Phosphoglyceraldehyde isomerase from a fungus traditional Chinese medicine and its coding gene and application

Abstract

The application discloses a phosphoglyceraldehyde isomerase from a fungus traditional Chinese medicine and an encoding gene and application thereof. The amino acid sequence of the phosphoglyceraldehyde isomerase is shown as SEQ ID No. 1, and the nucleotide sequence of the encoding gene is shown as SEQ ID No. 2. The phosphoglyceraldehyde isomerase is involved in the synthesis of a fungus traditional Chinese medicine polysaccharide precursor material phosphoglycero ketone from D-glyceraldehyde-3-phosphate, the nucleotide sequence of the encoding gene of the phosphoglyceraldehyde isomerase can be transferred into an engineering bacterium through a method of cloning, transduction, transformation and combination transfer, the expression of the phosphoglyceraldehyde isomerase gene is adjusted, the host is endowed with high expression of the phosphoglycero ketone, an effective way for expanding the yield of the phosphoglycero ketone and derivatives thereof is provided, and the application has a great application prospect.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and more specifically, to a glyceraldehyde-1-phosphate isomerase derived from fungal traditional Chinese medicine, its encoding gene, and its applications. Background Technology

[0002] As traditional Chinese medicinal fungi, Poria cocos, Cordyceps sinensis (also known as caterpillar fungus), Lentinus edodes, and Cicadae periostracum have all been found to contain various functional active ingredients such as polysaccharides, terpenes, nucleosides, and sterols, showing high potential for the development of new drugs. Polysaccharides, as the main active ingredient in these fungal medicinal materials, possess antioxidant, immunomodulatory, and antitumor biological activities. Fungal medicinal polysaccharides have shown good effects and broad clinical application prospects in antioxidation, immunomodulation, and antitumor activity.

[0003] *Hirsutella sinensis* is the only correct asexual line for *Cordyceps sinensis*, and it has been proven to have a high similarity to the active ingredients of wild *Cordyceps sinensis*, making it a suitable substitute in traditional Chinese medicine. Fungal polysaccharides are bioactive macromolecules composed of varying proportions of fructose, mannose, glucose, and other monosaccharides, as well as the precursor glycerol. They possess a wide range of physicochemical properties, and their immunomodulatory and antitumor activities have attracted widespread attention in the scientific community. Due to their broad biological characteristics, fungal polysaccharides with clinical application value have been applied in many specialized fields. Fungal polysaccharides also have anticancer effects and can enhance human immunity.

[0004] Because polysaccharides and their analogues have important pharmacological effects, numerous domestic and international publications have reported on the extraction and purification processes of polysaccharides. However, reports on functional genes related to polysaccharide biosynthesis are scarce. For example, Chinese patent CN113430186A discloses a fructose kinase from a fungal traditional Chinese medicine, which participates in the synthesis of D-fructose, a major precursor monosaccharide of Cordyceps polysaccharide, starting from D-fructose-6-phosphate. Chinese patent CN113897341A discloses a hexulose kinase from *Hymenochrysis pilosula*, which participates in the synthesis of D-fructose, a major precursor monosaccharide of Cordyceps polysaccharide, starting from D-fructose-1-phosphate. However, the functional enzymes or genes from fungal traditional Chinese medicines disclosed in the above-mentioned prior art are mainly related to the precursor monosaccharide D-fructose in fungal traditional Chinese medicine polysaccharides, while reports on functional genes from fungal traditional Chinese medicines related to the synthesis of the polysaccharide precursor glycerol phosphate are rare. Summary of the Invention

[0005] The purpose of this invention is to overcome the deficiencies and shortcomings of existing technologies in which there are few functional genes related to the synthesis of glycerol phosphate from fungal traditional Chinese medicine, and to provide a glycerol phosphate isomerase derived from fungal traditional Chinese medicine.

[0006] A second objective of this invention is to provide the encoding gene for the glyceraldehyde-3-phosphate isomerase of the fungus.

[0007] A third objective of this invention is to provide the application of the fungal glyceraldehyde phosphate isomerase and its encoding gene.

