A method for improving catalytic efficiency of ergothioneine synthase and an ergothioneine synthase mutant and application thereof

Abstract

The application discloses a method for improving catalytic efficiency of ergothioneine synthase and an ergothioneine synthase mutant and application, and belongs to the technical field of enzyme engineering and genetic engineering. In order to develop efficient ergothioneine synthase, the method provided by the application is to perform molecular modeling and sequence analysis on a wild-type ergothioneine synthase amino acid sequence, determine an amino acid sequence of a template protein, perform molecular docking on a substrate L-histidine betaine to obtain an amino acid site of substrate molecule combination, perform difference comparison on active pocket amino acids of the wild-type ergothioneine synthase amino acid sequence and amino acids in a range around the obtained amino acid site, determine a difference amino acid site as a key amino acid active site, and perform saturation mutation on the obtained key amino acid active site, and select an amino acid site with the largest mutation energy difference as a mutation site. The application has wide application space and market prospect in the field of functional nutritional product synthesis.

Description

Technical Field

[0001] This invention belongs to the fields of enzyme engineering and genetic engineering technology, specifically relating to a method for improving the catalytic efficiency of ergothioneine synthase and an ergothioneine synthase mutant and its application. Background Technology

[0002] Ergothioneine (EGT) is a natural amino acid derivative derived from histidine. As a natural antioxidant, it has multiple functions, including anti-aging, cancer prevention, maintaining DNA biosynthesis, maintaining normal cell growth, and maintaining cellular immune function. It is widely used in food, medicine, and cosmetics. Some bacteria, non-yeast fungi, and single-celled cyanobacteria can synthesize ergothioneine, while plants and animals cannot synthesize it and must obtain it through diet or the surrounding environment. Ergothioneine is mainly produced through chemical synthesis and natural extraction. Fermentation extraction of ergothioneine using edible fungi mycelium has a long fermentation cycle and low yield, and the chemically synthesized product has low safety. Microbial synthesis of ergothioneine has many advantages, including mild reaction conditions, renewable raw materials, natural products, and simple purification processes. Its safety has been recognized by various safety assessment agencies in the United States, the European Union, and China, which will accelerate its application and demand in the market. Ergothioneine biosynthesis has been achieved in various systems, including Escherichia coli, Bacillus, Aspergillus oryzae, and Saccharomyces cerevisiae. However, the synthesis efficiency and yield of ergothioneine remain uncompetitive and have not yet been mass-produced. Therefore, it is essential to find new genes and methods to further improve ergothioneine yield.

[0003] Researchers are constantly searching for new enzyme resources. For example, Egt1, derived from enoki mushrooms, oyster mushrooms, maitake mushrooms, and Reeschizon's enzyme, has been used for ergothioneine synthesis. However, naturally derived enzymes often have low catalytic efficiency, making it difficult to synthesize ergothioneine efficiently and in a targeted manner. Therefore, developing efficient ergothioneine synthases is crucial for the efficient synthesis of ergothioneine. However, no relevant research has been reported to date. Summary of the Invention

[0004] The purpose of this invention is to develop a highly efficient ergothionein synthase.

[0005] This invention provides a method for improving the catalytic efficiency of ergothionein synthase, the steps of which are as follows:

[0006] Step 1: Molecular modeling and sequence analysis were performed on the amino acid sequence of wild-type ergothioneine synthase to determine the amino acid sequence of the template protein and the three-dimensional structure of wild-type ergothioneine synthase.

[0007] Step 2: Molecular docking of the substrate L-histidine betaine is performed to obtain the amino acid sites that bind to the substrate molecule;

[0008] Step 3: The active pocket amino acids of the wild-type ergothioneine synthase amino acid sequence are compared with the amino acids within a 3.5 Å range around the amino acid sites obtained in Step 2 to determine the differential amino acid sites as key amino acid active sites.

