An engineered bacterium for producing monoglucuronyl glycyrrhetinic acid and / or glycyrrhizic acid and a construction method and application thereof

Abstract

The present invention provides an engineered bacterium for producing monoglucuronyl glycyrrhetinic acid and / or glycyrrhizic acid, and a construction method and application thereof. In a first aspect, the present invention provides an engineered bacterium for producing monoglucuronyl glycyrrhetinic acid and / or glycyrrhizic acid, wherein the engineered bacterium expresses an exogenous cellulose synthase, or the engineered bacterium simultaneously expresses an exogenous cellulose synthase and a glucuronyl transferase. The engineered bacterium provided by the present invention can achieve efficient production of monoglucuronyl glycyrrhetinic acid and / or glycyrrhizic acid. After batch fed fermentation, the engineered bacterium can produce 552.90 mg / L of monoglucuronyl glycyrrhetinic acid and 475.90 mg / L of glycyrrhizic acid, respectively, and the yields are 239.35 times and 79.58 times the highest levels reported in related work, respectively. It is a potential method for achieving large-scale industrial production of monoglucuronyl glycyrrhetinic acid and / or glycyrrhizic acid.

Description

Technical Field

[0001] The invention relates to an engineering bacterium for producing monoglucuronic acid glycyrrhetinic acid and / or glycyrrhizic acid, a construction method and application thereof, and relates to the technical field of bioengineering. Background Art

[0002] Glycyrrhizic acid is the main active ingredient of the traditional Chinese medicine licorice. It is a typical pentacyclic triterpenoid saponin compound with a molecular weight of 822.93; monoglucuronyl glycyrrhetinic acid is a pentacyclic triterpenoid saponin compound after the distal glucuronic acid is hydrolyzed from glycyrrhizic acid, with a molecular weight of 646.81. Both are products of different amounts of glucuronidation at the C-3 hydroxyl group of glycyrrhetinic acid. Monoglucuronyl glycyrrhetinic acid and glycyrrhizic acid not only have similar physiological activities to glycyrrhetinic acid, such as anti-inflammatory, antibacterial, antiviral, and liver protection, but are also natural sweeteners with sweetness 941 times and 170 times that of sucrose, respectively. Therefore, they are widely used in medicine, food, cosmetics, and health products.

[0003] At present, the main method for obtaining monoglucuronyl glycyrrhetinic acid and glycyrrhizic acid is "root digging and acid extraction", that is, extracting from the roots of licorice plants. The extraction process includes multiple steps such as drying, grinding, and crushing, and it also needs to be combined with auxiliary means such as ultrasound and microwave. The extraction process is complicated, the extraction efficiency is low, and the cost is high. In addition, licorice has a well-developed root system that can prevent wind and sand. It is mainly distributed in western deserts and semi-desertified areas such as Gansu, Xinjiang, and Inner Mongolia in my country, and has important ecological application value. The acquisition mode of "root digging and acid extraction" seriously damages the vegetation distribution of licorice and the local ecological environment, which is not conducive to environmental protection. Therefore, the licorice used at this stage mainly comes from imports and artificial cultivation, but artificially cultivated licorice has disadvantages such as a long growth cycle, occupying a large area of ​​arable land, and being easily affected by climatic conditions, which seriously limits the preparation of monoglucuronyl glycyrrhetinic acid and glycyrrhizic acid.

[0004] In recent years, the development of synthetic biology and metabolic engineering has endowed microorganisms with the ability to synthesize heterologous terpene compounds. At the same time, the advantages of using microorganisms to synthesize natural products have become increasingly obvious, becoming a potential green and environmentally friendly alternative method. Researchers have constructed prokaryotic microbial cell factories such as Escherichia coli, Corynebacterium glutamicum and Bacillus subtilis, as well as eukaryotic microbial cell factories such as Saccharomyces cerevisiae, Pichia pastoris and Yarrowia lipolytica to produce terpene compounds. At the same time, the development of iterative omics technology, gene editing technology and in vitro assembly technology has promoted the construction and optimization of cell factories, providing favorable conditions for further improving the production efficiency of terpene compounds. The Chinese invention patent with the authorization announcement number CN106566815B discloses an engineered yeast of Saccharomyces cerevisiae for producing glycyrrhetinic acid or its precursors. Based on the endogenous mevalonate pathway of Saccharomyces cerevisiae, β-amyrin synthase, cytochrome P450 oxidase and cytochrome P450 reductase are sequentially introduced to realize the heterologous de novo synthesis of glycyrrhetinic acid; the Chinese patent with the application publication number CN111411089A realizes the synthesis of five rare licorice triterpenoids in yeast; the Chinese invention patent with the authorization announcement number CN110106222B provides a glycosyltransferase that catalyzes the production of monoglucuronyl glycyrrhetinic acid and glycyrrhizic acid from glycyrrhetinic acid; according to the literature “O-glycosyltransferases from Homo sapiens contributes to the biosynthesis of Glycyrrhetic Acid 3-O-mono-β-D-glucuronide and Glycyrrhizin in Saccharomycescerevisiae" reported that the yields of monoglucuronyl glycyrrhetinic acid and glycyrrhizic acid were 2.31±0.21 mg / L and 5.98±0.47 mg / L, respectively. Seeking more efficient key enzymes and pathways to optimize the construction of new microbial cell factories and achieve efficient preparation of monoglucuronyl glycyrrhetinic acid and / or glycyrrhizic acid is one of the hot topics that technicians in this field continue to pay attention to. Summary of the invention

[0005] The present invention provides an engineering bacterium, which realizes the efficient production of monoglucuronyl glycyrrhetinic acid and / or glycyrrhizic acid by expressing exogenous cellulose synthase, or simultaneously expressing exogenous cellulose synthase and glucuronyl transferase.