[0008] The above-mentioned objective of this invention is achieved through the following technical solution:

[0009] This invention preliminarily obtained the triose-phosphate isomerase of the fungus *Hypericum sinense* based on genome and transcriptome sequencing. Further in-depth research confirmed that the triose-phosphate isomerase (EC: 5.3.1.1) and its encoding gene are involved in the synthesis of the polysaccharide precursor glycerol phosphate from D-glyceraldehyde-3-phosphate, thus providing a newly obtained triose-phosphate isomerase of the fungus *Hypericum sinense*, its encoding gene, and its applications.

[0010] This invention first provides a fungal glyceraldehyde phosphate isomerase, which has the following characteristics:

[0011] A. The amino acid sequence as shown in SEQ ID No. 1;

[0012] B. The amino acid sequence of a derivative protein that still possesses glyceraldehyde phosphate isomerase function, obtained by substituting and / or deleting and / or adding one or more amino acid residues of A above, having at least 70% to 99% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) homology to the amino acid sequence defined by A.

[0013] SEQ ID No. 1:

[0014] MGFDGPASPPRRRLIGLSTKMYFSLSRTRDFVSQFLQHLSAIPADKLSQVDVLVIPDFVSLAATAEQLKACPVPIWLGAQDCQWEDRGAYTGQVSPMVLSEAGVRVVVGHAERRRMFGEDDATVALKAAA VSRNSMVPLVCIGEQTRGAVAVAVDECRGQVEAVVGALGETPDVVLAYEPVWAIGASQPAGEEHVLGVVDGIRALECLRERKGSTRVVYGGSAGPGLYRRLSKGLDGLFLGRFGHDPERFVQTIHEVIEA.

[0015] This invention relates to a glyceraldehyde phosphate isomerase derived from the fungus *Hypericum sinense*, which participates in the synthesis of glycerol phosphate, a polysaccharide precursor, from D-glyceraldehyde-3-phosphate. The enzyme has 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more homology to the amino acid sequence shown in SEQ ID No. 1, preferably as shown in SEQ ID No. 1 (denoted as tpi protein). This enzyme catalyzes the preparation of glycerol phosphate from D-glyceraldehyde-3-phosphate. Due to the specificity of amino acid sequences, any fragment or variant of a peptide protein containing the amino acid sequence shown in SEQ ID No. 1, such as its conserved variants, bioactive fragments, or derivatives, is within the scope of protection of this invention, provided that the fragment or variant shares 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology with the amino acid sequence shown in SEQ ID No. 1 and still possesses glyceraldehyde-3-phosphate isomerase function. Specific modifications may include the deletion, insertion, or substitution of amino acids in the amino acid sequence; wherein, for conserved modifications of variants, the substituted amino acid has a similar structure or chemical properties to the original amino acid, such as replacing isoleucine with leucine; variants may also have non-conserved modifications, such as replacing glycine with tryptophan.

[0016] The pathway of the phosphoglyceraldehyde isomerase derived from fungal traditional Chinese medicine in the biosynthesis of phosphoglycerone from D-glyceraldehyde-3-phosphate is shown below:

[0017]

[0018] Therefore, the present invention also provides the application of the fungal glyceraldehyde isomerase in the biocatalytic preparation of glycerol phosphate from D-glyceraldehyde-3-phosphate.

[0019] Furthermore, the application involves using the lysate obtained by fermenting and culturing wet bacterial cells obtained from fungal glyceraldehyde isomerase, in vitro recombinant protein of fungal glyceraldehyde isomerase, or recombinant fungal glyceraldehyde isomerase as a catalyst, and D-glyceraldehyde-3-phosphate as a substrate, to prepare glycerol phosphate ketone.

[0020] This invention also provides the encoding gene for the glyceraldehyde-3-phosphate isomerase of the aforementioned fungal traditional Chinese medicine, which has the following characteristics:

[0021] A. A nucleotide sequence as shown in SEQ ID No. 2;

[0022] B. Hybridizes with the nucleotides defined in A and encodes a nucleotide sequence of the amino acid shown in SEQ ID No. 1;

[0023] C. A nucleotide sequence that is at least 70% to 99% (for example, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) homologous to the nucleotide sequence defined in A, and encodes the amino acid sequence as shown in SEQ ID No.1.