[0009] Step 4: Site-directed and saturation mutagenesis were performed on the key amino acid active sites obtained in Step 3. The amino acid site with the largest difference in mutation energy was selected as the mutation site and transferred into Saccharomyces cerevisiae. Activity was detected to obtain a mutant of ergothioneine synthase with enhanced enzyme activity.

[0010] Further specifying, the amino acid sequence of the wild-type ergothionein synthase is as shown in SEQ ID NO.1.

[0011] To further define the specific method of step 1, the SWISS-MODEL online server is used to search for template structures in the RCSB PDB protein crystal database by inputting the amino acid sequence of ergothionein synthase Egt1, based on sequence similarity, and the protein with the highest sequence similarity and crystal resolution is selected as the template protein for modeling.

[0012] To further define the specific method of step 2, the method is as follows: obtain the molecular structure file of the substrate L-histidine betaine, convert the molecular structure file of L-histidine betaine into a pdb file, input both the molecular pdb file of L-histidine betaine and the three-dimensional structure file of ergothioneine synthase Egt1 into the molecular docking software Autodock 4.2, obtain the binding energy of the binding site of the ergothioneine synthase Egt1 protein molecule, and obtain the amino acid sites of the substrate molecule based on the order of binding energy.

[0013] Furthermore, the amino acid sites for obtaining substrate molecule binding are selected as those with the highest binding energy.

[0014] A mutant of ergothioneine synthase, said mutant is obtained by mutating the amino acid at position 857, using the ergothioneine synthase amino acid sequence as shown in SEQ ID NO. 1 as the starting sequence.

[0015] To further refine the process, the aspartic acid at position 857 was mutated to glutamic acid.

[0016] The present invention provides the gene for the above-mentioned ergothionein synthase mutant.

[0017] The present invention provides a recombinant vector carrying the above-mentioned coding gene.

[0018] Further specifying, the starting vector of the recombinant vector is any one of the pET series, Duet series, pGEX series, pHY300, pHY300PLK, pPIC3K, pESC or pPIC9K series.

[0019] Further specifying, the recombinant vector carries the above-mentioned coding gene and nucleotide sequence as shown in SEQ ID NO. 3, which is the EGT2 gene.

[0020] The present invention provides a recombinant microbial cell that expresses the above-mentioned mutant, carries the above-mentioned gene, or carries the above-mentioned recombinant vector.

[0021] The present invention provides the application of the above-mentioned mutant, the above-mentioned gene, the above-mentioned recombinant vector or the above-mentioned recombinant microbial cell in the synthesis of ergothionein.

[0022] This invention provides a method for synthesizing ergothionein, wherein the above-mentioned mutant is fermented with shaking at 30°C for 48-72 h.

[0023] Further specifying the fermentation medium, the fermentation medium consisted of 4 g / L galactose, 3 g / L yeast extract, 7 g / L NH4H2PO4, 1 g / L KH2PO4, 0.5 g / L MgSO4•7H2O, 50 mM succinate, 12 mL / L vitamin solution, and 10 mL / L trace metal ion solution.

[0024] Further specified, the vitamin solution comprises 50 mg / L biotin, 200 mg / L p-aminobenzoic acid, 1 g / L nicotinic acid, 1 g / L calcium pantothenate, 1 g / L pyridoxine hydrochloride, 1 g / L thiamine hydrochloride, and 25 g / L inositol; the trace metal ion solution comprises 4.5 g / L CaCl2·2H2O, 4.5 g / L ZnSO4·7H2O, 3 g / L FeSO4·7H2O, 1 g / L H3BO3, 1 g / L LmnCl2·4H2O, 0.4 g / L Na2MoO4·2H2O, 0.3 g / L CoCl2·6H2O, 0.1 g / L CuSO4·5H2O, 0.1 g / L KI, and 15 g / L EDTA.

[0025] Beneficial Effects: This invention utilizes rational and semi-rational methods to optimize the performance of ergothioneine synthase, resulting in an ergothioneine synthase mutant with enhanced catalytic efficiency. This provides a new synthetic element for the biosynthesis of ergothioneine, and has broad application prospects and market potential in the field of functional nutrient synthesis. Attached Figure Description

[0026] Figure 1A schematic diagram of the three-dimensional structure after homology modeling of EGT1.