[0006] The present invention also provides a method for constructing the above-mentioned engineering bacteria.

[0007] The present invention also provides the use of the above-mentioned engineering bacteria in producing monoglucuronic acid glycyrrhizic acid and / or glycyrrhizic acid.

[0008] The first aspect of the present invention provides an engineered bacterium for producing monoglucuronyl glycyrrhetinic acid and / or glycyrrhizic acid, wherein the engineered bacterium expresses an exogenous cellulose synthase, or the engineered bacterium simultaneously expresses an exogenous cellulose synthase and a glucuronyl transferase;

[0009] The amino acid sequence of the cellulose synthase-like enzyme is shown in SEQ ID NO: 4;

[0010] The glucuronyl transferase is selected from one of (a) and (b):

[0011] (a) the amino acid sequence of glucuronyl transferase is shown in SEQ ID NO: 6;

[0012] (b) A mutant in which one or more amino acids in the amino acid sequence of (a) are mutated and has the same function.

[0013] In a specific embodiment, the mutant refers to a mutation in the 99th amino acid in the amino acid sequence shown in SEQ ID NO:6.

[0014] In a specific embodiment, the mutant refers to a mutant in which the 99th amino acid in the amino acid sequence shown in SEQ ID NO: 6 is mutated to leucine, and the amino acid sequence of the mutant is shown in SEQ ID NO: 16.

[0015] In a specific embodiment, the engineered bacteria uses Saccharomyces cerevisiae that produces glycyrrhetinic acid as a starting strain.

[0016] In a specific embodiment, the engineered bacteria also express exogenous UDP-glucose dehydrogenase, and the amino acid sequence of the UDP-glucose dehydrogenase is shown in SEQ ID NO:2.

[0017] In a specific embodiment, the engineered bacteria also overexpresses truncated hydroxymethylglutaryl-CoA reductase, dimethylallyl diphosphate isomerase, farnesyl pyrophosphate synthetase, farnesyl diphosphate farnesyltransferase, and squalene epoxidase;

[0018] The truncated hydroxymethylglutaryl-CoA reductase refers to the truncation of 1590 bp from the N-terminus of the hydroxymethylglutaryl-CoA reductase.

[0019] The second aspect of the present invention provides a method for constructing any of the above-mentioned engineering bacteria, comprising:

[0020] An expression cassette of a cellulose synthase-like enzyme is constructed, and the expression cassette of the cellulose synthase-like enzyme is constructed into the genome of the starting strain, or an expression cassette of a cellulose synthase-like enzyme and an expression cassette of a glucuronyl transferase are constructed, and the expression cassette of the cellulose synthase-like enzyme and the expression cassette of the glucuronyl transferase are constructed into the genome of the starting strain to obtain the engineered bacteria.

[0021] In a specific embodiment, the starting strain is Saccharomyces cerevisiae capable of producing glycyrrhetinic acid.

[0022] The third aspect of the present invention provides a method for producing monoglucuronyl glycyrrhetinic acid and / or glycyrrhizic acid, comprising fermenting any of the above-mentioned engineered bacteria to produce monoglucuronyl glycyrrhetinic acid and / or glycyrrhizic acid.

[0023] A fourth aspect of the present invention provides a kit for producing monoglucuronyl glycyrrhetinic acid and / or glycyrrhizic acid, the kit comprising any of the above-mentioned engineered bacteria.

[0024] The present invention constructs an engineered bacterium expressing an exogenous cellulose synthase, or constructs an engineered bacterium expressing an exogenous cellulose synthase and a glucuronyl transferase simultaneously, through metabolic engineering and synthetic biology, thereby achieving efficient production of monoglucuronyl glycyrrhetinic acid and / or glycyrrhizic acid, and the production process is simple, rapid and environmentally friendly, and is a potential method for achieving large-scale industrial production of monoglucuronyl glycyrrhetinic acid and / or glycyrrhizic acid. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The synthetic pathway diagram of monoglucuronyl glycyrrhetinic acid and glycyrrhizic acid in the engineered bacteria;

[0026] Figure 2 Liquid phase diagrams of the engineered bacteria GL01 and GAMG01 producing glycyrrhizic acid and monoglucuronyl glycyrrhetinic acid, respectively;

[0027] Figure 3 Liquid phase diagram of the engineered bacteria GAMG02 producing monoglucuronyl glycyrrhetinic acid;

[0028] Figure 4 Mass spectrum of monoglucuronyl glycyrrhetinic acid produced by engineered bacteria GAMG02;

[0029] Figure 5 Liquid phase diagram of glycyrrhizic acid production by engineered bacteria GL02;

[0030] Figure 6 Mass spectrum of glycyrrhizic acid produced by engineered bacteria GL02;

[0031] Figure 7 This is a test diagram of the relative yield of glycyrrhizic acid in the mutant provided by the present invention. DETAILED DESCRIPTION

[0032] In order to make the purpose, technical solutions and advantages of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0033] the term

[0034] As used herein, the "engineered bacteria" refers to a fungal cell strain that uses genetic engineering methods to efficiently express exogenous genes.

[0035] As used herein, "exogenous" refers to the relationship between two or more nucleic acid or protein sequences from different sources, or the relationship between a protein (or nucleic acid) and a host cell from different sources. For example, a nucleic acid is exogenous to a host cell if the combination of the nucleic acid and the host cell does not normally occur naturally. A particular sequence is "exogenous" to the cell or organism into which it is inserted.

[0036] As used herein, the "starting strain" refers to the original strain used for breeding.

[0037] As used herein, the "expression cassette" refers to a gene expression system that contains all the necessary elements required to express a target polypeptide, and generally includes the following elements: a promoter, a gene sequence encoding a polypeptide, and a terminator.