[0024] SEQ ID No.2:

[0025] ATGGGCTTCGACGGGCCAGCGTCACCGCCGCGGCGTCGGCTCATCGGGCTGTCGACCAAAATGTACTTTTCTCTGTCGCGCACCCGCGACTTTGTCAGTCAGTTCCTGCAGCATCTGTCGGCCATCCCGGCAGACAAACTCTCCCAGGTCGACGTCTTGGTCATCCCCGACTTTGTCTCTCTCGCGGCGACGGCCGAGCAGCTCAAGGCCTGTCCGGTGCCCATCTGGCTCGGCGCTCAGGACTGCCAGTGGGAGGACCGGGGCGCCTATACAGGCCAAGTCAGCCCCATGGTCCTGAGCGAGGCAGGCGTGCGCGTCGTCGAAGTCGGCCACGCGGAGCGGCGGCGCATGTTTGGCGAAGACGACGCGACGGTGGCGCTCAAGGCGGCGGCCGTGAGTCGCAACTCCATGGTGCCGCTCGTGTGCATCGGGGAGCAGACGCGCGGGGCCGTGGCCGTTGCCGTCGACGAGTGCCGCGGGCAGGTCGAGGCCGTGGTCGGCGCCCTGGGCGAGACGCCAGACGTGGTGCTCGCCTACGAGCCCGTCTGGGCCATTGGCGCGAGCCAGCCTGCGGGCGAGGAGCACGTGCTCGGCGTCGTCGACGGCATCCGCGCGCTTGAATGCCTGCGGGAGAGGAAGGGATCCACGAGGGTCGTGTACGGGGGCAGTGCAGGGCCGGGCCTGTATCGGCGCCTGAGCAAAGGCTTGGACGGCCTGTTTCTCGGCAGGTTTGGACACGACCCGGAGCGGTTTGTGCAGACGATTCACGAAGTCATCGAGGCTTGA.

[0026] This invention also provides the encoding gene for the above-mentioned enzyme, namely, the glyceraldehyde-3-phosphate isomerase gene, which has 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more homology to the nucleotide sequence shown in SEQ ID No. 2, preferably as shown in SEQ ID NO: 2 (denoted as the tpi gene). Due to the specificity of the nucleotide sequence, any variant of the polynucleotide shown in SEQ ID No. 2, as long as it has 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more homology to the polynucleotide and encodes the amino acid shown in SEQ ID No. 1, is within the scope of protection of this invention. The polynucleotide variant refers to a polynucleotide sequence with one or more nucleotide changes. This polynucleotide variant can be a live or non-live variant, including substitution variants, deletion variants, and insertion variants. As is known in the art, an allelic variant is a substitution of a polynucleotide, which may be a substitution, deletion, or insertion of a polynucleotide, but does not substantially alter the function of the peptide protein it encodes.

[0027] The gene can be used to construct a genetically engineered bacterium for phosphoglyceraldehyde isomerase to biocatalyze the preparation of phosphoglycerone from D-glyceraldehyde-3-phosphate, or to isolate and purify a recombinant protein from the genetically engineered bacterium and then biocatalyze the preparation of phosphoglycerone from D-glyceraldehyde-3-phosphate; thereby increasing the yield of phosphoglycerone or its derivatives.

[0028] The present invention also provides a recombinant expression vector containing the coding gene.

[0029] The present invention also provides recombinant bacteria containing the recombinant expression vector.

[0030] The present invention also provides the application of the encoding gene, the recombinant expression vector, or the recombinant bacteria in constructing a genetically engineered bacterium or fungal glyceraldehyde isomerase recombinant protein capable of biocatalyzing the preparation of glycerol phosphate from D-glyceraldehyde-3-phosphate.

[0031] Furthermore, the application involves constructing a recombinant expression vector containing the gene for glyceraldehyde isomerase of the fungus, transforming the recombinant expression vector into a host expression bacterium, inducing the culture of the obtained recombinant genetically engineered bacteria, and separating and purifying the culture medium to obtain bacterial cells containing glyceraldehyde isomerase of the fungus or further separating and purifying to obtain the recombinant protein of glyceraldehyde isomerase of the fungus.