[0027] Figure 2 This is a schematic diagram of multiple sequence alignment of ergothionein synthase.

[0028] Figure 3 This is a schematic diagram of the amino acid binding pocket for EGT1 substrates.

[0029] Figure 4 This is a schematic diagram of the substrate binding pocket after EGT1 docks with the substrate molecule hercynine.

[0030] Figure 5 This is a schematic diagram of the amino acid at a distance of 3.5 Å centered on hercynine.

[0031] Figure 6 This is a schematic diagram of the results of virtual saturation mutations of amino acids.

[0032] Figure 7 The image shows the plasmid pESC-HIS-EGT1-EGT2.

[0033] Figure 8 This is a comparison diagram of the catalytic activity of the mutant and wild-type proteins. Detailed Implementation

[0034] The present invention will be further described below with reference to specific embodiments, but the present invention is not limited to the embodiments. The computational methods, plasmids, etc. used can be obtained commercially or by conventional means known to those skilled in the art.

[0035] Example 1. Molecular modeling and sequence analysis

[0036] (1) Molecular homology modeling

[0037] Using the SWISS-MODEL online server, the amino acid sequence of ergothioneine synthase Egt1 (as shown in SEQ NO.1) was input. Template structures were searched in the RCSB PDB protein crystal database based on sequence similarity, and the protein with the highest sequence similarity and crystal resolution was selected as the template protein for modeling. This yielded a three-dimensional simulated structure of ergothioneine synthase Egt1, as shown below. Figure 1 As shown. The modeled protein structure was visualized using PyMOL and Discovery Studio.

[0038] (2) Protein sequence analysis is used to obtain conserved sequences.

[0039] In the NCBI website, the protein BLSAT sequence for ergothioneine synthase Egt1 (NCBI Sequence ID: XP_956324.3) was entered. The top five similarity sequences were selected, which were the amino acid sequences of other similar proteins (NCBI Sequence ID: 4X8B_A, NCBI Sequence ID: XP_956324.3, NCBI Sequence ID: NCBI WP_076930170.1, and NCBI Sequence ID: WP_003419806.1). The sequence identity and similarity of different enzymes were analyzed using multiple sequence alignment on the Clustal Omega web server.

[0040] like Figure 2 As shown, the template protein (Sequence ID: 4X8B_A) has good sequence homology with ergothionein synthase Egt1, with a sequence similarity of 25.25%, and is currently the only protein with a crystal structure.

[0041] Example 2. This example describes a method for screening amino acid sites that enhance the activity of ergothionein synthase.

[0042] 1. By comparing the structure of the template protein (RCSB PDB ID: 4X8B) with the target protein Egt1, the small molecule binding site of the target protein is obtained, so as to further obtain the binding mode between the protein and the substrate molecule through molecular docking.

[0043] (1) Comparison of amino acids at active sites

[0044] First, the obtained template protein crystal structure was visualized, and the active substrate molecule binding sites on the template protein were identified as active pocket amino acids as recorded in the PDB database.

[0045] like Figure 3 As shown, the protein small molecule binding pocket consists of 11 amino acids, including 6 polar amino acids (H506, H510, N450, D579, T827, and N857) and 5 relatively specific hydrophobic amino acids (Y823, Y826, Y863, W858, and Y859). The vast majority of the 5 hydrophobic amino acids are tyrosine (Y).