[0038] Engineering bacteria

[0039] The genes of cellulose synthase and / or glucuronyltransferase derived from Glycyrrhiza uralensis are transferred into yeast, so that the heterologous synthesis of monoglucuronyl glycyrrhetinic acid and / or glycyrrhizic acid can be achieved. Specifically, after yeast codon optimization, the nucleotide sequence of the gene encoding cellulose synthase is shown as SEQ ID No.3, and the amino acid sequence of cellulose synthase is shown as SEQ ID No.4, named GuCSyGT, which can efficiently catalyze glycyrrhetinic acid to obtain monoglucuronyl glycyrrhetinic acid; after yeast codon optimization, the nucleotide sequence of the gene encoding glucuronyltransferase is shown as SEQ ID No.5, and the amino acid sequence of glucuronyltransferase is shown as SEQ ID No.6, named UGT73P12, which can efficiently catalyze monoglucuronyl glycyrrhetinic acid to obtain glycyrrhizic acid; in addition, the inventors mutated the amino acids on the glucuronyltransferase and found that the partially mutated glucuronyltransferase also has the same catalytic function and helps to increase the yield of glycyrrhizic acid.

[0040] Based on the above research, in order to achieve efficient preparation of monoglucuronyl glycyrrhetinic acid and / or glycyrrhizic acid, the first aspect of the present invention provides an engineered bacterium, wherein the engineered bacterium expresses an exogenous cellulose synthase, or the engineered bacterium simultaneously expresses an exogenous cellulose synthase and a glucuronyl transferase;

[0041] The amino acid sequence of the cellulose synthase-like enzyme is shown in SEQ ID NO: 4;

[0042] The glucuronyl transferase is selected from one of (a) and (b):

[0043] (a) the amino acid sequence of glucuronyl transferase is shown in SEQ ID NO: 6;

[0044] (b) A mutant in which one or more amino acids in the amino acid sequence of (a) are mutated and has the same function.

[0045] Furthermore, the present invention carried out a saturation mutation on the glutamine at position 99 of glucuronyltransferase UGT73P12, and found that some mutants after the mutation also have the function of catalyzing monoglucuronic glycyrrhetinic acid to produce glycyrrhizic acid, among which the mutant in which the amino acid residue at position 99 is replaced by leucine has the best catalytic effect. The amino acid sequence of the mutant is shown in SEQ ID NO: 16, and the nucleotide sequence encoding the mutant is shown in SEQ ID No. 15.

[0046] In the present invention, the brewer's yeast that produces glycyrrhetinic acid is used as a starting strain, and the de novo synthesis of monoglucuronyl glycyrrhetinic acid and / or glycyrrhizic acid can be achieved using glucose as a substrate. The construction of the starting strain can be based on the construction method disclosed in the Chinese invention patent with authorization announcement number CN106566815B, specifically including: constructing a β-amyrin synthase expression cassette, a CYP450 oxidase expression cassette for oxidizing the carbon 11 position of β-amyrin, a CYP450 oxidase expression cassette for oxidizing the carbon 30 position of 11-oxy-β-amyrin, and a cytochrome P450 oxidoreductase expression cassette, and the above expression cassettes are jointly introduced into the genome of the brewer's yeast to obtain a starting strain that produces glycyrrhetinic acid using glucose as a substrate.

[0047] In addition, the starting strain used in the present invention, in addition to the Saccharomyces cerevisiae that produces glycyrrhetinic acid, can also be replaced by microbial production strains commonly used in the prior art, for example, Gram-positive bacteria such as Bacillus subtilis, Gram-negative bacteria such as Escherichia coli, actinomycetes such as Streptomyces coelicolour, yeasts such as Yarrowia lipolytica and Pichia pastoris, and fungi such as Aspergillus niger.

[0048] Since the sugar donor UDP-glucuronic acid is required in the process of catalyzing glycyrrhetinic acid to obtain monoglucuronyl glycyrrhetinic acid and glycyrrhizic acid, the synthesis of monoglucuronyl glycyrrhetinic acid and glycyrrhizic acid can be achieved by additionally adding the sugar donor UDP-glucuronic acid, but the cost is high, and the starting strain can only naturally synthesize UDP-glucose. Therefore, in order to achieve the de novo synthesis of monoglucuronyl glycyrrhetinic acid and / or glycyrrhizic acid, the engineered bacteria also express exogenous UDP-glucose dehydrogenase (UGDH) to catalyze UDP-glucose to produce UDP-glucuronic acid.

[0049] Furthermore, the present invention selects UDP-glucose dehydrogenase from Homo sapiens, the nucleotide sequence after yeast codon optimization is shown in SEQ ID No.1, the amino acid sequence of the encoded UDP-glucose dehydrogenase is shown in SEQ ID No.2, and it is named UGDH.

[0050] Figure 1 The synthetic pathway diagram of monoglucuronyl glycyrrhetinic acid and glycyrrhizic acid in the engineered bacteria is shown in FIG. Figure 1As shown, according to the endogenous mevalonate pathway of the starting strain and the introduced exogenous pathway, it is possible to synthesize glycyrrhetinic acid using glucose as a substrate, and then synthesize monoglucuronyl glycyrrhetinic acid and / or glycyrrhizic acid; in order to further enhance the endogenous mevalonate pathway of the starting strain, the engineered bacteria also overexpress truncated hydroxymethylglutaryl-CoA reductase, dimethylallyl diphosphate isomerase, farnesyl pyrophosphate synthetase, farnesyl diphosphate farnesyltransferase and squalene epoxidase, wherein the truncated hydroxymethylglutaryl-CoA reductase refers to the N-terminus of the hydroxymethylglutaryl-CoA reductase being truncated by 1590 bp, and the above sequences can all be retrieved from the database, which is the Saccharomyces Genome Database|SGD (yeastgenome.org).