[0032] The present invention also provides the application of the phosphoglyceraldehyde isomerase derived from fungal traditional Chinese medicine or its encoding gene or recombinant strain in the preparation of formulations that catalyze the preparation of phosphoglycerone from D-glyceraldehyde-3-phosphate.

[0033] The present invention also provides a formulation for catalyzing the preparation of glycerol phosphate from D-glyceraldehyde-3-phosphate, wherein the formulation contains the above-mentioned glyceraldehyde phosphate isomerase derived from fungal traditional Chinese medicine or its in vitro recombinant protein or its recombinant bacteria.

[0034] The key point of this invention is to provide the amino acid sequence shown in SEQ ID No. 1 and the nucleotide sequence shown in SEQ ID No. 2 and their functions; given that the amino acid sequence and nucleotide sequence are known, obtaining the amino acid sequence and nucleotide sequence, as well as obtaining the related vector and host cell, are conventional technical means for those skilled in the art.

[0035] Compared with the prior art, the present invention has the following beneficial effects:

[0036] This invention provides a glyceraldehyde-1-phosphate isomerase related to the synthesis of glyceraldehyde-1-phosphate, a polysaccharide precursor from fungi. Its amino acid sequence is shown in SEQ ID No. 1, and the nucleotide sequence of its encoding gene is shown in SEQ ID No. 2. This fungal glyceraldehyde-1-phosphate isomerase participates in the synthesis of glyceraldehyde-1-phosphate, a polysaccharide precursor, starting from D-glyceraldehyde-3-phosphate. The nucleotide sequence of its encoding gene can be transferred into engineered bacteria through cloning, transduction, transformation, and conjugation transfer. By regulating the expression of the glyceraldehyde-1-phosphate biosynthesis gene (glyceraldehyde-1-phosphate isomerase gene), the host is endowed with high expression of glyceraldehyde-1-phosphate, providing an effective way to increase the yield of glyceraldehyde-1-phosphate and its derivatives, and has significant application prospects. Attached Figure Description

[0037] Figure 1 This is a gel electrophoresis image of the PCR amplification product of the glyceraldehyde phosphate isomerase gene from a fungal traditional Chinese medicine.

[0038] Figure 2 Physical map of the recombinant expression plasmid pET-28a / tpi.

[0039] Figure 3 This is an SDS-PAGE image of glyceraldehyde-3-phosphate isomerase. Detailed Implementation

[0040] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0041] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0042] Example 1: Extraction and sequencing of total RNA from fungal traditional Chinese medicine

[0043] Total RNA was extracted from the fungus *Hypericum sinense* using TRIzol reagent according to the instructions. mRNA was enriched using Oligo(dT) magnetic beads, and fragmentation buffer was added to break the mRNA into short fragments (150-700 bp). The first cDNA strand was synthesized using hexabase random primers with the mRNA as a template. The second cDNA strand was then synthesized using the same hexabase random primers. The cDNA was purified using a QiaQuick PCR kit, end-repaired, polyA was added, sequencing adapters were ligated, fragment size was selected by agarose gel electrophoresis, and finally, PCR amplification was performed. The constructed library was sequenced using Illumina GA IIx.

[0044] Example 2: Assembly of short RNA read sequences from fungal traditional Chinese medicines and functional annotation of the Unigene.

[0045] De novo transcriptome assembly was performed using the short-read assembly software SOAPdenovo (Li, Zhu et al. De novo assembly of human genomes with massively parallel short read sequencing[J]. Genome Res, 2010, 20:265-272.). After gap filling, the Unigene sequence was compared with the protein databases COG, Swiss-Prot, nr, and KEGG using blastx (evalue < 0.00001). The protein with the best comparison result was selected to determine the sequence orientation of the Unigene.