[0046] II. Molecular docking method for ergothionein synthase

[0047] L-histidine betaine was selected as the substrate and docked into the protein ergothionein synthase Egt1 to obtain the binding mode between the two:

[0048] First, download the molecular structure file of the substrate Hercynine from the small molecule structure database PubChem (https: / / pubchem.ncbi.nlm.nih.gov / 440727), and convert it to a PDB file using Open Babel. Input both the Hercynine PDB file and the 3D structure file of ergothioneine synthase Egt1 obtained in Example 1 into the molecular docking software Autodock 4.2 to dock Hercynine into the binding site of the ergothioneine synthase Egt1 protein molecule. The protein is set as the receptor, and Hercynine as the ligand. The specific molecular docking parameters are as follows:

[0049] (1) A 1.2 nm grid box is formed around the substrate binding sites on the receptor as the geometric center to encompass all the extra space at the center of the binding sites. Different types of atoms are used as probes to scan the grid and calculate the grid energy; this part of the task is completed by the AutoGrid program.

[0050] (2) Using the AutoDock program, the small molecule ligand Hercynine is subjected to a conformational search within the grid box. Finally, the results are scored based on the different conformations, orientations, positions, and energies of the ligands, and then ranked.

[0051] (3) The Autodock docking software identified 50 interaction phases in the molecular systems. Using the built-in clustering analysis module of the Autodock software, the structures were clustered and ranked according to the molecular docking energy scores. The docking phase with the most clusters and the best energy scores was selected as the binding mode complex system between ergothionein synthase Egt1 protein and L-histidine betaine.

[0052] The results are as follows Figure 3 As shown, L-histidine betaine forms good polar hydrogen bonds with amino acids H506, H510, and N857 on the protein. Meanwhile, the -N(CH3)3 terminal of the small molecule forms good hydrophobic bonds with neighboring amino acids Y823, Y826, W858, Y859, and Y863. The binding energy for both polar hydrogen bonds and hydrophobic bonds is -5.8 kcal / mol, indicating good binding ability.

[0053] III. Virtual screening and saturation mutagenesis of amino acids near the substrate binding pocket

[0054] Amino acids within a 3.5 Å range surrounding the substrate molecule obtained in step two (amino acids H506, H510, and N857 on the protein in Example 2, and neighboring Y823, Y826, W858, Y859, and Y863) were selected. The differences between the template protein and the active pocket structure amino acids of ergothioneine synthase EGT1 (11 amino acids obtained in Example 1) were compared using PyMOL software. The amino acids with differences in ergothioneine synthase EGT1 were then subjected to site-directed saturation mutagenesis to determine whether the site was a key site for improving activity.

[0055] Figure 5 As shown, there are two different amino acid sites on the Egt1 protein, namely MET509 and ASN857.

[0056] Using Discovery Studio software, virtual saturation mutations were performed on key amino acids MET509 and ASN857. The mutation energies obtained from these virtual saturation mutations were used to determine the optimal combination of amino acid mutations.

[0057] like Figure 6 As shown, among the 38 mutation sites corresponding to amino acids MET509 and ASN857, the ASN857GLU mutation resulted in the largest difference in mutation energy. Therefore, the ASN857GLU mutation site was selected for further experimental verification.

[0058] Example 3. Construction of recombinant plasmids

[0059] The amino acid sequence of wild-type ergothionein synthase Egt1 is shown in SEQ ID NO.1.

[0060] Construction of ergothioneine synthase expression vector: Ergothioneine synthase gene EGT1 (N) 857E (its sequence is shown in SEQ ID NO.2) and EGT2 (Its sequence is shown in SEQ ID NO.3) cloned into the pESC-HIS plasmid, as follows: Figure 7 As shown.

[0061] EGT1 (N857E) (sequence shown in SEQ NO.2) and EGT2 (sequence shown in SEQ NO.3) were obtained by PCR, and the primers used are shown in Table 1. The pESC-HIS plasmid was linearized using restriction endonucleases BamHI and SalI, and then the EGT1 (N857E) fragment was ligated into the pESC-HIS plasmid using the Hieff Clone® Plus Multi One Step Cloning Kit (10912ES10) to obtain the recombinant plasmid pESC-HIS-EGT1 (N857E). The pESC-HIS-EGT1 plasmid was linearized using EcoRI and SpeI, and then the EGT2 fragment was ligated into the pESC-HIS-EGT1 plasmid using the Hieff Clone® Plus Multi One Step Cloning Kit (10912ES10) to obtain the recombinant plasmid pESC-HIS-EGT1(N857E)-EGT2.