[0051] Methods for constructing engineered bacteria

[0052] The present invention provides a method for constructing the above-mentioned engineered bacteria, comprising: constructing an expression cassette of a cellulose synthase-like enzyme, and constructing the expression cassette of the cellulose synthase-like enzyme into the genome of a starting strain, or constructing an expression cassette of a cellulose synthase-like enzyme and an expression cassette of a glucuronyl transferase, and constructing the expression cassette of the cellulose synthase-like enzyme and the expression cassette of the glucuronyl transferase into the genome of a starting strain to obtain the engineered bacteria.

[0053] In a specific embodiment, using Saccharomyces cerevisiae producing glycyrrhetinic acid as the starting strain, those skilled in the art can construct an expression cassette of the corresponding enzyme based on the nucleotide sequence of the corresponding enzyme provided by the present invention, and construct it into the genome of the starting strain. The construction method is described in detail below:

[0054] Step 1: Obtaining the target gene

[0055] According to the nucleotide sequences encoding the corresponding enzymes and their sources provided by the present invention, the corresponding target genes are obtained by PCR amplification, recombination or artificial synthesis. Specifically, the nucleotide sequence encoding UDP-glucose dehydrogenase is shown in SEQ ID No. 1, the nucleotide sequence encoding cellulose synthase is shown in SEQ ID No. 3, and the nucleotide sequence encoding glucuronyl transferase is shown in SEQ ID No. 5.

[0056] Step 2: Construction of expression cassette

[0057] In order to stably express the target gene in yeast, an expression cassette including the target gene is constructed. According to different needs, a promoter and a terminator are connected to the upstream and downstream of the target gene, respectively. The selection of the promoter and the terminator and the connection method with the target gene can be carried out according to conventional technical means in the art. For example, the promoter P GAL10, UDP-glucose dehydrogenase gene UGDH and terminator T TPI1 The UDP-glucose dehydrogenase UGDH expression cassette was obtained by overlapping extension PCR. GAL2 , cellulose synthase-encoding gene GuCSyGT and terminator T ADH1 The expression cassette of cellulose synthase-like GuCSyGT was obtained by overlapping extension PCR. GAL7 , glucuronyl transferase gene UGT73P12 and terminator T PGK1 The overlapping extension PCR method was used to connect and obtain the expression cassette of glucuronyl transferase UGT73P12.

[0058] The sequences of the promoter and terminator used in the present invention are all known sequences and can be retrieved from a database, which is Saccharomyces Genome Database|SGD (yeastgenome.org).

[0059] In addition, in order to facilitate the screening of positive transformed strains, a screening marker expression cassette needs to be constructed. Based on the yeast used in the present invention, the screening is usually based on auxotrophic mutant strains. Specifically, yeast cannot grow in the absence of a certain specific medium component, while yeast transformed with recombinant genes can grow normally in the absence of the special medium component, thereby screening the successfully transformed positive transformants. In one embodiment of the present invention, a leucine expression cassette is constructed as a screening marker gene, which is transferred into the starting strain together with the target gene, and the transformant is cultured in a medium lacking leucine. The strain that can grow normally is the positive transformant, and the engineered bacteria can be obtained by continuing to culture.

[0060] When the starting strain is yeast, left and right homology arms can be constructed, and the target gene can be inserted into the yeast genome through the homologous recombination ability of the yeast itself, so that the target gene is stably expressed in the yeast. The selection of the insertion site cannot destroy the normal growth and metabolism of the chassis strain itself. YPRCtau3 is a more commonly used integration site. Therefore, the present invention uses YPRCtau3 to insert the target gene. Subsequently, according to the insertion site of the target gene, the left and right homology arms are designed and inserted into the upstream and downstream of the expression cassette. The specific operation can be performed according to conventional technical means in the art.

[0061] When the engineered bacteria also overexpress truncated hydroxymethylglutaryl-CoA reductase, dimethylallyl diphosphate isomerase, farnesyl pyrophosphate synthetase, farnesyl diphosphate farnesyltransferase and squalene epoxidase, the same method can be used to construct an expression cassette including the target gene encoding the above enzymes, and transfer it into the genome of the starting strain to obtain an engineered bacteria capable of overexpressing the above enzymes. The specific method can be carried out according to conventional technical means in the art.

[0062] Step 3: Insert the constructed expression cassette into the genome of the starting strain to obtain an engineered bacterium.

[0063] The starting strain may be a yeast capable of producing glycyrrhetinic acid disclosed in the Chinese invention patent with authorization announcement number CN106566815B.

[0064] In addition, the starting strain used in the present invention can also be replaced by a microbial production strain commonly used in the prior art, for example, Gram-positive bacteria such as Bacillus subtilis, Gram-negative bacteria such as Escherichia coli, actinomycetes such as Streptomyces coelicolour, yeasts such as Yarrowia lipolytica and Pichia pastoris, and fungi such as Aspergillus niger. The construction of a specific recombinant plasmid / expression cassette can be carried out according to conventional technical means in the art.

[0065] The insertion of the expression cassette can be carried out according to conventional techniques in the art. In one embodiment of the present invention, the lithium acetate method can be used, that is, using alkaline Li + Changing the permeability of yeast cell membranes promotes the formation of competent states and makes it easier for cells to absorb external DNA.

[0066] Method for producing monoglucuronyl glycyrrhetinic acid and / or glycyrrhizic acid

[0067] The present invention also provides a method for producing monoglucuronyl glycyrrhetinic acid and / or glycyrrhizic acid, which comprises fermenting any of the above-mentioned engineering bacteria to produce monoglucuronyl glycyrrhetinic acid and / or glycyrrhizic acid.