[0046] Secondly, the Unigene sequence was compared with Swiss-Prot, the protein database nr, KEGG, and COG (evaluation value <0.00001) using blastx to identify the protein with the highest similarity to the given Unigene sequence, thus obtaining the protein functional annotation information of the Unigene. By statistically analyzing the GO functional classification of all Unigenes, the gene functional distribution characteristics of this species can be understood macroscopically. The pathway annotation of the Unigene can be further obtained from KEGG annotation information.

[0047] Example 3: Analysis of the biosynthetic pathway of polysaccharides in fungal traditional Chinese medicine

[0048] Based on the fructose and mannose metabolic pathways annotated in the KEGG metabolic pathway annotation (map00051), the annotated enzymes are those related to the fructose and mannose metabolic pathways of fungal traditional Chinese medicine that have been detected. One unigene, glyceraldehyde phosphate isomerase, which synthesizes the polysaccharide precursor glycerol phosphate from D-glyceraldehyde-3-phosphate, was detected. Using online detection with NCBI's ORF Finder software, the open reading frame (SEQ ID No. 2) of this gene was identified, and the corresponding protein sequence (SEQ ID No. 1) was obtained.

[0049] Example 4: Primer design for the tpi gene of glyceraldehyde phosphate isomerase in fungal Chinese medicine

[0050] Primers were designed using the GENE RUNNER primer design software based on the predicted open reading frame DNA sequence of the gene. These primers were used to clone the phosphoglyceraldehyde isomerase gene (SEQ ID No. 2) of phosphoglycerone, a precursor of polysaccharides synthesized by fungi in traditional Chinese medicine. The primers were synthesized by Suzhou Hongxun Biotechnology Co., Ltd., and the primer sequences are listed below:

[0051] tpi gene: forward primer 5'-ATA GAA TTC ATG GGC TTC GAC GGG-3'

[0052] Reverse primer 5'-AGG AAG CTT TCA AGC CTC GATGAC-3'

[0053] The tpi gene is 786 bp in length.

[0054] Example 5: Preparation of the first strand of cDNA from fungal traditional Chinese medicine

[0055] Total RNA was extracted from the fungal Chinese herbal medicine *Hypericum sinense* according to the method provided in Example 1. After obtaining the total RNA, the first strand of cDNA from the fungal Chinese herbal medicine was synthesized as follows.

[0056] First-strand cDNA was synthesized from Total RNA using the PrimeScript 1st Strand cDNA Synthesis Kit (TaKaRa). The experimental steps are as follows:

[0057] [1] Prepare the following mixture in a Microtube tube: Oligo dT Primer (1 μL), Total RNA (0.5 μg), dNTP Mixture (1 μL), RNase Free dH2O (up to 10 μL).

[0058] [2] Denaturation and annealing are beneficial for the denaturation of template RNA and the specific annealing of reverse transcription primers and templates, which can improve the efficiency of reverse transcription reaction. Therefore, denaturation and annealing reactions are performed on a PCR instrument with the following conditions: 65℃ for 5 min.

[0059] [3] After annealing, centrifuge for a few seconds to allow the mixture of template RNA / primers, etc. to accumulate at the bottom of the microtube.

[0060] [4] Prepare the following reverse transcription reaction solution in the above Microtube tube. Total Volume (20 μL): the above denaturing and annealing reaction solution (10 μL), 5× PrimeScript Buffer (4 μL), RNase Inhibitor (0.5 μL), RNase Free dH2O (5 μL), PrimeScript RTase (0.5 μL).

[0061] [5] The reverse transcription reaction was performed on the PCR instrument under the following conditions: 42℃ for 15-30 min, 70℃ for 15 min.

[0062] The first strand of cDNA can be synthesized using the PolyA structure at the 3' end of eukaryotic mRNA with the help of reverse transcriptase, and the Oligo(dT) primer can be used as a template to synthesize cDNA.

[0063] Example 6: Cloning, expression, and protein activity detection of the tpi gene, an enzyme of glyceraldehyde-3-phosphate isomerase, which is a precursor of glycerol phosphate in the synthesis and metabolism of polysaccharides from fungal traditional Chinese medicine.