[0062] Table 1 Primers

[0063]

[0064] Wild-type plasmids: EGT1 (sequence shown in SEQ NO.1) and EGT2 (sequence shown in SEQ NO.3) were obtained by PCR. The pESC-HIS plasmid was linearized using restriction endonucleases BamHI I and Sal I. Then, the EGT1 fragment was ligated into the pESC-HIS plasmid using the Hieff Clone® Plus Multi One Step Cloning Kit (10912ES10) to obtain the recombinant plasmid pESC-HIS-EGT1. The pESC-HIS-EGT1 plasmid was linearized using EcoRI and SpeI. The EGT2 fragment was then ligated into the pESC-HIS-EGT1 plasmid using the Hieff Clone® Plus Multi One Step Cloning Kit (10912ES10) to obtain the recombinant plasmid pESC-HIS-EGT1-EGT2.

[0065] Example 4. Construction of recombinant bacteria

[0066] The pESC-HIS-EGT1(N857E)-EGT2 plasmid from Example 3 was transformed into a yeast strain by chemical transformation to obtain the pESC-HIS-EGT1(N857E)-EGT2 recombinant strain.

[0067] Example 5. Expression and purification of ergothionein synthase

[0068] The catalytic activity was described by detecting the most abundant product in the pESC-HIS-EGT1(N857E)-EGT2 recombinant strain obtained in Example 4.

[0069] Example 6. Application of ergothioneine synthase mutant in the synthesis of ergothioneine.

[0070] The application of synthesized ergothioneine described in this embodiment refers to the use of the ergothioneine synthase Egt1 mutant (N obtained by the method of this invention) 857E The proteins pESC-HIS-EGT1(N857E)-EGT2 and EGT2 are transferred into yeast. Under conditions containing suitable carbon sources, such as sucrose and galactose as raw materials, nitrogen sources, and other growth factors, the genetically engineered yeast strain can convert sugars in the fermentation broth to produce ergothionein. Ergothionein can be detected from the fermentation broth using liquid chromatography and other detection equipment.

[0071] Strain activation: Take the strain stored at -80℃, streak it on a YPD plate, and incubate it in a 30℃ incubator for 2 days. Pick a single colony and inoculate it into liquid YPD medium. Incubate the strain at 30℃ and 180 rpm on a shaker to activate the strain and obtain the primary seed.

[0072] The yeast genetically engineered strain for synthesizing ergothioneine described in this invention is suitable for any liquid culture medium for medium to large-scale culture of engineered yeasts, preferably YPD liquid culture medium, which has the following formulation: 20 g / L glucose, 10 g / L yeast extract, 20 g / L peptone, and the remainder being water.

[0073] Cell fermentation: Well-grown activated bacterial strains were transferred to Erlenmeyer flasks containing 50 mL of YPD liquid medium and cultured at 180 rpm and 30 ℃ for 24 h to obtain secondary seed culture. The secondary seed culture was inoculated into the fermentation medium at 5% of the medium volume. The preferred secondary seed culture medium consisted of: 4 g / L galactose, 3 g / L yeast extract, 7 g / L NH4H2PO4, 1 g / L KH2PO4, 0.5 g / L MgSO4•7H2O, 50 mM succinate (pH 5.0), 12 mL / L vitamin solution (50 mg / L biotin, 200 mg / L para-aminobenzoic acid, 1 g / L nicotinic acid, 1 g / L calcium pantothenate, 1 g / L pyridoxine hydrochloride, 1 g / L thiamine hydrochloride, 25 g / L inositol), and 10 mL / L trace metal ion solution (4.5 g / L CaCl2·2H2O). 4.5 g / L ZnSO4·7H2O, 3 g / L FeSO4·7H2O, 1 g / L H3BO3, 1 g / L MnCl2·4H2O, 0.4 g / L Na2MoO4·2H2O, 0.3 g / L CoCl2·6H2O, 0.1 g / L CuSO4·5H2O, 0.1 g / L KI, 15 g / L EDTA, with the remainder being water.