[0068] Specifically, when the engineered bacteria are capable of expressing exogenous cellulose synthase, glycyrrhetinic acid can be used as a substrate to produce monoglucuronyl glycyrrhetinic acid.

[0069] When the engineered bacteria can simultaneously express exogenous cellulose synthase and glucuronyl transferase, glycyrrhetinic acid can be used as a substrate to produce monoglucuronyl glycyrrhetinic acid and glycyrrhizic acid.

[0070] When the brewer's yeast that produces glycyrrhetinic acid is used as the starting strain and the exogenous UDP-glucose dehydrogenase gene UGDH, the cellulose synthase gene GuCSyGT and the glucuronyl transferase gene UGT73P12 are expressed at the same time, monoglucuronyl glycyrrhetinic acid and glycyrrhizic acid can be produced using glucose as a substrate, and the fermentation conditions can be carried out according to conventional technical means in the art.

[0071] The present invention also provides a kit for producing monoglucuronic acid glycyrrhetinic acid and / or glycyrrhizic acid, wherein the kit comprises any one of the above-mentioned engineering bacteria.

[0072] In a specific embodiment, the kit further comprises glucose for providing a substrate for the production of monoglucuronyl glycyrrhetinic acid and / or glycyrrhizic acid.

[0073] In summary, the present invention constructs an engineered bacterium capable of expressing exogenous cellulose synthase, or constructs an engineered bacterium capable of simultaneously expressing exogenous cellulose synthase and glucuronyl transferase through metabolic engineering and synthetic biology, thereby achieving efficient production of monoglucuronyl glycyrrhetinic acid and / or glycyrrhizic acid, and the production process is simple, rapid and environmentally friendly, which is a potential method for achieving large-scale industrial production of monoglucuronyl glycyrrhetinic acid and / or glycyrrhizic acid.

[0074] The present invention is further described below in conjunction with specific examples. Unless otherwise specified, the various materials and reagents used in the following specific examples are commonly used materials and reagents in the art and can be obtained through regular commercial channels; the methods used are conventional methods known to those skilled in the art.

[0075] Example 1 Obtaining genes encoding related enzymes

[0076] (1) Obtaining the gene encoding UDP-glucose dehydrogenase (UGDH)

[0077] The UDP-glucose dehydrogenase gene UGDH from Homo sapiens was selected, and the nucleotide sequence after yeast codon optimization was SEQ ID No.1, and the amino acid sequence of the encoded protein was SEQ ID No.2.

[0078] (2) Obtaining the gene encoding cellulose synthase (GuCSyGT)

[0079] The cellulose synthase-like gene GuCSyGT from Glycyrrhiza uralensis was selected, and the nucleotide sequence after yeast codon optimization was SEQ ID No.3, and the amino acid sequence of the encoded protein was SEQ ID No.4.

[0080] (3) Obtaining the gene encoding glucuronyltransferase (UGT73P12)

[0081] The glucuronyl transferase gene UGT73P12 from Glycyrrhiza uralensis was selected, and the nucleotide sequence after yeast codon optimization was SEQ ID No.5, and the amino acid sequence of the encoded protein was SEQ ID No.6.

[0082] (4) Obtaining screening marker genes

[0083] According to the leucine (LEU2) expression cassette sequence in the pRS415 vector, primers SEQ ID No.7: 5'-TTCTGCGTTCGTTAACTGTGGGAATACTCAGGTATCG-3' and SEQ ID No.8: 5'-GACGTCCCATCCCTCCAATATCAAATTAGGAATCGTAG-3' were designed, and PCR amplification was performed using Phanta Max Super-Fidelity DNA Polymerase (purchased from Novazonics), and cloned into the pUC19 vector. Sequencing confirmed that no mutation occurred.

[0084] (5) Obtaining genomic homology arms

[0085] According to the sequence of the left homologous arm YPRC3L (SEQ ID No.9) and the right homologous arm YPRC3R (SEQ ID No.10) of the YPRCtau3 site in the Saccharomyces cerevisiae genome, primers SEQ ID No.11: 5'-CAGTGAATTCGAAAAGGAGGTGCACGCATTAT-3' and SEQ ID No.12: 5'-AGGAGAAAAAACTTCCAAGGAGGTGAAGAACGTC-3' were designed to amplify the left homologous arm YPRC3L of YPRCtau3; primers SEQ ID No.13: 5'-GATATTGGAGGGATGGGACGTCAGCACTG-3' and SEQ ID No.14: 5'-CTCTAGAGGATCCCCGGGTACGGTATTACTCGAGCCCG-3' were designed to amplify the right homologous arm YPRC3R.

[0086] The BY4741 genome was used as a template and PCR amplified using Phanta Max Super-Fidelity DNA Polymerase (purchased from Novazonics), and cloned into the pUC19 vector. Sequencing confirmed that no mutation occurred.

[0087] Experimental Example 2 Obtaining the expression cassette

[0088] (1) Construction of UDP-glucose dehydrogenase UGDH expression cassette

[0089] The left homology arm YPRC3L, yeast promoter P GAL10 , UDP-glucose dehydrogenase gene UGDH and yeast terminator T TPI1 The overlapping extension PCR method was used to connect the two fragments to obtain the expression cassette of UDP-glucose dehydrogenase.

[0090] (2) Construction of the cellulose synthase-like GuCSyGT expression cassette

[0091] The yeast terminator T TPI1 , yeast promoter P GAL2 , cellulose synthase-encoding gene GuCSyGT and yeast terminator T ADH1 The overlap extension PCR method was used to connect the two fragments to obtain the expression cassette of cellulose synthase-like enzyme GuCSyGT.

[0092] (3) Construction of glucuronyltransferase UGT73P12 expression cassette

[0093] The yeast terminator T ADH1 , yeast promoter P GAL7 , glucuronyl transferase gene UGT73P12 and yeast terminator T PGK1 The overlapping extension PCR method was used to connect the two fragments to obtain the expression cassette of glucuronyl transferase UGT73P12.