[0064] (1) PCR amplification of the glyceraldehyde phosphate isomerase tpi gene

[0065] Using the first strand of cDNA obtained in Example 5 as a template, PfuDNA polymerase PCR amplification was performed using the tpi gene primers 5'-ATAGAA TTC ATG GGC TTC GAC GGG-3' and 5'-AGG AAG CTT TCA AGC CTC GATGAC-3' synthesized in Example 4. The reaction conditions were set as follows:

[0066] Pfu PCR amplification reaction system. Total system (100 μL): nucleic acid-free water (85 μL), 10× buffer (10 μL), 4× dNTP (1 μL), Pfu (1 μL), cDNA first strand (1 μL), forward primer (50 μM, 1 μL), reverse primer (50 μM, 1 μL).

[0067] Pfu DNA Ploymerase PCR amplification conditions: denaturation (95℃, 5 min), 35 cycles (denaturation: 95℃, 45 s; annealing: 55-70℃, 1 min; extension: 72℃, 2 min), post-extension (72℃, 20 min).

[0068] (2) Gel electrophoresis detection of PCR products of glyceraldehyde phosphate isomerase tpi gene

[0069] First, heat the prepared 0.9% agarose gel in a microwave oven until it dissolves evenly. Next, take 15 mL of gel and, when it cools to approximately 50°C, add 1 μL of Goldview staining solution. Mix well and pour the mixture onto an electrophoresis gel plate. Remove any air bubbles and insert the sample comb. After the gel solidifies, carefully remove the sample comb and place the gel plate into the electrophoresis tank. Add TAE electrophoresis buffer to the electrophoresis tank. Then, take 5 μL of sample and add 1.5 μL of 6× Loading Buffer and 4 μL of ddH2O. Mix well and pipette the mixture, loading 10 μL of sample. Next, connect the power cord between the electrophoresis tank and the electrophoresis apparatus; the positive terminal is red, and the negative terminal is black. Finally, turn on the power and begin electrophoresis. The maximum voltage should not exceed 5V / cm. Stop electrophoresis when the sample has passed 2 / 3 of the gel plate. After disconnecting the power, remove the gel and place it in a gel imaging system for observation and photography.

[0070] Transcriptome sequencing predicted the size of the glyceraldehyde-1,4-phosphate isomerase (TPI) gene to be 786 bp. Agarose gel electrophoresis results showed that the glyceraldehyde-1,4-phosphate isomerase (TPI) gene was successfully amplified, and the actual size was consistent with the predicted size. (See attached image.) Figure 1 Sequencing showed that the sequence was identical to SEQ ID No. 2.

[0071] (3) Treatment with base A and purification of PCR products of glyceraldehyde phosphate isomerase tpi gene

[0072] Because the Pfu DNA polymerase PCR product has blunt ends, it needs to be treated with base A and purified after gel recovery before it can be used for T-vector ligation. The base A addition system for the gel recovery product is as follows: Total system (50 μL): gel recovery product (25 μL), 10×Taq DNA Polymerase Buffer (5 μL), 4×dNTPs (2 μL), Taq DNA Polymerase (1 μL), sterile water (17 μL).

[0073] Add A bases to the PCR instrument at 72℃ for 20 min, and finally purify using the AxyPrep PCR cleaning kit.

[0074] (4) Ligation of the glyceraldehyde phosphate isomerase tpi gene with the cloning vector

[0075] The cloning vector pMD18-T Vector was purchased from TaKaRa (TaKaRa code D101A). The glyceraldehyde-1-phosphate isomerase (tpi) gene was ligated into the cloning vector to construct the recombinant plasmid pMD18-T / tpi. The ligation system and conditions are as follows: Total volume (10 μL): T Vector (1 μL), T4 DNA Ligase (1 μL), 2×Buffer (5 μL), target gene (3 μL).

[0076] Connection conditions: 16℃, 16h; Inactivation: 65℃, 15min.

