-month-old licorice plants were higher than those in the control; at 5 and 12 months of age, the contents of glycyrrhizic acid and glycyrrhizin in the roots of Z6 and Z15 plants were significantly higher than those in the control. Further investigation revealed a positive correlation between the accumulation patterns of glycyrrhizic acid and glycyrrhizin and changes in root fresh weight and dry weight.

[0211] In summary, the results of this invention show that endophytic fungi Z6 and Z15, which have in vitro glycyrrhizic acid conversion activity, have a more significant growth-promoting and quality-improving effect on licorice after re-inoculation.

[0212] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. A method for screening endophytic fungi possessing glycyrrhizic acid biotransformation activity, characterized in that: The method includes primary screening and secondary screening steps: The initial screening involves culturing candidate endophytic fungi in a solid medium with glycyrrhizic acid as the sole carbon source, and selecting candidate endophytic fungi that show no morphological differences and grow well on a control solid medium with other carbon sources replacing glycyrrhizic acid as candidate endophytic fungi to enter the secondary screening. The secondary screening involves liquid fermentation of the candidate endophytic fungi that have entered the secondary screening stage using a liquid culture medium supplemented with glycyrrhizic acid, and selecting candidate endophytic fungi that can convert glycyrrhizic acid in the liquid culture medium as endophytic fungi with biotransformation activity of glycyrrhizic acid.

2. The endophytic fungus screened by the method of claim 1, which has the activity of bioconversion of glycyrrhizinic acid, characterized in that: The endophytic fungus possessing the activity of biotransforming glycyrrhizic acid is Z15 and / or Z6; Z15 is Aspergillus neoterreus, strain number Z15, and its registration number at the China General Microbiological Culture Collection Center is CGMCC No. 41706. Z6 is Aspergillus keveioide, strain number Z6, and its registration number at the China General Microbiological Culture Collection Center is CGMCC No. 41704.

3. A culture characterized by: The culture is either the Z15 culture obtained by culturing the Z15 described in claim 2 in a microbial culture medium, or the Z6 culture obtained by culturing the Z6 described in claim 2 in a microbial culture medium.

4. A product characterized by: Contains the Z15 culture of claim 2 and / or the Z6 culture of claim 3 and / or the Z15 culture of claim 3.

5. Use of Z15 and / or Z6 and / or Z15 culture and / or Z6 culture according to claim 2, characterized in that: The application is at least one or more of X1-X3: X1. Application in the catalytic hydrolysis of glycyrrhizic acid; X2. Application in increasing licorice yield; X3. Application in improving the quality of licorice.

6. Use of a substance that enhances the activity of a GH2 protein and / or increases the expression amount of a gene of a GH2 protein and / or increases the content of a GH2 protein, characterized in that: The application is at least one or more of Y1-Y3: Y1. Application in the catalytic hydrolysis of glycyrrhizic acid; Y2. Application in increasing licorice yield; Y3. Application in improving the quality of licorice; The GH2 protein is derived from Z15 or Z6 as described in claim 2; The GH2 protein is a protein that is, as shown in A1), A2), or A3): A1) The amino acid sequence is that of the protein listed in SEQ ID No. 6 or SEQ ID No. 4; A2) A protein obtained by substituting and / or deleting and / or adding amino acid residues of the protein in A1) that has more than 90% identity with the protein shown in A1) and has the same activity. A3) is a fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of A1) or A2).

7. Use according to claim 6, characterized in that, The substance is a biomaterial related to the GH2 protein, and is any one of the following: B1) The nucleic acid molecule encoding the GH2 protein; B2) An expression cassette containing the nucleic acid molecule described in B1); B3) A recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2); B4) Recombinant microorganisms containing the nucleic acid molecules described in B1), or recombinant microorganisms containing the expression cassette described in B2), or recombinant microorganisms containing the recombinant vector described in B3).

8. The application according to claim 7, characterized in that, The nucleic acid molecule encoding the GH2 protein is a nucleic acid molecule with a coding sequence of SEQ ID No. 5 or SEQ ID No.

3.

9. The GH2 protein as described in claim 6.

10. The biomaterial as described in claim 7 or 8.

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