[0094] (4) Construction of screening marker expression cassette

[0095] The yeast terminator T PGK1 , the related gene of the screening marker leucine expression cassette, and the right homologous arm YPRC3R were connected using the overlapping extension PCR method to obtain the expression cassette of the screening marker LUE2.

[0096] Experimental Example 3 Obtaining engineered bacteria

[0097] Utilizing the homologous recombination ability of Saccharomyces cerevisiae itself, the lithium acetate method was used to transfer the UDP-glucose dehydrogenase UGDH expression cassette and the cellulose synthase GuCSyGT expression cassette into the YPRCtau3 site of the starting strain (using the yeast capable of producing glycyrrhetinic acid disclosed in the Chinese invention patent with authorization announcement number CN106566815B).

[0098] The transformed bacterial solution was spread on a selective medium SD-LEU plate lacking leucine, and the transformants were screened for positive clones by colony PCR after 48 hours of culture, and sequenced for verification. The verified transformants were transferred to liquid selective medium SD-LEU and cultured for 1 to 2 days to obtain the engineered bacteria, named GAMG01.

[0099] Using the same method, the expression cassettes of UDP-glucose dehydrogenase UGDH, cellulose synthase GuCSyGT and glucuronyl transferase UGT73P12 were transferred into the YPRCtau3 site of the starting strain to construct an engineered bacterium named GL01.

[0100] Experimental Example 4 Obtaining the key enzyme expression cassette in the mevalonate pathway

[0101] The following yeast endogenous sequences, such as left and right homology arms, yeast promoter, endogenous gene and yeast terminator, were obtained by PCR amplification using Phanta Max Super-Fidelity DNA Polymerase (purchased from Novazonics) with the genome of Saccharomyces cerevisiae BY4741 as a template.

[0102] (1) Construction of the truncated hydroxymethylglutaryl-CoA reductase tHMG1 expression cassette

[0103] The left homology arm rDNA, yeast promoter P TDH3 , truncated hydroxymethylglutaryl-CoA reductase encoding gene tHMG1 and yeast terminator T ECM10 The overlapping extension PCR method was used to connect the two fragments to obtain the expression cassette of hydroxymethylglutaryl-CoA reductase.

[0104] (2) Construction of the farnesyl pyrophosphate synthase ERG20 expression cassette

[0105] The yeast terminator T ECM10 , yeast promoter P TEF1 , farnesyl pyrophosphate synthase gene ERG20 and yeast terminator T AIP1 The overlap extension PCR method was used to connect the two fragments to obtain the expression cassette of farnesyl pyrophosphate synthetase.

[0106] (3) Construction of the farnesyl diphosphate farnesyl transferase ERG9 expression cassette

[0107] The yeast terminator T AIP1 , yeast promoter P HTA2 , farnesyl diphosphate farnesyltransferase gene ERG9 and yeast terminator T YHI9 The overlap extension PCR method was used to connect the two fragments to obtain the expression cassette of farnesyl diphosphate farnesyl transferase.

[0108] (4) Construction of squalene epoxidase ERG1 expression cassette

[0109] The yeast terminator T YHI9 , yeast promoter P CHO1 , squalene epoxidase gene ERG1 and yeast terminator T RPL41B The overlapping extension PCR method was used to connect the two fragments to obtain the expression cassette of squalene epoxidase.

[0110] (5) Construction of the expression cassette of dimethylallyl diphosphate isomerase IDI1

[0111] The yeast terminator T RPL41B , yeast promoter P FBA1 , dimethylallyl diphosphate isomerase gene IDI1 and yeast terminator T TDH1 The overlapping extension PCR method was used to connect the two fragments to obtain the expression cassette of dimethylallyl diphosphate isomerase.

[0112] (6) Construction of screening marker expression cassette

[0113] The yeast terminator T TDH1 , the related gene of the selection marker hygromycin expression cassette, and the right homologous arm rDNA are connected using the overlapping extension PCR method to obtain the expression cassette of the selection marker Hyg.

[0114] Experimental Example 5 Obtaining an engineered yeast strain that overexpresses key enzymes in the mevalonate pathway

[0115] Using the homologous recombination ability of yeast itself, the above-constructed expression cassettes were respectively transferred into the rDNA sites of the genomes of the engineered bacteria GL01 and GAMG01 by the lithium acetate method. The bacterial solution after transformation was spread on the YPD medium supplemented with hygromycin, and the transformants were screened for positive clones by colony PCR after 48 hours of culture, and sequenced for verification. The verified correct transformants were transferred to the liquid YPD medium supplemented with hygromycin and cultured for 1 to 2 days, and then streaked on plates or preserved with glycerol. The engineered yeasts that successfully integrated the expression cassettes related to precursor synthesis were named GL02 and GAMG02, respectively.

[0116] Experimental Example 6 Fermentation and Detection of Monoglucuronyl Glycyrrhetinic Acid and Glycyrrhizic Acid

[0117] (i) The engineered bacteria constructed in Example 3 were inoculated into 5 mL of YPD medium and cultured overnight at 30° C. and 200 rpm with shaking to obtain seed solution. Then, the inoculum was inoculated into 30 mL of YPD medium at a rate of 1% and fermented at 30° C. and 200 rpm for 5 days.

[0118] The product obtained after fermentation was centrifuged at 5000 rpm for 10 minutes to separate the supernatant and the bacteria. The centrifuged bacteria were resuspended with 0.5 mL of saturated sodium chloride and broken by beads. The broken bacteria and the separated supernatant were extracted with equal volumes of ethyl acetate, and the obtained organic phase was evaporated to dryness and re-dissolved with methanol. Detected by ultra-high performance liquid chromatography (UHPLC) and liquid chromatography-mass spectrometry (LC-MS).