[0077] (5) Transformation of glyceraldehyde-1-phosphate isomerase tpi recombinant plasmid pMD18-T / tpi

[0078] First, the recombinant plasmid pMD18-T / tpi was transformed into *E. coli* JM109 to construct recombinant bacteria *E. coli* JM109 / pMD18-T / tpi carrying the glyceraldehyde-1,5-phosphate isomerase (tpi) gene. Second, 10 μL of the reaction mixture was transferred to competent *E. coli* JM109 cells and incubated on ice for 30 min; followed by heat shock at 42°C for 90 s. Then, the cells were incubated on ice for 2–3 min; 800 μL of LB broth was added, and the cells were incubated at 37°C for 250 rpm for 1 h. Finally, the mixture was plated (containing Amp resistance) and incubated overnight at 37°C.

[0079] (6) Screening of E. coli JM109 / pMD18-T / tpi positive recombinant bacteria for glyceraldehyde-3-phosphate isomerase

[0080] Colony PCR eliminates the need for genomic DNA extraction, allowing direct PCR amplification using DNA exposed after bacterial cell pyrolysis as a template. This method is simple, rapid, and quickly identifies positive colonies containing the target plasmid, making it commonly used in transformation identification. In this experiment, single colonies inoculated into liquid culture medium were subjected to colony PCR to verify the transfer of the target gene. First, a single colony was picked up with a toothpick and added to a 1.5 mL centrifuge tube containing 100 μL of sterile water, then incubated in a boiling water bath for 30 min. Next, the supernatant was centrifuged and used as a template for PCR amplification, with the PCR program set to the standard Taq enzyme amplification procedure. Finally, the colony PCR products were detected by 0.9% agarose gel electrophoresis.

[0081] (7) Sequencing of the recombinant plasmid pMD18-T / tpi containing glyceraldehyde-3-phosphate isomerase

[0082] After culturing the positive recombinant bacteria identified by colony PCR overnight in liquid LB medium, 4 mL of the bacterial culture was used to extract plasmids, following the instructions provided with the AxyPrep plasmid DNA miniature kit. Sequencing was performed by Suzhou Hongxun Biotechnology Co., Ltd. Sequencing verification confirmed that sequence SEQ ID NO:2 had been recombined into pMD18-T / tpi.

[0083] (8) Construction of the recombinant expression plasmid pET-28a / tpi for glyceraldehyde-28 phosphate isomerase

[0084] Based on the principle of exogenous gene expression in Escherichia coli and the comparison of restriction sites between the expression vector pET-28a and the glyceraldehyde-28 phosphate isomerase tpi gene, the EcoRI and HindIII double restriction sites were determined. Recombinant E. coli JM109 / pMD18-T / tpi was cultured in liquid LB tubes in a shaker and the recombinant plasmid was extracted.

[0085] The recombinant plasmid pMD18-T / tpi and expression vector pET-28a of the glyceraldehyde-1-phosphate isomerase (tpi) gene were digested with EcoRI / HindIII restriction endonucleases at 37°C for 6 hours, respectively. The digestion system is shown below:

[0086] EcoRI / HindIII double digestion system. Total system (50μL): vector / target gene (25μL), 10×TangoBuffer (5μL), ddH2O (18μL), EcoRI (1μL), HindIII (1μL).

[0087] After enzyme digestion, the DNA was inactivated at 65°C for 15 min, and then recovered and purified using the Axygen DNA Gel Extraction Kit.

[0088] The glyceraldehyde-28 phosphate isomerase tpi gene and expression vector pET-28a were double-digested and purified, then ligated overnight at 16°C using T4 ligase to construct the recombinant expression plasmid pET-28a / tpi. The resulting glyceraldehyde-28 phosphate isomerase recombinant expression plasmid pET-28a / tpi is shown in the image. Figure 2 The ligation system consisted of the following components: Total volume (20 μL): expression vector pET-28a (5 μL), T4 DNA Ligase (1 μL), target gene (12 μL), and 10×Ligation Buffer (2 μL).

[0089] (9) Transformation of the recombinant expression plasmid pET-28a / tpi for glyceraldehyde-28 phosphate isomerase and screening of positive single clones

[0090] The constructed glyceraldehyde-28a / tpi recombinant expression plasmid was heat-shocked into E. coli BL21 host bacteria, then plated onto LB agar plates containing kanamycin (Kan) resistance (50 mg / L) and incubated overnight at 37°C. Single colonies were randomly selected from the plates, and PCR amplification was performed using primers for each functional gene. Positive clones were selected.