[0074] The liquid volume in the fermentation flask should not exceed 1 / 10 to 1 / 5 of the total volume to ensure aerobic conditions for cell growth. The fermentation flask is then transferred to an incubator at 30℃ and 160 to 180 rpm for 72 hours with shaking. The product is then quantitatively detected using liquid chromatography.

[0075] The ergothioneine synthase mutant exhibits approximately 10% increased enzyme catalytic activity, such as... Figure 8 As shown.

[0076] Formula for the enzyme catalytic activity of ergothioneine synthase mutant: Ergothioneine yield in mutant strains - Ergothioneine yield in wild-type egt1 strains / Ergothioneine yield in wild-type egt1 strains.

[0077] SEQ ID NO.1:

[0078] MPSAESMTPSSALGQLKATGQHVLSKLQQQTSNADIIDIRRVAVEINLKTEITSMFRPKDGPRQLPTLLLYNERGLQLFERITYLEEYYLTNDEIKILTKHATEMASFIPSGAMIIELGSGNLRKVNLLLEALDNAGKAIDYYALDLSREELERTLAQVPSYKHVKCHGLLGTYDDGRDWLKAPENINKQKCILHLGSSIGNFNRSDAATFLKGFTDVLGPNDKMLIGVDACNDPARVYHAYNDKVGITHEFILNGLRNANEIIGETAFIEGDWRVIGEYVYDEEGGRHQAFYAPTRDTMVMGELIRSHDRIQIEQSLKYSKEESERLWSTAGLEQVSEWTYGNEYGLHLLAKSRMSFSLIPSVYARSALPTLDDWEALWATWDVVTRQMLPQEELLEKPIKLRNACIFYLGHIPTFLDIQLTKTTKQAPSEPAHFCKIFERGIDPDVDNPELCHAHSEIPDEWPPVEEILTYQETVRSRLRGLYAHGIANIPRNVGRAIWVGFEHELMHIETLLYMMLQSDKTLIPTHIPRPDFDKLARKAESERVPNQWFKIPAQEITIGLDDPEDGSDINKHYGWDNEKPPRRVQVAAFQAQGRPITNEEYAQYLLEKNIDKLPASWARLDNENISNGTTNSVSGHHSNRTSKQQLPSSFLEKTAVRTVYGLVPLKHALDWPVFASYDELAGCAAYMGGRIPTFEETRSIYAYADALKKKKEAERQLGRTVPAVNAHLTNNGVEITPPSSPSSETPAESSSPSDSNTTLITTEDLFSDLDGANVGFHNWHPMPITSKGNTLVGQGELGGVWEWTSSVLRKWEGFEPMELYPGYTADFFDEKHNIVLGGSWATHPRIAGRKSFVNWYQRNYPYAWVGARVVRDL。

[0079] SEQ ID NO.2:

[0080]

[0081] SEQ ID NO.3:

[0082]

Claims

1. An ergothioneine synthase mutant, characterized in that, The mutant is based on the ergothionein synthase Egt1 from Neurospora crassa, whose amino acid sequence is shown in SEQ ID NO.1, with the aspartic acid at position 857 mutated to glutamic acid.

2. The encoding gene of the ergothionein synthase mutant according to claim 1.

3. A recombinant vector, characterized in that, The recombinant vector carries the coding gene described in claim 2.

4. A recombinant microbial cell, characterized in that, Microbial cells expressing the mutant of claim 1, carrying the gene of claim 2, or carrying the recombinant vector of claim 3.

5. The use of the mutant of claim 1, the encoding gene of claim 2, the recombinant vector of claim 3, or the recombinant microbial cell of claim 4 in the synthesis of ergothionein.

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