[0119] Figure 2 The liquid phase diagrams of the engineered bacteria GL01 and GAMG01 producing glycyrrhizic acid and monoglucuronyl glycyrrhetinic acid, respectively, are shown in Figure 2 As shown, the engineered bacteria GL01 and GAMG01 can produce glycyrrhizic acid and monoglucuronyl glycyrrhetinic acid, respectively, and the yield of monoglucuronyl glycyrrhetinic acid is 28.33 mg / L, and the yield of glycyrrhizic acid is 27.62 mg / L.

[0120] (ii) The same method was used to perform fermentation verification on the engineered bacteria constructed in Example 5. Figure 3 Liquid phase diagram of the engineering bacteria GAMG02 producing monoglucuronyl glycyrrhetinic acid. Figure 4 The mass spectrum of monoglucuronyl glycyrrhetinic acid produced by the engineered bacteria GAMG02. Figure 5 Liquid phase diagram of glycyrrhizic acid produced by engineered bacteria GL02. Figure 6 The mass spectrum of glycyrrhizic acid produced by the engineered bacteria GL02 is shown in Figure 2. Figure 3-6 As shown, the engineered bacteria GAMG02 and GL02 can synthesize monoglucuronyl glycyrrhetinic acid and glycyrrhizic acid, respectively, and the yield of monoglucuronyl glycyrrhetinic acid of GAMG02 is 51.49 mg / L, and the yield of glycyrrhizic acid of GL02 is 40.78 mg / L.

[0121] Experimental Example 7 Obtaining UGT73P12 mutants and mutant recombinant engineering strains

[0122] The crystal structure of UGT73P12 was rationally analyzed, and the glutamine at the key residue 99 was selected for saturation mutation. ADH1 -P GAL7 -UGT73P12-T PGK1 As templates, PCR amplification was performed using corresponding primers (see Table 1), colony PCR and sequencing were performed on the positive clones, and finally the recombinant expression cassettes of 19 mutants were obtained.

[0123] PCR amplification system (50 μL): template 0.5 μL, upstream and downstream primers 2 μL each, Phanta Max Super-Fidelity DNA Polymerase 1 μL, 2x Buffer 25 μL, ddH2O 19.5 μL.

[0124] Table 1 Primer sequences of UGT73P12 mutant

[0125]

[0126] The obtained PCR product was purified using Tiangen's common DNA product purification kit, and then the template in the product was digested with DpnI. The digested product was then transferred into competent E. coli DH5α, and the transformants were picked for colony PCR and sequencing verification. Finally, a recombinant expression cassette containing 19 mutants was successfully constructed.

[0127] The recombinant expression cassette of the mutant was transferred into the YPRCtau3 site of the Saccharomyces cerevisiae genome that was previously constructed to produce the precursor glycyrrhetinic acid together with the UDP-glucose dehydrogenase UGDH expression cassette, the cellulose synthase GuCSyGT expression cassette and the selection marker expression cassette by the lithium acetate method. The bacterial solution after transformation was spread on the selective medium SD-LEU plate lacking leucine. After 48 hours of culture, the transformants were screened for positive clones by colony PCR and sequenced for verification. The correctly verified transformants were transferred to the liquid selective medium SD-LEU for 1 to 2 days, and the plates were streaked or glycerol was used for bacterial preservation. The engineered yeasts that successfully integrated the UGT73P12 mutant were named GL03-GL21, and the corresponding relationship between the strain number and the UGT73P12 mutant is shown in Table 2.

[0128] According to the method provided in Example 6, the engineered bacteria GL02-GL21 were fermented respectively. After 5 days of fermentation, the fermentation products were analyzed. The results are as follows: Figure 7 As shown, the strain GL03 had the highest yield, indicating that the mutant provided by the present invention is helpful to further increase the yield of glycyrrhizic acid.

[0129] Table 2 Correspondence between strain numbers and mutants

[0130]

[0131] Experimental Example 8 Production of Monoglucuronyl Glycyrrhetinic Acid and Glycyrrhizic Acid by Fed-Batch Fermentation

[0132] The production strain GAMG02 of monoglucuronyl glycyrrhetinic acid and the production strain GL03 of glycyrrhizic acid were subjected to batch feeding fermentation respectively, and the processes of the two were the same. The specific method includes: taking out the engineered bacteria frozen in glycerol, thawing it into liquid, inoculating it into a test tube containing 5mL YPD liquid culture medium at a ratio of 10%, culturing it at 30°C, 200rpm overnight, and then transferring it to a 250mL triangular flask containing 50mL YPD liquid culture medium at a ratio of 10%, culturing it at 30°C, 200rpm for 24h, and obtaining seed liquid. The seed liquid was inoculated into a fermenter, the fermentation temperature was controlled at 30°C, the fermentation pH was controlled at 5.5, and the feed concentration of glucose was controlled within 20g / L.

[0133] The fermentation medium is composed of: glucose 40g / L, (NH4)2SO4 15g / L, KH2PO4 8g / L, MgSO4·7H2O 6.15g / L, vitamin solution 12mL / L, trace metal salt solution 10mL / L. The vitamin solution is composed of: biotin 0.05g / L, calcium pantothenate 1g / L, niacin 1g / L, inositol 25g / L, thiamine 1g / L, pyridoxal 1g / L, p-aminobenzoic acid 0.2g / L. The composition of the trace metal salt solution is: EDTA 15g / L, ZnSO4·7H2O 5.75g / L, MnCl2·4H2O 0.32g / L, anhydrous CuSO4 0.5g / L, CoCl2·6H2O0.47g / L, Na2MoO4·2H2O 0.48g / L, CaCl2·2H2O2.9g / L, FeSO4·7H2O 2.8g / L.