[0091] (10) Inducible expression of glyceraldehyde-28a / tpi phosphate isomerase by recombinant E. coli BL21 / pET-28a / tpi

[0092] Positive monoclonal antibodies were inoculated into 5 mL of LB liquid medium containing Kan resistance (50 mg / L) and incubated overnight at 37°C and 250 rpm. One mL of the culture was then transferred to 50 mL of LB liquid medium containing Kan resistance (50 mg / L) and incubated at 37°C and 250 rpm until the bacterial cell concentration (OD600) reached approximately 0.6–0.8. IPTG at a specific concentration (240 mg / L) was then added to the culture for induction culture for 8 hours. Bacterial cells were collected for electrophoretic analysis and enzyme activity assay.

[0093] (11) SDS-PAGE analysis of the expression product of glyceraldehyde-28a / tpi recombinant E. coli BL21 / pET-28a / tpi.

[0094] E. coli BL21 transformed with an empty vector and recombinant bacteria without IPTG induction were used as controls. After IPTG induction and culture for a certain period (8 h), 1.0 mL of the induced culture was collected by centrifugation, resuspended in 100 μL of distilled water, and 100 μL of loading buffer was added. After mixing and boiling for 10 min, SDS-PAGE electrophoresis analysis was performed. Figure 3 SDS-PAGE images of glyceraldehyde-3-phosphate isomerase tpi expressed by recombinant E. coli BL21 / pET-28a / tpi (its amino acid sequence was verified by sequencing as shown in SEQ ID NO:1) and a control sample of recombinant bacteria without the addition of the inducer IPTG.

[0095] (12) Detection of protein activity of recombinant E. coli BL21 / pET-28a / tpi containing glyceraldehyde-2-phosphate isomerase

[0096] Enzyme solution preparation: Weigh 1.0 g (0.2 g dry weight) of collected recombinant E. coli BL21 / pET-28a / tpi wet cells, suspend in 30 mL of phosphate buffer (50 mM, pH 8.0), and sonicate (350 W power, 2 s of disruption, 2 s interval, 5 min of total disruption) to be used as the enzyme for catalysis.

[0097] phosphoglyceraldehyde isomerase tpi conversion system: Add 20 mL of E. coli BL21 / pET-28a / tpi ultrasonically disrupted bacterial cells and 0.2 g of D-glyceraldehyde-3-phosphate to a 50 mL conversion flask. Invert at 30 °C and 150 rpm for 2–3 h. After conversion, centrifuge and collect the supernatant for subsequent detection.

[0098] After testing and calculation, the results showed that the maximum substrate conversion rate of the glyceraldehyde-3-phosphate isomerase expressed by the recombinant E. coli BL21 / pET-28a / tpi was approximately 29.6%. This indicates that the present invention has obtained a novel enzyme related to the synthesis of glycerol phosphate, a precursor of fungal traditional Chinese medicine polysaccharides. This enzyme participates in the biocatalysis of D-glyceraldehyde-3-phosphate to glycerol phosphate. Glycerol phosphate can be prepared by constructing a genetically engineered bacterium to biocatalyze the production of D-glyceraldehyde-3-phosphate, or by isolating and purifying the recombinant protein from this genetically engineered bacterium and then biocatalyzing the production of glycerol phosphate from D-glyceraldehyde-3-phosphate; thereby increasing the yield of glycerol phosphate or its derivatives.

Claims

1. The application of glyceraldehyde-3-phosphate isomerase derived from fungal traditional Chinese medicine in the biocatalytic preparation of glycerol phosphate from D-glyceraldehyde-3-phosphate, characterized in that, The amino acid sequence of the phosphoglyceraldehyde isomerase derived from fungal traditional Chinese medicine is shown in SEQ ID No.

1.

2. The application according to claim 1, characterized in that, The application involves using fungal glyceraldehyde-3-phosphate isomerase as a catalyst and D-glyceraldehyde-3-phosphate as a substrate to prepare glycerol phosphate ketone.

Images