[0134] After 5 days of fermentation, the fermentation products were analyzed according to the method provided in Example 6. The strain GAMG02 could synthesize 552.90 mg / L of monoglucuronyl glycyrrhetinic acid, and the strain GL03 could synthesize 475.90 mg / L of glycyrrhizic acid. The yields were 239.35 times and 79.58 times the highest levels reported in related work, respectively.

[0135] Comparative Example 1

[0136] This comparative example uses glycosyltransferase (GuGT14) and glycosyltransferase (UGT1A3) to produce monoglucuronyl glycyrrhetinic acid and glycyrrhizic acid, specifically including:

[0137] (1) Obtaining genes encoding glycosyltransferase (GuGT14) and glycosyltransferase (UGT1A3)

[0138] A glycosyltransferase gene from Glycyrrhiza uralensis was selected, and the nucleotide sequence after yeast codon optimization was shown as SEQ ID No. 17, and the amino acid sequence of glycosyltransferase GuGT14 was shown as SEQ ID No. 18. A glycosyltransferase gene from Homo sapiens was selected, and the nucleotide sequence after yeast codon optimization was shown as SEQ ID No. 19, and the amino acid sequence of glycosyltransferase UGT1A3 was shown as SEQ ID No. 20.

[0139] (2) Construction of glycosyltransferase (GuGT14) expression cassette and glycosyltransferase (UGT1A3) expression cassette

[0140] The yeast terminator T TPI1 , yeast promoter P GAL2 , glycosyltransferase gene GuGT14 and yeast terminator T ADH1 The overlapping extension PCR method was used to connect the two fragments to obtain the expression cassette of glycosyltransferase GuGT14.

[0141] The yeast terminator T TPI1 , yeast promoter P GAL2 , glycosyltransferase gene UGT1A3 and yeast terminator T ADH1 The overlapping extension PCR method was used to connect the two fragments to obtain the expression cassette of glycosyltransferase UGT1A3.

[0142] (3) Acquisition and fermentation verification of engineered bacteria

[0143] By using the lithium acetate method, the UDP-glucose dehydrogenase UGDH expression cassette and the glycosyltransferase GuGT14 expression cassette were transferred into the YPRCtau3 site of the starting strain (the yeast capable of producing glycyrrhetinic acid disclosed in the Chinese invention patent with the authorization announcement number CN106566815B was used) to obtain an engineered bacterium named GAMG03. A 5-day fermentation experiment was conducted on the strain GAMG03, and 5.36 mg / L of the target compound monoglucuronyl glycyrrhetinic acid could be synthesized. The synthetic ability of the product was weaker than that of the cellulose synthase GuCSyGT, indicating that the cellulose synthase GuCSyGT used in the present invention is helpful to increase the yield of monoglucuronyl glycyrrhetinic acid.

[0144] By using the lithium acetate method, the UDP-glucose dehydrogenase UGDH expression cassette and the glycosyltransferase UGT1A3 expression cassette were transferred into the YPRCtau3 site of the starting strain (the yeast capable of producing glycyrrhetinic acid disclosed in the Chinese invention patent with the authorization announcement number CN106566815B was used) to obtain an engineered bacterium named GL22. A 5-day fermentation experiment was conducted on the strain GL22, and 2.01 mg / L of monoglucuronyl glycyrrhetinic acid and 5.17 mg / L of glycyrrhizic acid were synthesized. The synthetic ability of the product was weaker than that of the cellulose synthase GuCSyGT and the glycosyltransferase UGT73P12, indicating that the enzyme used in the present invention helps to increase the yield of glycyrrhizic acid.

[0145] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. An engineered bacterium for producing glycyrrhizic acid, characterized in that: The engineered bacteria simultaneously express exogenous cellulose synthase and glucuronyl transferase; The amino acid sequence of the cellulose synthase-like enzyme is shown in SEQ ID NO: 4; The glucuronyl transferase is a mutant in which the amino acid at position 99 in the amino acid sequence shown in SEQ ID NO: 6 is mutated to leucine, and the amino acid sequence of the obtained mutant is shown in SEQ ID NO: 16; The engineering bacteria uses the brewer's yeast producing glycyrrhetinic acid as a starting strain.

2. The engineered bacteria according to claim 1, characterized in that The engineered bacteria also express exogenous UDP-glucose dehydrogenase, and the amino acid sequence of the UDP-glucose dehydrogenase is shown in SEQ ID NO:

2.

3. The engineered bacteria according to claim 1, characterized in that The engineered bacteria also overexpress truncated hydroxymethylglutaryl-CoA reductase, dimethylallyl diphosphate isomerase, farnesyl pyrophosphate synthetase, farnesyl diphosphate farnesyltransferase and squalene epoxidase; The truncated hydroxymethylglutaryl-CoA reductase refers to the truncation of 1590 bp from the N-terminus of the hydroxymethylglutaryl-CoA reductase.

4. The method for constructing the engineered bacteria according to any one of claims 1 to 3, characterized in that: It includes: Constructing an expression cassette of a cellulose synthase-like enzyme and an expression cassette of a glucuronyl transferase, and constructing the expression cassette of the cellulose synthase-like enzyme and the expression cassette of the glucuronyl transferase into the genome of a starting strain to obtain the engineered bacteria; The starting strain is Saccharomyces cerevisiae capable of producing glycyrrhetinic acid.

5. A method for producing glycyrrhizic acid, characterized in that: The engineered bacteria according to any one of claims 1 to 3 are fermented to produce glycyrrhizic acid.

6. A kit for producing glycyrrhizic acid, characterized in that: The kit comprises the engineered bacteria according to any one of claims 1 to 3.

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